Semisynthesis, biological activity, and molecular modeling studies of C-ring-modified camptothecins

Article abstract of DOI:10.1021/jm801153y

The synthesis, biological activity, and molecular modeling studies of C-ring-modified camptothecins are reported. A general synthetic protocol, based on "C-5 camptothecin (C-5-CPT) enolate chemistry", allows one to obtain various C5-substituted analogues.

Full text of DOI:10.1021/jm801153y

J. Med. Chem. 2009, 52, 1029–1039
1029
Semisynthesis, Biological Activity, and Molecular Modeling Studies of C-Ring-Modified
Camptothecins†
Cristian Samor`ı,‡ Andrea Guerrini,‡ Greta Varchi,*,‡ Gabriele Fontana,§ Ezio Bombardelli,§ Stella Tinelli,|
Giovanni Luca Beretta,| Serena Basili, Stefano Moro, Franco Zunino,| and Arturo Battaglia‡
Istituto CNR per la Sintesi Organica e FotoreattiVita` “I.S.O.F.”, Area della Ricerca di Bologna, Via P. Gobetti 101, 40129 Bologna, Italy,
Indena SPA, Viale Ortles 12, 20139 Milano, Italy, Diparitmento di Oncologia Sperimentale e Laboratori, Fondazione IRCCS Istituto Nazionale
Tumori, 20133 Milano, Italy, and Molecular Modelling Section (MMS), Dipartimento di Scienze Farmaceutiche, UniVersita` di PadoVa, Via
Marzolo 5, I-35131 PadoVa, Italy
ReceiVed September 15, 2008
The synthesis, biological activity, and molecular modeling studies of C-ring-modified camptothecins are
reported. A general synthetic protocol, based on “C-5 camptothecin (C-5-CPT) enolate chemistry”, allows
one to obtain various C5-substituted analogues. All new compounds, obtained as 1:1 epimeric mixtures,
were tested for their antiproliferative activity. Experimental data showed that all novel derivatives are less
active than the reference compounds and that one of the two epimers is more active than the other. Molecular
docking simulations were performed to achieve more insight into the interactions between the new C5-
modified CPTs and Topo I. A good correlation was observed when the data of cytotoxicity and the values
calculated for the free binding energy were combined.
Introduction
Camptothecin (CPT,a 1, Figure 1) is a naturally occurring
quinoline alkaloid from Camptotheca acuminata, having re-
markable antitumoral and antileukemic activity.1 The FDA has
approved two water soluble semisynthetic camptothecin ana-
logues, namely, topotecan 22 and irinotecan 33 for the clinical
treatment of the ovarian, small-cell lung, and refractory col-
orectal cancers. The molecular target of the CPT family is the
DNA topoisomerase I (Topo I), an enzyme that catalyzes the
relaxation of supercoiled DNA during DNA replication.4
The drug acts by forming a reversible and stable enzyme-
DNA-drug complex, thus preventing the religation step of the
enzyme action. SAR studies on CPT derivatives led to the
synthesis of several analogues with improved pharmacological
properties, mostly regarding synthetic modifications of the
quinoline system5 (A/B rings, Figure 1) and especially positions
Figure 1. Camptothecin 1 and its carboxylate 1a and their analogues
topotecan 2 and irinotecan 3.
C7, C9, and C10. Additional studies have been made on the
E-ring modification; in particular the 20-OH esterification has
substituted camptothecins was generally lower with respect to
been intensively studied to overcome poor solubility and
improve delivery of CPT analogues.6 Moreover, quite recently,
CPT except for the methylamine and hydroxylamine substitu-
ents, which showed good biological activity and water solubil-
a new family of very active seven-member ꢀ-hydroxy lactone
ity.10
derivatives were synthesized by Pommier and co-workers.7
So far, very few studies have been made on C-ring synthetic
modifications, probably because of the low antineoplastic
activity observed for the analogues so far reported, such as the
hydroxy, alkoxy, acyloxy,8 and hydroxymethyl9 5-substituted
derivatives. Similarly, the anticancer activity of 5-amino
Accordingly, it appears quite clear that ABCD system
planarity is required for an efficient Topo I inhibition. Since
the 5-position is adjacent to the pyridone moiety, the docking
to the receptor active site might be inhibited when substituents
are present at this position or when hydrogen bonding, favored
by the presence of a hydroxy or amino group, occurs between
the 5-position and the pyridone ring.9 These findings seem to
be confirmed by the behavior of 5-ethylene CPT analogues, in
fact, where the presence of the ethylene moiety, assumed to lie
in the molecular plane, does not reduce its antitumor activity.11
However, further studies on 5-substituted CPT derivatives gave
contradictory results. For instance, C5-propionyl moieties12 and
different 5-ethoxy derivatives bearing hydroxy, methoxy, or
fluoro groups at the 2-position13 showed good antitumoral
activity. One of these analogues, DRF-1042, is currently in phase
II clinical trials.14 Finally, it is worth noting that 5-amino-
substituted CPT inhibitory activity against hypoxia inducible
†
Dedicated to the memory of Dr. Arturo Battaglia. He committed his
life to the organic synthesis of biologically active molecules and to teaching
his alumni the great importance of chemistry for life.
* To whom correspondence should be addressed. Phone: (+39)
0516398283. Fax: (+39) 051 6398349. E-mail: g.varchi@isof.cnr.it.
‡
Istituto CNR per la Sintesi Organica e Fotoreattivita` “I.S.O.F.”.
Indena SPA.
Fondazione IRCCS Istituto Nazionale Tumori.
§
|
Universita` di Padova.
a Abbreviations: CPT, camptothecin; Topo I, topoisomerase I; TESCl,
triethylsilyl chloride; DMF, dimethylformamide; DMAP, dimethylaminopy-
ridine; LHMDS, lithium bis(trimethylsilyl)amide; TBDMS, tert-butyldi-
methylsilyl group; QPLD, quantum mechanics polarized ligand docking.
10.1021/jm801153y CCC: $40.75
2009 American Chemical Society
Published on Web 02/03/2009

1030 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Samor`ı et al.
