Total synthesis and biological evaluation of 22-hydroxyacuminatine

Article abstract of DOI:10.1021/jm051116e

A total synthesis of 22-hydroxyacuminatine, a cytotoxic alkaloid isolated from Camptotheca acuminata, is reported. The key step in the synthesis involves the reaction of 2,3-dihydro-1H-pyrrolo[3,4-b]quinoline with a brominated phthalide to generate a substituted pentacyclic 12H-5,11a-diazadibenzo[b,h] fluoren-11-one intermediate. Despite its structural resemblance to camptothecin and luotonin A, a biological evaluation of 22-hydroxyacuminatine in a topoisomerase I-deficient cell line P388/CPT45 has confirmed that the observed cytotoxicity is not due to topoisomerase I inhibition, even though 22-hydroxyacuminatine has a hydroxyl group that can theoretically hydrogen bond to Asp533. This result is consistent with the hypothesis that π-π stacking is more important than hydrogen-bonding interactions in determining topoisomerase I inhibitor binding in the ternary cleavage complex.

Full text of DOI:10.1021/jm051116e

1408
J. Med. Chem. 2006, 49, 1408-1412
Total Synthesis and Biological Evaluation of 22-Hydroxyacuminatine
Xiangshu Xiao,† Smitha Antony,‡ Yves Pommier,‡ and Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology and the Purdue Cancer Center, School of Pharmacy and Pharmaceutical
Sciences, Purdue UniVersity, West Lafayette, Indiana 47907, and Laboratory of Molecular Pharmacology, Center for Cancer Research,
National Cancer Institute, Bethesda, Maryland 20892-4255
ReceiVed NoVember 6, 2005
A total synthesis of 22-hydroxyacuminatine, a cytotoxic alkaloid isolated from Camptotheca acuminata, is
reported. The key step in the synthesis involves the reaction of 2,3-dihydro-1H-pyrrolo[3,4-b]quinoline with
a brominated phthalide to generate a substituted pentacyclic 12H-5,11a-diazadibenzo[b,h]fluoren-11-one
intermediate. Despite its structural resemblance to camptothecin and luotonin A, a biological evaluation of
22-hydroxyacuminatine in a topoisomerase I-deficient cell line P388/CPT45 has confirmed that the observed
cytotoxicity is not due to topoisomerase I inhibition, even though 22-hydroxyacuminatine has a hydroxyl
group that can theoretically hydrogen bond to Asp533. This result is consistent with the hypothesis that
π-π stacking is more important than hydrogen-bonding interactions in determining topoisomerase I inhibitor
binding in the ternary cleavage complex.
Scheme 1. Retrosynthetic Analysis of Aromathecin 2c
Introduction
Camptothecin (CPT, 1) is a potent topoisomerase I (Top1)
inhibitor with significant cytotoxicity against a variety of
different cancer cell lines.1 Top1 is a nuclear enzyme that relaxes
the DNA supercoils generated during DNA replication and
transcription. The enzyme operates by carrying out a trans-
esterification reaction involving a nucleophilic attack of the
Tyr723 hydroxyl group on a DNA phosphodiester bond, thus
creating a transient nick that is rapidly resealed by the reverse
transesterification reaction.2 CPT inhibits the reverse reaction
by intercalating between the base pairs at the DNA cleavage
site, thus stabilizing the ternary DNA cleavage complex
consisting of the drug, DNA, and the enzyme. Collision of the
DNA replication fork with the single-stranded DNA break then
results in a double-stranded break and cancer cell apoptosis.
Two clinically useful CPT derivatives, topotecan (Hycamtin)
and irinotecan (Camptosar), are presently in clinical use as
anticancer agents.3 However, lactone ring hydrolysis of the CPT
system produces the hydroxy carboxylate which has a high
affinity to human serum albumin protein, and this limits the
efficacy of CPT derivitaves.4 As a consequence, there is interest
in developing metabolically stable non-CPT Top1 inhibitors as
novel anticancer drugs.5
stabilized DNA-Top1 cleavage complex, which revealed that
the crucial 20-OH of CPT is hydrogen bonded with Asp533,7
introduction of an additional hydrogen bond donor to the E-ring
of the aromathecins capable of interacting with Asp533 would
be expected to increase the Top1 inhibitory potency. Therefore,
aromathecin 2b was considered. Interestingly, a review of the
literature revealed that compound 2b had been previously
isolated from Camptotheca acuminata as a rare natural product
(0.000006% yield) named 22-hydroxyacuminatine.8 Although
2b exhibited cytotoxicity toward P388 and KB cell lines, it has
remained to be determined whether this cytotoxicity is due to
Top1 inhibition. This question prompted us to investigate the
total synthesis of 22-hydroxyacuminatine9 as well as its biologi-
cal properties. We also wanted to establish a flexible route that
would open up a way to synthesize analogues for investigation
of structure-activity relationships.
Results and Discussion
Bromide 2c was envisioned as a precursor to 22-hydroxy-
acuminatine (2b) in that a variety of different transformations
are possible with the bromide, which is considered as an
advantage in terms of potential analogue syntheses. By analogy
to the synthesis of CPT10 and aromathecin 2a,6 the bromide 2c
was dissected in the D-ring to give fragments 311 and 4 (Scheme
1).
The synthesis of fragment 4 is presented in Scheme 2.
Methylation of commercially available benzoic acid derivative
5 provided methyl ester 6a. Oxidation of the methyl group in
6a to the corresponding aldehyde proved to be problematic. No
reaction was observed with CrO3/Ac2O/HOAc,12 or ceric
ammonium nitrate (CAN),13 or selenium dioxide (SeO2),14
presumably due to the steric hindrance. Consistent with this
hypothesis, oxidation of 5 with CrO3/Ac2O/HOAc resulted in
overoxidation of the methyl group to a carboxylic acid followed
by cyclization to the anhydride. On the other hand, EÄ tard
As a strategy to enhance stability, replacement of the E-ring
present in CPT with an aromatic ring has been investigated.
The resulting compounds are collectively referred to “aroma-
thecins” as opposed to camptothecins. We have previously
shown that aromathecin 2a is a weak Top1 inhibitor with low
cytotoxicity.6 On the basis of the crystal structure of the CPT-
* Corresponding author. Phone: 765-494-1465. Fax: 765-494-6790.
E-mail: cushman@pharmacy.purdue.edu.
† Purdue University.
‡ National Cancer Institute.
10.1021/jm051116e CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/24/2006

Synthesis and EValuation of 22-Hydroxyacuminatine
Scheme 2. Synthesis of Bromide 4
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1409
Figure 1. AM1-optimized structure of 11.
reaction of methyl ester 6a with chromyl chloride15 provided
lactone 7 instead of the expected aldehyde derivative.16 When
less than 2 equiv of chromyl chloride was used, another product
6b was also generated from the EÄ tard reaction. Radical
bromination of lactone 7 gave the requisite bromide 4 unevent-
fully.
require the preparation of nitrile 13 as one of the building blocks.
To this end, Rosenmund-von Braun reaction18 of bromide 7
with CuCN in refluxing DMF gave cyanide 12 in quantitative
yield leaving the lactone intact (Scheme 4). No reaction was
observed at a temperature of 120 °C or lower. Radical
bromination of the new lactone 12 delivered bromide 13, which
reacted with the tricyclic amine hydrobromide 3 to yield cyanide
14. DIBAL-H reduction followed by hydrolysis and further
DIBAL-H reduction of the resulting aldehyde afforded 22-
hydroxyacuminatine (2b) in 67% yield.