Scheme 1. CPT Resonance System after Base-Induced Removal
of the 5-CH
pound 8 could be more resistant toward enzymatic degradation
than DRF-1042.
(b) Oxygenation. As an alternative to this acidic oxidation
and in line with the enolate chemistry, 5-OH-CPT was
synthesized through a direct oxidation of the enolate with
2-benzenesulfonyl-3-phenyloxaziridine.21,22 The reaction af-
forded, after silyl group removal, the desired compound 10 as
a 1:1 inseparable R/ꢀ-epimeric mixture (Scheme 4).
(c) Fluorination. Electrophilic fluorination with N-fluoroben-
zenesulfonimide (NFSI) of 4a followed by 20-OTES deprotec-
tion gave the 5-fluorocamptothecin 12 as a 1:1 R/ꢀ-epimeric
mixture, which was separated by silica gel flash chromatography.
To the best of our knowledge, the synthesis of CPT analogues
bearing a halo substituent at C-5 has not been previously
reported (Scheme 4).
(d) Amination. So far, the 5-nitrogen functionalization has
been obtained from the corresponding 5-OH derivatives via a
two-step procedure, comprising the nucleophilic substitution of
the OH with an amino function.10,15 Instead, we chose the
dibenzyl- and di-tert-butyl-azodicarboxylate23 as suitable re-
agents for the 4a enolate amination. The reaction provided the
corresponding 5-hydrazino derivatives 13 and 14, respectively;
these compounds were obtained as a 1:1 R/ꢀ epimeric mixture,
which was separated by silica gel column chromatography,
affording the pure epimers 13a and 13b and pure epimers 14a
and 14b. Although the hydrazino derivatives are potential
precursors of the 5-aminocamptothecin, their conversion was
not attempted.
factors HIF-1R15,16 was better than that displayed by 1 and
topotecan. For these reasons, more studies were needed to better
understand the role of modification at the C-5 position.
The facile removal of a proton from C-5 of CPT has been
well established by H1/H2 and H1/H3 hydrogen exchange
experiments,17 as well as by the synthesis of a number of
5-alkylated CPT analogues performed under basic conditions.9,18
These derivatizations are successfully achieved because of the
formation of a stable tautomeric enolate through an extended
four-ring resonance system, which stabilizes the C ring as a
heteroaromatic pyrrole system (Scheme 1). However, such
reactivity has only been exploited for a few selected alkylation
reactions, while other derivatizations such as hydroxylation13
and amination19 were achieved under oxidative conditions,
whereas alkylations, such as C5-propionyl substitution, were
achieved through a Wittig reaction on the 5-OH camptothecin.12
Thus, C-5 CPT enolate chemistry deserves further investigation
in order to develop a general synthetic protocol able to provide
more diversified C-5 CPT analogues. In particular, besides
alkylation, electrophilic amination, oxygenation, and fluorination
were also considered.
The hydrolysis of the 20-OTES substituent of the pure
epimers 13a and 13b yielded the corresponding CPT analogues
15a and 15b, whereas the hydrolysis of 14a and 14b gave
compounds 16a and 16b (Scheme 5).
Chemistry
Among the electrophilic azides usually employed for reaction
with enolates (phenylsulfonyl-, tosyl-, and 2,4,6-triisopropyl-
benzenesulfonyl azide),24 we selected tosyl azide as the less
sterically demanding. Moreover, reaction parameters should be
carefully examined, since this azide could serve for diazo or
azide transfer.25 Nevertheless, in our case, the azide transfer
occurred almost quantitatively and the 5-azido derivative 17 was
obtained with only trace amounts of the diazide derivative 19
(Table 1). Chromatographic workup of the mixture gave the
pure epimers 17a and 17b, which were deprotected giving the
CPT derivatives 18a and 18b, respectively (Scheme 5). Treat-
ment of the azido epimer 18a with hydrogen over 10%
palladium on activated carbon gave the 5-amino-camptothecin
22 as a 1:1 unseparable epimeric mixture (Scheme 5).
With the di-Boc-5-hydrazino compound 14 in our hands, we
envisioned the possibility of synthesizing a new six-membered
ring CPT derivative in which the amido substituents of the
pyridone moiety and the nitrogen atom at the 5-position are
joined into an additional heteroaromatic triazole ring. To this
end, sequential TFA-induced removal of the BOC protecting
groups, followed by cyclization of the corresponding trifluoro
acetate salt, yielded a 1:1 epimeric mixture of 5,17-dihydro,
4,17,18-triazole derivatives 20 whose oxidation with DDQ
provided the corresponding CPT analogue 21 in almost quan-
titative yield (Scheme 6).
The C-5 CPT functionalization initially required suitable
protection of the 20-OH group, which was achieved by treatment
of 1 with TESCl in DMF in the presence of an excess of
imidazole and DMAP according to a modified Corey protocol.20
As shown in Scheme 2, the 20-OTES camptothecin 4 was
obtained in quantitative yield. The silyl protective group highly
enhanced the solubility of 4 with respect to 1 in ethereal
solvents, allowing the heterofunctionalization of the 5-position
under mild conditions and low temperatures. Accordingly,
treatment of 4 with LHMDS at -78 °C provided the corre-
sponding lithium enolate. After reaction with suitable electro-
philes, a number of 5-substituted derivatives were obtained as
a mixture of R/ꢀ epimers, which were separated, where possible,
by silica gel flash column chromatography. Removal of the
silicon group with the complex Et3N·3HF afforded the 5-sub-
stituted CPT analogues (Scheme 2).