Due to the structural similarity of 22-hydroxyacuminatine (2b)
with CPT (1) and luotonin A (15), both of which are Top1
inhibitors exhibiting Top1-dependent growth inhibition in
yeast,19 a Top1-mediated DNA cleavage assay20 was performed
to examine the Top1 inhibitory potency. In this assay, only
marginal DNA cleavage was observed with 2b, indicating that
it is a weak Top1 inhibitor. To further verify whether the
reported cytotoxicity of 2b8 is due to the weak Top1 inhibition,
the cytotoxicity of 2b in a P388 leukemia cell line and its Top1-
deficient subclone P388/CPT4521 was investigated.22 The IC50
observed for 22-hydroxyacuminatine (2b) in P388 cells is 35.5
( 0.5 µM, while in P388/CPT45 cells (a Top1-deficient cell
line) it is 43.0 ( 1.0 µM. The IC50 in P388 cells is roughly an
order of magnitude higher than the originally reported value.8
More importantly, these values demonstrate that the cytotoxicity
of 2b is not due to Top1 inhibition. It seems unlikely that
differences between the camptothecins and 22-hydroxyacumi-
natine (2b) in their hydrogen bonding to the protein are
responsible for the observed difference in Top1 inhibitory
activities, since docking and energy minimization of 2b in the
ternary complex indicates that, like the lactone form of the
Coupling of fragments 311 and 4 afforded bromide 2c in 20%
yield (Scheme 3). Treatment of 2c with n-BuLi to undergo
bromine-lithium exchange to give the corresponding anion 8,
followed by trapping with paraformaldehyde, would give 22-
hydroxyacuminatine (2b). However, the product formed from
the reaction was 11, while 2b was not formed at all. Employ-
ment of excess n-BuLi (2 equiv) for the reaction did not result
in the formation of any 2b. This result indicated that the
bromine-lithium exchange occurred at a faster rate than
deprotonation at C-5 (see the CPT numbering system in structure
1). However, the resulting anion 8 evidently undergoes rapid
proton transfer involving the 5 position to give benzylic anion
1
9. The H NMR spectrum of the product 11 is interesting in
that the methine proton next to the hydroxymethyl side chain
is presented as a doublet instead of the expected triplet. This
1
unusual H NMR feature is presumably due to intramolecular
hydrogen bond formation between the hydroxyl group and the
lactam oxygen, which results in the H1-C2-C3-H4 dihedral
angle being close to 90°. This dihedral angle was found to be
91.6° after the structure was energetically minimized by AM1
in Gaussian03 (Figure 1).17
The failure to transform 2c to 2b suggests that it might be
advantageous to install the one-carbon unit before the coupling
reaction. Therefore, the possible intermediate 14 with a cyano
group at the desired position was considered, which would
Scheme 3. Synthesis of 2c and Its Reaction with n-BuLi

1410 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Scheme 4. Synthesis of 22-Hydroxyacuminatine (2b)
Xiao et al.
camptothecins,7,23 the hydroxyl group of 2b can form a direct
hydrogen bond with the Asp533 side chain carboxylate of Top1
acid at 0 °C and the reaction mixture was extracted with CHCl3 (3
× 30 mL). The combined organic layers were washed with H2O
(2 × 5 mL) and brine (2 × 5 mL). The organic solution was dried
over Na2SO4, filtered, concentrated, and the residue was subjected
to a flash column chromatography, eluting with CHCl3, yielding a
light yellow solid 9.0 mg (76%): 1H NMR (300 MHz, CDCl3) δ
10.52 (s, 1 H), 8.97 (s, 1 H), 8.81 (d, J ) 8.4 Hz, 1 H), 8.38 (s, 1
H), 8.29 (t, J ) 8.4 Hz, 1 H), 8.22 (d, J ) 8.4 Hz, 1 H), 7.91 (d,
J ) 7.8 Hz, 1 H), 7.82 (t, J ) 7.8 Hz, 1 H), 7.71 (t, J ) 7.8 Hz,
1 H), 7.64 (t, J ) 7.8 Hz, 1 H), 5.39 (s, 2 H). DIBAL-H (1.0 M in
CH2Cl2, 58 µL, 58 µmol) was added to a stirred solution of aldehyde
obtained as above (9.0 mg, 29 µmol) in CH2Cl2 (2 mL) at -78 °C.
The reaction mixture was stirred at -78 °C for 2 h, when MeOH
(0.1 mL) was added to quench the reaction at -78 °C. A saturated
aqueous solution of potassium sodium tartrate (5 mL) was added,
and the solution was stirred at room temperature for 1 h. CHCl3 (3
× 20 mL) was used to extract the product. The combined organic
layers were washed with H2O (2 × 5 mL) and brine (2 × 5 mL).
The organic solution was dried over Na2SO4, filtered, concentrated,
and the residue was subjected to a flash column chromatography,
eluting with CHCl3 and CHCl3-MeOH (10:1), yielding a light
yellow solid 6.0 mg (67%): mp >255 °C (dec) [lit.,8 mp 258-
(Figure 2). On the other hand, the biological evaluation results
are consistent with the hypothesis that π-π stacking is more
important than hydrogen-bonding interactions in determining
inhibitor binding in the ternary cleavage complex,24,25 and the
results are also in agreement with an earlier report that
compound 2a does not exhibit its cytotoxicity by poisoning Top1
in yeast.26 The inefficient π-π stacking between 22-hydroxy-
acuminatine (2b) and the neighboring base pairs is most likely
due to the nonpolar nature of the E-ring, where no heteroatoms
are included or are directly attached, resulting in an electrostati-
cally neutral potential surface (Figure 3) that does not comple-
ment the electrostatics of the adjacent DNA base pairs as well
as the E-ring of camptothecin does. According to the energy-
minimized hypothetical model, the E-ring of 22-hydroxyacumi-
natine is not well positioned to overlap with the adjacent base
pairs (see Figure 2). This further demonstrates that the electronic
properties of the E-ring of the aromathecins and camptothecins
are critical determinants of enzyme inhibitory activity.27
In conclusion, a highly convergent total synthesis of 22-
hydroxyacuminatine (2b) has been achieved. The biological
properties of 22-hydroxyacuminatine were characterized, dem-
onstrating that Top1 inhibition is not a significant factor in its
cytotoxicity despite its structural resemblance to CPT.
1
260 °C (dec)]. H NMR (500 MHz, DMSO-d6) δ 8.66 (s, 1 H),
8.29 (d, J ) 7.5 Hz, 1 H), 8.19 (d, J ) 8.5 Hz, 1 H), 8.11 (d, J )
7.5 Hz, 1 H), 7.83 (td, J ) 8.5, 1.5 Hz, 1 H), 7.81 (d, J ) 7.0 Hz,
1 H), 7.73 (s, 1 H), 7.69 (td, J ) 8.0, 1.5 Hz, 1 H), 7.57 (t, J ) 7.5
Hz, 1 H), 5.51 (t, J ) 5.0 Hz, 1 H), 5.36 (s, 2 H), 4.95 (d, J ) 5.0
Hz, 2 H); ESIMS m/z (rel intensity) 315 (MH+, 100); HRESIMS
m/z Calcd for C20H14N2O2 + H 315.1134, found 315.1136. Anal.
(C20H14N2O2‚1.0H2O) C, H, N.