(a) Alkylation. For our purpose methyl iodide (MeI) and tert-
butyl(3-iodopropoxy)dimethylsilane were used as alkylating
agents. The addition was carried out under more demanding
reaction conditions with respect to those reported above. The
reaction was performed according to a slightly modified
literature procedure,18 using potassium tert-butoxide (tBuOK)
in DMF at 0 °C. Compounds 5 and 6 were obtained as
inseparable 1:1 R/ꢀ-epimeric mixtures in moderate overall yields
(40% and 41%, respectively) (Scheme 3). Partial deprotection
of the 20-OH group was observed for compound 6 under these
conditions. Quantitative removal of the TES and the TBDMS
protecting groups from 5 and 6, respectively, afforded the target
5-Me and 5-(CH2)3OH CPT analogues 7 and 8, as an inseparable
1:1 R/ꢀ-epimeric mixture. Interestingly, the 5(RS)-(3-hydroxy-
propionyl)-20(S)-CPT analogue 8 represents the carbon isostere
of DRF-1042 [5(RS)-(2-hydroxyethoxy)-20(S)-CPT].14 Com-
Cytotoxic Activity
Antiproliferative Activity. The new 5-modified CPT ana-
logues were tested for their antiproliferative activity against the
human non-small-cell lung cancer carcinoma NCI-H460 cell
line with a short-term exposure (1 h).26 Topotecan, CPT (1),
and SN38, the active metabolite of irinotecan, were used as

C-Ring-Modified Camptothecins
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1031
Scheme 2. Synthetic Protocol of 5-Substituted CPT Derivatives via Enolate Routea
a (i) TESCl, imidazole, DMAP, DMF; (ii) LHMDS, THF, -78 °C; (iii) electrophile, 4-5 h; (iv) Et3N·3HF, THF, 20 °C, 35-40 h.
Scheme 3. Synthesis of 5-Alkylcamptothecinsa
The bulky dimension of the substituents could produce a steric
hindrance at the target level and thus might explain this feature.
The presence of an additional ring (CPTs 20 and 21)
influenced the antiproliferative activity of CPTs, with compound
21 about 5-fold more active than the corresponding compound
20.
DNA Cleavage Assays. Topoisomerase I-mediated DNA
cleavage assays with purified human topoisomerase I were
performed to investigate the capability of the C5-modified CPTs
to stimulate DNA damage.27 SN38 was used as reference
compound (Figure 3). The cleavage pattern was found to be
similar to that of SN38 for all the tested CPT derivatives.
Compound 18b exhibited an intensity of DNA damage similar
to that of SN38. A slightly reduced capability to damage DNA
was observed for 12a, 12b, 21, and 7, whereas 15b, 20, and 22
were substantially less efficient.
a (i) tBuOK, DMF, 0 °C, then R-I; (ii) Et3N·3HF, THF, 20 °C, 35-40 h.
Scheme 4. Synthesis of 5-Hydroxycamptothecin 10 and
5-Fluorocamptothecin 12a
Theoretical Calculations. To achieve more insight into the
interactions of the new C-5 CPT analogues with Topo I, a
molecular modeling study of the structures of the potential
ternary cleavage complexes of Topo I, DNA, and both R/ꢀ-
epimeric C-5 CPT analogues was carried out using molecular
docking simulations. In particular, it is now well recognized
that electrostatic interactions play a critical role in noncovalent
interactions between DNA/ligand, protein/ligand, and DNA/
protein complexes. Because of the highly charged nature of
DNA, we assumed that the electrostatic interactions between
DNA and its ligand might play a critical role in the accurate
estimation of the binding free energy. In order to estimate the
binding free energy with improved accuracy, we carried out
molecular docking simulations based on the quantum mechanics
(QM) polarized ligand docking protocol using QPLD-derived
partial charges (see Experimental Section for details).
a (i) LHMDS, THF, -78 °C; (ii) THF, -78 °C; (iii) Et3N·3HF, 20 °C.
In general, all ternary complexes with Topo I and DNA are
characterized by a negative free energy of binding ∆Gbind, as
shown in Table 3. Thus, local energy minima were observed
for structures in which all the new C-5 CPT analogues are
accommodated into the DNA intercalation site (Figure 4)
adopting a binding motif which is similar to the crystallographic
pose of topotecan for most derivatives, although slightly
different in some cases, as will be discussed later in detail.
In particular, smaller C-5 substituents such as 5-methyl (7),
5-hydroxy (8), 5-fluorine (12), 5-azido (18), and 5-amino (22)
are apparently accepted in both epimeric configurations, with a
slight preference for the ꢀ-epimer (up configuration) in the case
of the 5-azido (18) derivative. Consistently, the 5-diazido
derivative (19) is also accepted. In each case, the large overlap
of the planar ABCD ring system with the nucleic bases
reference drugs. The collected data (Table 2) show that all new
compounds are less active than the reference CPTs. Moreover,
it appears that, except for compounds 12a and 12b, one of the
two epimers is more active than the other, the 5-N3 (18a and
18b) derivatives (entries 12-13) being the most active of the
series.
The presence of two N3 in position 5 did not produce a
substantial change in cytotoxic activity (19 vs 18a and 18b).
Methyl substituent (7) partially increased the potency, while
the introduction of hydrophilic groups (22, 8) was detrimental
to the antiproliferative activity. Although the capability to cross
the plasma membrane is strongly influenced by the lipophilic
properties of the compounds, a reduced potency was observed
for the most hydrophobic derivatives (15a, 15b, 16a, and 16b).

1032 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Scheme 5. Amination of Compound 4a
Samor`ı et al.
a (i) LHMDS, THF, -78 °C; (ii) THF, -78 °C; (iii) Et3N·3HF, 20 °C; (iv) Pd/C.
Table 1
entry
electrophyle
E
producta yield(%)
1
2
3
CbzNdNCbz
BOCNdNBOC
Me(C6H4)SO2N3 N3
(Cbz)N-NH(Cbz)
13
14
17
>95
>95
>95
(BOC)N-NH(BOC)
b
a a and b diastereoisomer formation. b Diazidation occurs.
Scheme 6. Synthesis of the Six-Membered Ring CPT Derivative
21a
Figure 2. Chemical structures for all tested compounds.
a (i) TFA, 20 °C, 20 h, 80%; (ii) DDQ, THF, 60 °C, 3 h, 95%.
Also, the stabilizing interactions with the Arg364 and the
Asp533 are maintained, as shown in Figure 5 (panel A). In
contrast, bulkier C-5 substituents such as 5-NBoc-NHBoc (15)
and 5-NCbz-NHCbz (16) significantly modify the occupancy
of these derivatives into the intercalation site. In fact, to permit
a sterically favorable allocation of these bulky substituents inside
the major groove of the DNA, the planar ABCD ring system
shifts back its position with respect to that observed in the crystal
structure of topotecan (Figure 5, panel B). This movement
reduces the intensity of the π-π stacking interactions and
abolishes the formation of both stabilizing interactions with the
Arg364 and Asp533. Moreover, the ꢀ-epimer at C-5 position
of both derivatives 15 and 16 seems to better stabilize the ternary
complex compared to the R-epimer (down stereochemistry)
(∆Gbind(15R) ) -5.79 kcal/mol; ∆Gbind(15ꢀ) ) -6.95 kcal/
mol). Our results indicate that the introduction of substituents
in position 5 of CPT resulted in derivatives with reduced
cytotoxic potency compared to the reference drugs. In general,
the presence of a small lipophilic substituent is well tolerated
while the modification with hydrophilic groups resulted in
reduced potency (e.g.,7 vs 22). In contrast, the presence of a
bulky group is detrimental for cytotoxic potency (15, 16, and
20). This observation is not a general feature, since the
expansion of the CPT structure with an additional aromatic ring
(compound 21) produced a derivative with good activity. The
Table 2. Cytotoxic Activity for C5-Substituted Camptothecins on a
Human H460 Cell Linea
entry
compd
E
IC50 (µM), 1 h
1
2
3
4
CPT (1)
topotecan
SN38
7
H
H
H
CH3
0.33
0.61
0.13
5.9
5
8
(CH2)3OH
16
6
7
8
9
10
11
12
13
14
15
16
17
12a
12b
15a
15b
16a
16b
18a
18b
19b
F
F
56
56
>173
77
85
60
11.5
6.7
9.3
NBoc-NHBoc
NBoc-NHBoc
NCbz-NHCbz
NCbz-NHCbz
N3
N3
(N3)2
20b
46.8
10.6
30
21b
22
NH2
a Sensitivity was assessed by growth inhibition assay after 1 h of drug
exposure. Cells were counted 72 h after drug-free medium incubation.
b Formulas are reported in Figure 2.
guarantees the significant influence of the π-π stacking
interactions on the orientation of the ligand within the intercala-
tion site and on the overall binding energy.

C-Ring-Modified Camptothecins
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1033
Figure 3. Topoisomerase I-mediated DNA cleavage by SN38 and different camptothecin derivatives. Samples were reacted with 1, 10, and 50 µM
drug at 37 °C for 30 min. The reaction was then terminated by adding 0.5% SDS and 0.3 mg/mL proteinase K and incubating for 45 min at 42 °C
before loading on a denaturing 8% polyacrylamide gel: C, control DNA; T, reaction without drug; M, purine markers. The experiment was repeated
three times, and the results of a representative value are reported.
Table 3. Free Energy of Binding ∆Gbind Calculated for All R/ꢀ-Epimeric C-5 CPT Analogues with the QM-Polarized Ligand Docking Protocol
entry
compd
5-CPT substituent
G score (kcal/mol)
1
2
3
4
5
6
7
8
CPT (1)
7Ra
7ꢀb
H
CH3
CH3
(CH2)3OH
(CH2)3OH
F
F
NBoc-NHBoc
NBoc-NHBoc
NCbz-NHCbz
NCbz-NHCbz
N3
-11.82
-10.63
-10.98
-9.90
-10.02
-8.93
-9.08
-5.79
-6.95
-8.17
-8.40
-10.82
-11.12
-10.76
-7.94
-7.35
-10.50
-9.12
-9.74
-8.04
-10.95
-9.86
-10.25
-7.98
-9.92
8R
8ꢀ
12R
12ꢀ
15R
15ꢀ
16R
16ꢀ
18R
18ꢀ
19
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
N3
(N3)2
20R
20ꢀ
21c
22R
22ꢀ
23R
23ꢀ
24R
24ꢀ
25R
25ꢀ
O(CH2)2OH
O(CH2)2OH
CH2COOMe
CH2COOMe
NHCH3
NHCH3
a R-Epimer: down stereochemistry. b ꢀ-Epimer: up stereochemistry. c Formulas are reported in Figure 2.
finding that the ꢀ-epimer is more active than the corresponding
R-epimer suggests a possible steric hindrance between the Topo
I-DNA structure and the R-oriented group.
In the present docking study, we have also included three
known 5-substitued CPT derivatives (compounds 23-25, entries
20-24, Table 3) that have shown, experimentally, appreciable
antitumoral activity.10,12,14 These molecules fit into the Topo
I-DNA complex in a binding mode, which is comparable to
that of the new 5-substituted CPTs synthesized herein, and their
predicted ∆Gbind values fall within the range of ∆Gbind calculated

1034 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Samor`ı et al.
Figure 4. Docking results for the C-5 modified CPTs. In panels 1A (side view) and 1B (top view) the binding conformation of topotecan (yellow)
from the crystal structure of the Topo I-DNA-topotecan ternary complex (PDB code 1k4t) is shown as a reference. Panels 2A (side view) and
2B (top view) show the superimposition of the best conformations for all C-5 modified CPTs, from docking simulations into the Topo I-DNA
binding site. Topo I and DNA are represented by cartoon structure, colored in blue and pink, respectively.
Figure 5. Binding conformation of 5-N3 and 5-NBocNHBoc derivatives from docking simulations. Panels 1A and 2A: binding conformations of
5-N3 and 5-NBocNHBoc, respectively (colored in orange). Panels 2A and 2B: conformations of 5-N3 and 5-NBocNHBoc, respectively, superimposed
on the topotecan binding conformation (in gray) for comparison. Polar interactions are represented by green dashed lines.
for our compounds. Moreover, also for these derivatives we
observed that the ꢀ-epimer is better accepted than the R-epimer.
Finally, the 4,17,18-triazole derivative 21 increasing the
extension of the aromaticity of the planar ring system is also
properly accommodated within the intercalation site as dem-
onstrated by the calculated ∆Gbind value (∆Gbind(21) ) -10.50
kcal/mol).