7-Bromo-12H-5,11a-diazadibenzo[b,h]fluoren-11-one (2c). A
solution of bromide 4 (193 mg, 0.66 mmol) in THF (7 mL) was
added to a stirred suspension of tricyclic amine hydrobromide 3
(220 mg, 0.66 mmol) and Et3N (3.0 mL) in THF (7 mL). The
resulting mixture was stirred at room temperature for 24 h. CHCl3
(100 mL) was added to dilute the reaction mixture, which was then
washed with water (2 × 10 mL) and brine (2 × 10 mL). The organic
solution was dried over anhydrous Na2SO4, filtered, and concen-
trated, yielding a brown residue, which was used for the next
operation without further purification. The product obtained as
above was dissolved in acetic acid (7 mL), and sodium acetate (0.7
g) was added to the reaction mixture, which was then stirred at
room temperature for 36 h. Then acetic acid was removed under
Experimental Section
22-Hydroxyacuminatine (2b). DIBAL-H (1.0 M in CH2Cl2, 57
µL, 57 µmol) was added to a stirred solution of nitrile 14 (11.7
mg, 38 µmol) in toluene (3 mL) at -78 °C. The resulting reaction
mixture was stirred at -78 °C for 3 h, when MeOH (0.1 mL) was
added to quench the reaction. The reaction mixture was poured
into a 1.0 M solution of oxalic acid (5 mL) and stirred at room
temperature for 2 h. Solid Na2CO3 was added to neutralize the oxalic
Figure 2. Hypothetical model of the binding of 22-hydroxyacuminatine (2b) in the Top1-DNA-inhibitor ternary complex. The cleaved DNA
strand is on the left, while the uncleaved strand is on the right. The DNA minor groove side is facing forward, while the major groove is on the
back. The model is programmed for walleyed (relaxed) viewing.

Synthesis and EValuation of 22-Hydroxyacuminatine
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1411
was dried over anhydrous Na2SO4, filtered, and concentrated,
1
yielding a colorless powder (356 mg, 75%): mp 88-89 °C. H
NMR (300 MHz, CDCl3) δ 7.67 (d, J ) 7.8 Hz, 1 H), 7.63 (d, J
) 7.8 Hz, 1 H), 7.31 (t, J ) 7.8 Hz, 1 H), 5.03 (s, 2 H); 13C NMR
(75 MHz, CDCl3) δ 169.4, 146.2, 136.3, 130.5, 127.4, 124.0, 116.1,
69.4; EIMS m/z (rel intensity) 214 (81BrM+, 31), 212 (79BrM+, 30),
185 (100), 183 (98), 157 (27), 155 (29), 75(63). Anal. (C8H5BrO2)
C, H, Br.
12-Hydroxymethyl-12H-5,11a-diaza-dibenzo[b,h]fluoren-11-
one (11). n-BuLi (16 µL, 2.5 M in hexane, 0.04 mmol) was added
to a stirred solution of bromide 2c (10 mg, 0.027 mmol) in THF (2
mL) at -78 °C. The resulting dark red solution was stirred at -78
°C for 15 min, when a suspension of paraformaldehyde (4 mg, 0.135
mmol) in THF (0.5 mL) was added. The reaction mixture was then
allowed to warm to room temperature and stirred at room
temperature for 1 h. H2O (2 mL) was added to quench the reaction.
CHCl3 (50 mL) was added to dilute the reaction mixture, which
was then washed with H2O (2 × 5 mL) and brine (2 × 5 mL). The
organic solution was dried over anhydrous Na2SO4, filtered, and
concentrated. The residue was subjected to silica gel flash column
chromatography, eluting with CHCl3-MeOH (20:1), yielding a light
Figure 3. Electrostatic potential surfaces of 22-hydroxyacuminatine
(top) and camptothecin (bottom) mapped on their total electron densities
calculated at the HF/6-31G** level of theory. Both electrostatic potential
surface maps are scaled to a range of -38 to +38 kcal/mol.
1
reduced pressure, and the residue was dissolved in CHCl3 (100 mL),
and washed with saturated NaHCO3 (2 × 10 mL), water (2 × 10
mL), and brine (2 × 10 mL). The organic solution was dried over
Na2SO4, filtered, concentrated, and the residue was subjected to
silica gel (40 g) flash column chromatography, eluting with CHCl3,
yielding a light yellow solid (49 mg, 20%): mp >270 °C (dec).