To make such a correlation, we assumed that the antiproliferative
activity data are mostly pharmacodynamically driven, since the IC50
values and the cleavage potency show a similar trend for
compounds that were tested in the cleavage assay. The cleavage
potency was defined by comparing the intensity of cleavage bands
of the tested compounds with that of the reference drug (SN38).
As shown in Figure 6, a linear correlation between antiproliferative
potency and ∆Gbind was observed (r2 ) 0.76).
Interestingly, we have also found that an acceptable correla-
tion was observed when the data of cytotoxicity and the values
calculated for the free energy of binding were combined (Figure
6).
Conclusions
A general synthetic protocol, based on the “C-5-CPT enolate
chemistry”, allowed the synthesis of various C5-substituted

C-Ring-Modified Camptothecins
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1035
or by correlated spectroscopy. IR spectra were recorded on a FT-
IR ESP spectrometer as thin films on NaCl plates. Mass spectra
were recorded on an ion trap spectrometer with an ionization
potential of 70 eV. High resolution mass spectra (HRMS) were
performed with a resolution of 10 000 against a suitable mass
standard. The commercially available reagents were used as received
without further purification.
20-OTES-camptothecin (4). To a suspension of camptothecin
(1) (100 mg, 0.29 mmol) in dry DMF (3 mL), imidazole (98 mg,
1.44 mmol) was added. After 10 min triethylsilyl chloride (0.19
mL, 1.15 mmol) was introduced dropwise, followed by DMAP (40
mg, 0.29 mmol). The mixture was stirred at room temperature until
TLC analysis (CH2Cl2-MeOH, 30:1) showed complete disappear-
ance of the starting material (60 h). Excess of solvents was removed
under diminished pressure, and water was added to the residue.
The aqueous layer was extracted with CH2Cl2 (20 mL × 3), and
the organic phases were dried over Na2SO4, filtered, and concen-
trated under vacuum. The resulting solid residue was purified by
silica gel flash chromatography (CH2Cl2-MeOH, 30:1) to afford
1
4 (130 mg, 98%) as a pale-yellow solid. H NMR (CDCl3, 400
MHz) δ 8.37 (s, 1 H, Ar, H-7), 8.25 (d, 1 H, J ) 8.4 Hz, Ar), 7.92
(d, 1 H, J ) 8.0 Hz, Ar), 7.82 (t, 1 H, J ) 8.0 Hz, Ar), 7.65 (t, 1
H, J ) 8.4 Hz, Ar), 7.57 (s, 1 H, H-14), 5.67 (d, 1 H, J ) 16.4 Hz,
H-17), 5.29 (s, 2 H, H-5), 5.25 (d, 1 H, J ) 16.4 Hz, H-17),
2.00-1.84 (m, 2 H, H-19), 1.03-0.93 (m, 12 H), 0.80-0.71 (m, 6
H). 13C NMR (CDCl3, 100 MHz) δ 171.7, 157.6, 152.5, 151.5,
149.0, 145.9, 130.9, 130.4, 130.0, 128.4, 128.1, 128.0, 127.9, 118.9,
94.4, 75.3, 66.0, 50.0, 33.2, 7.9, 7.2, 6.4. MS (70 eV): m/z (rel
intensity), 433 [M+ - 29] (89), 390 [M+ - 72] (100). HRMS m/z
calcd for C26H30N2O4Si 462.197, m/z found 462.193.
General Procedure Electrophilic Addition to 20-OTES-CPT
(4). Method A. A solution of 20-OTES-Camptothecin (4) (1 equiv)
in dry THF (6 mL) was cooled to -78 °C, and 1.0 M LHMDS in
THF (1.2 equiv) was added dropwise. The solution was stirred at
this temperature for 30 min, and then a solution of the electrophile
(1.3 equiv) in THF (2 mL) was introduced dropwise. The solution
was stirred at -78 °C for 2 h and then warmed to 20 °C and stirred
for additional 2 h. The reaction mixture was treated with saturated
NH4Cl and extracted with CH2Cl2, dried over Na2SO4, and filtered,
and the solvent was removed under vacuum. The crude mixture
was purified by silica gel flash chromatography employing the
above-reported eluents.
Figure 6. Correlation between cytotoxicity and ∆Gbind: red, the most
active compound of the series; green, compounds with slightly reduced
DNA damage activity; blue, less active compounds; black, compounds
not tested in the cleavage assay.
analogues. In particular, the reported methodology provided,
besides C5-alkylated derivatives, new C5-fluorinated and ami-
nated analogues, along with the first six-membered ring CPT
derivative 21. All compounds were obtained as 1:1 epimeric
mixtures, which were separated by silica gel flash chromatog-
raphy where possible. The new C5-modified CPT analogues
were tested for their antiproliferative activity against the human
non-small-cell lung cancer carcinoma NCI-H460. The collected
data showed that all new compounds are less active than the
reference compounds and that, except for compounds 12a and
12b, one of the two epimers is more active than the other, the
C5-N3 18 derivatives being the most active of the series.
Molecular docking simulations were performed to achieve more
insight into the interactions between the new C5-modified CPTs
and Topo I. The experiments were carried out on the structures
of the potential ternary cleavage complexes of Topo I, DNA,
and both R/ꢀ-epimeric C5-CPTs in order to estimate the binding
free energy values. Interestingly, in this study a good correlation
was observed when the data of cytotoxicity and the values
calculated for the free energy of binding were combined. In
particular, a good correlation was identified for compound 18b,
the most active in the cleavage assay, which was effective in
reducing cell proliferation and in producing a stable, cleavable
complex.
General Procedure for He Electrophilic Addition to 20-OTES-
CPT (4). Method B. A solution of 20-OTES-camptothecin (4) (1.0
t
equiv) in dry DMF (4 mL) was cooled to 0 °C, and BuOK (1.3
equiv) was added. The solution was stirred at 0 °C for 5 min, and
then a solution of the electrophile (5.0 equiv) in dry DMF (0.2
mL) was introduced dropwise. The solution was stirred for 15 min
at this temperature and allowed to warm to 20 °C. After an
additional 30 min, the reaction mixture was diluted with water (10
mL) and extracted with EtAcO. The organic layers were washed
with brine, dried over Na2SO4, and filtered, and the solvents were
removed under vacuo. The mixture of diastereoisomers was purified
by flash chromatography employing the above-reported eluents.
20-OTES-5-fluorocamptothecin (11). Method A was employed.
The crude mixture was purified by silica gel flash chromatography.
Step gradient elution with n-hexane-AcOEt, 3:1 followed by 2:1
and 1:1, allowed us to separate the 1:1 mixture of diastereoisomers
11a and 11b (overall yield 97%). 11a: 1H NMR (CDCl3, 400 MHz)
δ 8.52 (s, 1 Η, Αr, H-7), 8.25 (d, 1 H, J ) 8.4 Hz, Ar), 7.96 (d,
1 H, J ) 8.4 Hz, Ar), 7.87 (t, 1 H, J ) 8.4 Hz, Ar), 7.69 (t, 1 H,
J ) 8.4 Hz, Ar), 7.47 (d, 1 H, 1JHF ) 61.2 Hz, H-5), 7.45 (s, 1 H,
H-14), 5.62 (d, 1 H, J ) 16.8 Hz, H-17), 5.22 (d, 1 H, J ) 16.8
Hz, H-17), 2.02-1.84 (m, 2 H, H-19), 1.03-0.93 (m, 12 H),
0.80-0.71 (m, 6 H). 13C NMR (CDCl3, 100 MHz) δ 171.4, 157.5,
152.3, 151.2, 150.3, 143.6, 133.7, 131.7, 130.2, 128.9, 128.4, 127.9,
126.3, 121.8, 98.9, 93.8 (d, 1JCF ) 213.2 Hz, C-5), 75.1, 65.7, 33.1,
7.8, 7.2, 6.4. HRMS m/z calcd for C26H29FN2O4Si 480.188, m/z
Experimental Section
Chemistry. General Remarks. All reactions were performed
under an atmosphere of dry nitrogen using oven-dried glassware.
Tetrahydrofuran, toluene, and ethyl ether were distilled from sodium
benzophenone ketal. Dichloromethane and DMF were distilled from
calcium hydride. All other solvents were HPLC grade. Reactions
were magnetically stirred and monitored by thin layer chromatog-
raphy (TLC) with E. Merck silica gel 60-F254 plates. Flash column
chromatography was performed with Merck silica gel (0.04-0.63
µm, 240-400 mesh) under high pressure. NMR spectra were
recorded on 400 MHz spectrometer. Unless otherwise stated, all
NMR spectra were measured in CDCl3 solutions and referenced to
1
the CHCl3 signal. All H and 13C shifts are given in ppm (s )
singlet; d ) doublet; t ) triplet; dd ) quadruplet; dt ) doublet of
triplets, m ) multiplet; br s ) broad signal). Coupling constants J
are given in Hz. Assignments of proton resonances were confirmed,
when possible, by selective homonuclear decoupling experiments
1
found 480.195. 11b: H NMR (CDCl3, 400 MHz) δ 8.51 (s, 1 Η,
Αr, H-7), 8.25 (d, 1 H, J ) 8.4 Hz, Ar), 7.96 (d, 1 H, J ) 8.4 Hz,
Ar), 7.87 (t, 1 H, J ) 8.4 Hz, Ar), 7.68 (t, 1 H, J ) 8.4 Hz, Ar),

1036 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Samor`ı et al.
7.51 (d, 1 H, 1JHF ) 60.8 Hz, H-5), 7.42 (s, 1 H, H-14), 5.62 (d, 1
H, J ) 17.2 Hz, H-17), 5.20 (d, 1 H, J ) 17.2 Hz, H-17), 2.02-1.82
(m, 2 H, H-19), 1.04-0.93 (m, 12 H), 0.80-0.71 (m, 6 H). 13C
NMR (CDCl3, 100 MHz) δ 171.2, 157.8, 152.5, 151.2, 150.3, 143.7,
133.7, 131.7, 130.2, 128.9, 128.3, 127.9, 126.3, 121.8, 99.0, 93.8
) 7.6 Hz, Ar), 7.63 (br t, 1 H, J ) 7.6 Hz, Ar), 7.56-6.90 (m, 11
H, Ar + H-14), 6.52 (br s, 1 H, H-5), 5.55 (d, 1 H, J ) 16.8 Hz,
H-17), 5.44-4.71 (m, 5 H), 1.98-1.80 (m, 2 H, H-19), 1.05-0.90
(m, 12 H), 0.81-0.70 (m, 6 H). 13C NMR (CDCl3, 100 MHz) δ
171.5, 157.9, 156.4, 156.4, 152.9, 152.4, 150.9, 149.4, 144.8, 135.3,
132.1, 130.6, 129.9, 128.6-127.8 (11 C), 119.9, 98.5, 79.3, 75.2,
68.4, 67.8, 65.6, 32.9, 7.8, 7.2, 6.4. HRMS m/z calcd for
C42H44N4O8Si 760.293, m/z found 760.297.
1
(d, JCF ) 214.8 Hz, C-5), 75.0, 65.8, 33.3, 7.9, 7.1, 6.4. HRMS
m/z calcd for C26H29FN2O4Si 480.188, m/z found: 480.192.
20-OTES-5-azidocamptothecin (17). Method A was employed.
The crude mixture was purified by silica gel flash chromatography.