1H NMR (300 MHz, CDCl3) δ 8.50 (d, J ) 8.4 Hz, 1 H), 8.37 (s,
1 H), 8.31 (d, J ) 8.4 Hz, 1 H), 8.10 (s, 1 H), 7.97 (d, J ) 7.5 Hz,
1 H), 7.91 (d, J ) 7.8 Hz, 1 H), 7.82 (t, J ) 7.2 Hz, 1 H), 7.64 (t,
J ) 6.9 Hz, 1 H), 7.40 (t, J ) 7.8 Hz, 1 H), 5.37 (s, 2 H); ESIMS
m/z (rel intensity) 365 (81BrMH+, 100), 363 (79BrMH+, 100). Anal.
(C19H11N2OBr) C, H, N.
yellow solid (2.5 mg, 29%): mp >250 °C (dec). H NMR (300
MHz, CDCl3) δ 8.53 (d, J ) 8.1 Hz, 1 H), 8.32 (s, 1 H), 8.25 (d,
J ) 8.7 Hz, 1 H), 7.92 (d, J ) 7.8 Hz, 1 H), 7.73-7.83 (m, 4 H),
7.58-7.66 (m, 2 H), 5.95 (d, J ) 7.5 Hz, 1 H), 4.38 (d, J ) 12.3
Hz, 1 H), 4.05 (dd, J ) 12.6, 7.5 Hz, 1 H); 13C NMR (75 MHz,
CDCl3) δ 163.0, 149.1, 139.6, 137.4, 133.1, 131.2, 130.7, 129.9,
129.5, 128.8, 128.5, 128.2, 127.9 (2 C), 127.6 (2 C), 127.1, 102.3,
66.8, 66.0; ESIMS m/z (rel intensity) 315 (MH+, 100); HREIMS
m/z Calcd for C20H14N2O2 314.1055, found 314.1046.
3-Cyano-phthalide (12). A mixture of bromide 7 (1.0 g, 4.72
mmol) and CuCN (634 mg, 7.1 mmol) in anhydrous DMF (15 mL)
was heated under reflux for 12 h. The reaction mixture was cooled
to room temperature and quenched by slow addition of brine (20
mL). EtOAc (3 × 60 mL) was used to extract the product. The
combined organic layers were washed with H2O (2 × 20 mL) and
brine (2 × 20 mL). The organic solution was dried over anhydrous
Na2SO4, filtered, and concentrated. The residue was subjected to
silica gel flash column chromatography, eluting with hexanes-
EtOAc (4:1), yielding a white powder (750 mg, 100%): mp 173-
174 °C. 1H NMR (300 MHz, CDCl3) δ 8.13 (d, J ) 7.5 Hz, 1 H),
7.97 (d, J ) 7.8 Hz, 1 H), 7.69 (t, J ) 7.5 Hz, 1 H), 5.45 (s, 2 H);
13C NMR (75 MHz, CDCl3) δ 168.8, 149.3, 137.3, 130.1 (2 C),
127.4, 114.9, 107.4, 68.6; IR (film) 3077, 2235, 1770, 1267, 1020,
754 cm-1; CIMS m/z (rel intensity) 160 (MH+, 100). Anal. (C9H5-
NO2) C, H, N.
3,4-Dibromo-3H-isobenzofuran-1-one (4). A mixture of lactone
7 (906 mg, 4.27 mmol), NBS (818 mg, 4.7 mmol), and AIBN (71
mg, 0.43 mmol) in CCl4 (40 mL) was heated at reflux for 15 h.
CCl4 was removed under reduced pressure. The residue was
extracted with hot n-hexane (3 × 100 mL). Removal of hexane
1
yielded a white powder (1.25 g, 100%): mp 89-90 °C. H NMR
(300 MHz, CDCl3) δ 7.89 (d, J ) 7.8 Hz, 1 H), 7.87 (d, J ) 7.8
Hz, 1 H), 7.50 (t, J ) 7.8 Hz, 1 H), 7.22 (s, 1 H); 13C NMR (75
MHz, CDCl3) δ 166.2, 147.3, 138.7, 132.6, 126.3, 125.0, 117.8,
74.5; CIMS m/z (rel intensity) 295 (81Br81BrMH+, 29), 293
(81Br79BrMH+, 63), 291 (79Br79BrMH+, 31), 100 (100). Anal. (C8H4-
Br2O2) C, H, N.
Methyl 3-Bromo-2-methylbenzoate (6a). Concentrated H2SO4
(0.5 mL) was added to a stirred solution of 3-bromo-2-methylben-
zoic acid (5) (2.15 g, 10 mmol) in MeOH (20 mL) at room
temperature. The resulting mixture was then heated under reflux
for 12 h. Methanol was partially removed under reduced pressure,
and Et2O (150 mL) was added to dilute the residue, which was
then washed with a saturated aqueous solution of NaHCO3 (2 ×
10 mL), H2O (2 × 10 mL), and brine (2 × 10 mL). The organic
solution was dried over anhydrous Na2SO4, filtered, and concen-
trated, yielding a colorless liquid oil (2.15 g, 94%): 1H NMR (300
MHz, CDCl3) δ 7.64 (d, J ) 7.8 Hz, 1 H), 7.60 (d, J ) 7.8 Hz, 1
H), 7.00 (t, J ) 7.8 Hz, 1 H), 3.98 (s, 3 H), 2.55 (s, 3 H); 13C
NMR (75 MHz, CDCl3) δ 167.8, 138.7, 135.9, 132.6, 129.1, 127.1,
126.9, 52.2, 20.6; IR (film) 2997, 2951, 1725, 1434, 1285, 1255,
1096, 1010, 753 cm-1; EIMS m/z (rel intensity) 230 (81BrM+, 19),
228 (79BrM+, 19), 89 (78), 63 (100). Anal. (C9H9BrO2) C, H, Br.
3-Bromo-phthalide (7). A solution of methyl ester 6a (510 mg,
2.23 mmol) in CCl4 (3 mL) was placed in a round-bottomed flask
immersed in an ice-cold bath. A solution of chromyl chloride (0.36
mL, 4.45 mmol) in CCl4 (3 mL) was added over a period of 1 h.
The resulting mixture was stirred at room temperature for 2 h and
then slowly heated to reflux for 20 h. The reaction mixture was
cooled to room temperature and poured into to an ice-cold saturated
solution of Na2SO3 (5 mL) at 0 °C. EtOAc (3 × 30 mL) was used
to extract the product. The combined organic layers were washed
with H2O (2 × 10 mL) and brine (2 × 10 mL). The organic solution
3-Bromo-4-cyano-3H-isobenzofuran-1-one (13). A mixture of
lactone 12 (705 mg, 4.43 mmol), NBS (868 mg, 4.88 mmol), and
AIBN (73 mg, 0.443 mmol) in CCl4 (30 mL) was heated under
reflux for 60 h. CCl4 was removed under reduced pressure, and
the residue was extracted with Et2O. Evaporation of Et2O resulted
in a residue that was subjected to silica gel flash column chroma-
tography, eluting with hexanes-EtOAc (4:1), yielding a white
1
powder 922 mg (87%): mp 118-119 °C. H NMR (300 MHz,
CDCl3) δ 8.16 (d, J ) 7.8 Hz, 1 H), 8.05 (d, J ) 7.8 Hz, 1 H),
7.78 (t, J ) 7.8 Hz, 1 H), 7.50 (s, 1 H); 13C NMR (75 MHz, CDCl3)
δ 177.7, 150.5, 138.8, 131.8, 130.2, 125.6, 113.8, 108.3, 71.9; CIMS
m/z (rel intensity) 240 (81BrMH+, 18), 238 (79BrMH+, 20), 158
(100).