Step gradient elution with n-hexane-AcOEt, 3:1 followed by 2:1
and 1:1, allowed us to separate the 1:1 mixture of diastereoisomers
17a and 17b (overall yield 97%). 17a: 1H NMR (CDCl3, 400 MHz)
δ 8.45 (s, 1 Η, Αr, H-7), 8.25 (d, 1 H, J ) 8.4 Hz, Ar), 7.95 (d,
1 H, J ) 8.4 Hz, Ar), 7.86 (t, 1 H, J ) 8.4 Hz, Ar), 7.68 (t, 1 H,
J ) 8.4 Hz, Ar), 7.49 (s, 1 H, H-14), 6.97 (s, 1 H, H-5), 5.65 (d,
1 H, J ) 16.8 Hz, H-17), 5.26 (d, 1 H, J ) 16.8 Hz, H-17),
2.01-1.84 (m, 2 H, H-19), 1.03-0.94 (m, 12 H), 0.80-0.71 (m, 6
H). 13C NMR (CDCl3, 100 MHz) δ 171.6, 158.3, 152.2, 150.8,
150.0, 144.0, 132.9, 131.4, 130.1, 128.6, 128.3, 128.2, 128.1, 120.8,
98.7, 75.4, 75.2, 65.7, 33.1, 7.9, 7.2, 6.4. HRMS m/z calcd for
C26H29N5O4Si 503.199, m/z found: 503.191. 17b: 1H NMR (CDCl3,
400 MHz) δ 8.45 (s, 1 Η, Αr, H-7), 8.24 (d, 1 H, J ) 8.4 Hz, Ar),
7.95 (d, 1 H, J ) 8.4 Hz, Ar), 7.86 (t, 1 H, J ) 8.4 Hz, Ar), 7.68
(t, 1 H, J ) 8.4 Hz, Ar), 7.46 (s, 1 H, H-14), 6.99 (s, 1 H, H-5),
5.66 (d, 1 H, J ) 16.8 Hz, H-17), 5.22 (d, 1 H, J ) 16.8 Hz,
H-17), 2.02-1.84 (m, 2 H, H-19), 1.03-0.94 (m, 12 H), 0.80-0.71
(m, 6 H). 13C NMR (CDCl3, 100 MHz) δ 171.4, 158.4, 152.3, 150.9,
150.0, 144.0, 132.9, 131.4, 130.1, 128.6, 128.3, 128.2, 128.1, 120.8,
98.7, 75.3, 75.1, 65.8, 33.3, 7.9, 7.2, 6.4. HRMS m/z calcd for
C26H29N5O4Si 503.199, m/z found 503.192.
20-OTES-5-OH-camptothecin (9). Method A was employed.
The crude mixture was purified by silica gel flash chromatography.
Elution with n-hexane-AcOEt, 3:1 followed by 7:2, afforded 9 as
1
pale-yellow solids in 95% yield. H NMR (CDCl3, 400 MHz) δ
8.48 (s, 0.5 Η, Αr, H-7), 8.47 (s, 0.5 H, Ar, H-7), 8.25 (d, 1 H, J
) 8.8 Hz, Ar), 7.96 (d, 1 H, J ) 8.8 Hz, Ar), 7.85 (t, 1 H, J ) 8.8
Hz, Ar), 7.67 (t, 1 H, J ) 8.8 Hz, Ar), 7.49 (s, 0.5 H, H-14), 7.48
(s, 0.5 H, H-14), 7.09 (d, 0.5 H, J ) 2.8 Hz, H-5), 7.05 (d, 0.5, J
) 2.8 Hz, H-5), 5.63 (d, 0.5 H, J ) 16.8 Hz, H-17), 5.62 (d, 0.5
H, J ) 16.8 Hz, H-17), 5.55 (bs, 0.5 H, OH), 5.51 (bs, 0.5 H,
OH), 5.23 (d, 0.5 H, J ) 16.8 Hz, H-17), 5.21 (d, 0.5 H, J ) 16.8
Hz, H-17), 1.94-1.86 (m, 2 H, H-19), 0.98-0.92 (m, 12 H),
0.78-0.72 (m, 6 H). HRMS m/z calcd for: C26H30N2O5Si 478.192,
m/z found: 478.187.
20-OTES-5-methylcamptothecin (5). Method B was employed.
For the electrophile, the crude mixture was purified by silica gel
flash chromatography. Elution with n-hexane-EtOAc, 1:3, afforded
5 as an inseparable 1:1 mixture of epimers (pale-yellow solid, 40%
yield). 1H NMR (CDCl3) δ 8.25 (d, 1 H, J ) 8.4 Hz, Ar), 8.25 (s,
1 H, Ar), 7.92 (d, 1 H, J ) 8.4 Hz, Ar), 7.81 (t, 1 H, J ) 8.4 Hz,
Ar), 7.64 (t, 1 H, J ) 8.4 Hz, Ar), 7.54 (s, 0.5 H, Ar), 7.52 (s, 0.5
H, Ar), 5.74 (m, 1 H, H-5), 5.66 (d, 0.5 H, J ) 16.8 Hz), 5.63 (d,
0.5 H, J ) 16.8 Hz), 5.24 (d, 0.5 H, J ) 16.8 Hz), 5.22 (d, 0.5 H,
J ) 16.8 Hz), 1.98-1.84 (m, 5 H, H-19 + Me), 1.04-0.96 (m, 12
H), 0.82-0.74 (m, 6 H). 13C NMR (CDCl3) δ 171.9, 158.1, 151.7,
151.2, 149.3, 145.4, 134.7, 130.3, 130.1, (130.0-130.0), 128.3,
128.1, 127.8, 119.9, (98.1-98.0), 75.3, (66.2-66.0), (59.2-59.1),
(33.2-33.2), (18.9-18.5), (7.9-7.9), 7.2, 6.5. HRMS m/z calcd
for C27H32N2O4Si 476.213, m/z found 476.209.