7-Cyano-12H-5,11a-diazadibenzo[b,h]fluoren-11-one (14). A
solution of bromide 13 (150 mg, 0.63 mmol) in CH3CN (2 mL)
was added to a stirred suspension of tricyclic amine hydrobromide
3 (105 mg, 0.31 mmol) and pyridine (1.5 mL) in CH3CN (2 mL).
The resulting mixture was stirred at room temperature for 24 h.
CHCl3 (100 mL) was added to dilute the reaction mixture, which
was then washed with water (2 × 10 mL) and brine (2 × 10 mL).
The organic solution was dried over anhydrous Na2SO4, filtered,
and concentrated, yielding a brown residue, which was used for
the next operation without further purification. The product obtained
as above was dissolved in acetic acid (5 mL), and sodium acetate
(0.5 g) was added to the reaction mixture, which was then heated

1412 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Xiao et al.
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S.; Kamassah, A.; Morin, P. E.; Projean, D.; Ducharme, J.; Roberts,
E. New Diarylmethylpiperazines as Potent and Selective Nonpeptidic
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at 50 °C for 24 h. Then acetic acid was removed under reduced
pressure, and the residue was dissolved in CHCl3 (100 mL) and
washed with saturated NaHCO3 (2 × 10 mL), water (2 × 10 mL),
and brine (2 × 10 mL). The organic solution was dried over Na2-
SO4, filtered, concentrated, and the residue was subjected to silica
gel (40 g) flash column chromatography, eluting with CHCl3,
yielding a light yellow solid (18 mg, 18%): mp >262 °C (dec).
1H NMR (300 MHz, CDCl3) δ 8.73 (d, J ) 8.4 Hz, 1 H), 8.36 (s,
1 H), 8.25 (d, J ) 8.4 Hz, 1 H), 8.05 (dd, J ) 8.4, 1.2 Hz, 1 H),
7.98 (s, 1 H), 7.90 (d, J ) 7.5 Hz, 1 H), 7.81 (td, J ) 7.2, 1.2 Hz,
1 H), 7.63 (td, J ) 7.2, 1.2 Hz, 1 H), 7.59 (t, J ) 7.8 Hz, 1 H),
5.37 (s, 2 H); ESIMS m/z (rel intensity) 310 (MH+, 100). Anal.
(C20H11N3O‚0.75H2O) C, H, N.
Molecular Modeling Procedure. The structure of the ternary
complex, containing topoisomerase I, DNA, and topotecan, was
downloaded from the Protein Data Bank (PDB code 1K4T). One
molecule of PEG and the topotecan carboxylate form were deleted.
All of the atoms were then fixed according to the Sybyl atom types.
Hydrogens were added and minimized using MMFF94s force field
and MMFF94 charges. The structure of the 22-hydroxyacuminatine,
constructed in Sybyl and energy minimized with the Tripos force
field and Gasteiger-Hu¨ckel charges, was overlapped with the
structure of topotecan using the A-, B-, C-rings, and the structure
of topotecan was then deleted. The new whole complex was
subsequently subjected to energy minimization using MMFF94s
force field with MMFF94 charges. During the energy minimization,
the structure of the 22-hydroxyacuminatine was allowed to move,
while the structures of the protein, nucleic acids, and the surrounding
water molecules were frozen. The energy minimization was
performed using the Powell method with a 0.05 kcal/mol Å energy
gradient convergence criterion and a distance-dependent dielectric
function.
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Acknowledgment. This work was made possible by the
National Institutes of Health (NIH) through support of this work
with Research Grant UO1 CA89566. This research was
conducted in a facility constructed with support from Research
Facilities Improvement Program Grant Number C06-14499 from
the National Center for Research Resources of the National
Institutes of Health.
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Supporting Information Available: Elemental analysis data
and 1H NMR spectra of compounds 2b, 2c, 11, and 39. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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