20-OTES-5-DTBAC-camptothecin (14). Method A was em-
ployed. The crude mixture was purified by silica gel flash
chromatography. Elution with n-hexane-AcOEt, 3:1, allowed the
separation of the 1:1 mixture of diastereoisomers 14a and 14b as
pale-yellow solids (overall yield 97%). 14a: 1H NMR (CDCl3, 400
MHz) δ 8.80 (br s, 1 Η, Αr), 8.23 (d, 1 H, J ) 8.4 Hz, Ar), 8.01
(br d, 1 H, Ar), 7.90-7.71 (m, 2 H, Ar), 7.70-7.45 (m, 2 H, Ar +
H-14), 6.52 (br s, 1 H, H-5), 5.61 (d, 1 H, J ) 16.8 Hz, H-17),
5.23 (d, 1 H, J ) 16.8 Hz, H-17), 2.03-1.81 (m, 2 H, H-19),
1.79-1.08 (br s, 18 H), 1.06-0.92 (m, 12 H), 0.80-0.70 (m, 6
H). 13C NMR (CDCl3, 100 MHz) δ 171.7, 157.8, 155.5, 155.5,
152.0, 152.0, 151.2, 149.4, 145.0, 132.1, 130.6, 130.0, 128.7, 128.4,
127.9, 119.9, 98.2, 82.7, 81.5, 79.7, 75.2, 65.7, 33.2, 28.3, 27.6,
7.7, 7.2, 6.4. HRMS m/z calcd for C36H48N4O8Si 692.324, m/z found
692.319. 14b: 1H NMR (CDCl3, 400 MHz) δ 8.79 (br s, 1 Η, Αr),
8.23 (d, 1 H, J ) 8.4 Hz, Ar), 8.01 (br d, 1 H, Ar), 7.85-7.76 (m,
2 H, Ar), 7.65 (br t, 1 H, J ) 8.4 Hz, Ar), 7.52 (s, 1 H, H-14), 6.54
(br s, 1 H, H-5), 5.61 (d, 1 H, J ) 16.8 Hz, H-17), 5.22 (d, 1 H,
J ) 16.8 Hz, H-17), 2.03-1.82 (m, 2 H, H-19), 1.76-1.08 (br s,
18 H), 1.04-0.92 (m, 12 H), 0.80-0.70 (m, 6 H). 13C NMR
(CDCl3, 100 MHz) δ 171.5, 157.9, 155.5, 155.5, 152.3, 152.0,
151.2, 149.4, 145.1, 132.1, 130.6, 130.0, 128.7, 128.4, 127.9, 119.9,
98.2, 82.9, 81.5, 79.6, 75.2, 65.8, 33.3, 28.3, 27.4, 7.8, 7.2, 6.4.
HRMS m/z calcd for C36H48N4O8Si 692.324, m/z found 692.317.
20-OTES-5DBAC-camptothecin (13). Method A was em-
ployed. The crude mixture was purified by silica gel flash
chromatography. Step gradient elution with n-hexane-AcOEt, 4:1
followed by 7:2, allowed the separation of the 1:1 mixture of
20-OTES-5-(3-hydroxypropyl)camptothecin (6). Method B
was employed. For the electrophile, the crude mixture was purified
by silica gel flash chromatography. Elution with n-hexane-EtOAc,
1:3 followed by 1:1, afforded 6 as an inseparable 1:1 mixture of
1
epimers (pale-yellow solid, 41% yield). H NMR (CDCl3) δ 8.29
(s, 1 H, Ar), 8.22 (d, 1 H, J ) 8.4 Hz, Ar), 7.91 (d, 1 H, J ) 8.4
Hz, Ar), 7.82 (t, 1 H, J ) 8.4 Hz, Ar), 7.65 (t, 1 H, J ) 8.4 Hz,
Ar), 7.63 (s, 0.5 H, Ar), 7.61 (s, 0.5 H, Ar), 5.86 (dd, 0.5 H, J1 )
6.0 Hz, J2 ) 2.8 Hz, H-5), 5.82 (dd, 0.5 H, J1 ) 6.0 Hz, J2 ) 2.8
Hz, H-5), 5.72 (d, 0.5 H, J ) 16.4 Hz, H-17), 5.70 (d, 0.5 H, J )
16.4 Hz, H-17), 5.29 (d, 0.5 H, J ) 16.4 Hz, H-17), 5.27 (d, 0.5
H, J ) 16.4 Hz, H-17), 3.75 (s, 1 H, OH), 3.55-3.49 (m, 2 H),
2.68-2.58 (m, 1 H), 2.52-2.44 (m, 1 H), 1.98-1.74 (m, 2 H,
H-19), 1.25-1.10 (m, 2 H), 1.05 (t, 1.5 H, J ) 7.6 Hz, Me), 1.04
(t, 1.5 H, J ) 7.6 Hz, Me), 0.82 (s, 9 H), -0.03 (s, 3 H), -0.07 (s,
3H).13CNMR(CDCl3)δ(174.0-173.9),157.9,152.1,(149.8-149.8),
149.2, (146.5-146.5), (133.0-133.0), 130.5 (2C), 129.8, 128.2,
127.9, 119.4, (97.6-97.6), (72.7-72.7), (66.5-66.4), 62.8,
(62.4-62.3), 31.6, (27.6-27.1), (26.3-26.1), 25.9, 18.2, (7.9-7.8),
(-5.4 to -5.4). HRMS m/z calcd for C29H36N2O5Si 520.239, m/z
found 520.231.
1
diastereoisomers 13a and 13b (overall yield 98%). 13a: H NMR
General Procedure for Silyl Group Removal. To a solution
of 20-OTES-CPT 5-substituted derivatives (1 equiv) in dry THF
(5 mL), Et3N·3HF (7.5 equiv) was added dropwise. The mixture
was reacted at room temperature until TLC analysis (n-hexane-
AcOEt, 1:1) showed the complete conversion of the starting material
(35 h). The solvent was removed under diminished pressure, and
the residue was purified by silica gel flash chromatography.
20-OH-5-fluorocamptothecin (12a, 12b). Reaction of 11a (11b)
with Et3N·3HF afforded, after silica gel flash chromatography
purification (n-hexane/EtOAc, 1:1), 12a (12b) in 97% yield as a
(CDCl3, 400 MHz) δ 8.70 (br s, 1 Η, Αr), 8.39 (br s 1 H, Ar),
8.22 (br d, 1 H, J ) 7.6 Hz, Ar), 7.95 (br d, 1 H, J ) 7.6 Hz, Ar),
7.83 (br t, 1 H, J ) 7.6 Hz, Ar), 7.65 (br t, 1 H, J ) 7.6 Hz, Ar),
7.64-7.00 (m, 11 H, Ar + H-14), 6.49 (br s, 1 H, H-5), 5.57 (d,
1 H, J ) 16.4 Hz, H-17), 5.47-4.44 (m, 5 H), 1.98-1.82 (m, 2 H,
H-19), 1.02-0.89 (m, 12 H), 0.80-0.70 (m, 6 H). 13C NMR
(CDCl3, 100 MHz) δ 171.6, 158.0, 156.3, 156.3, 153.0, 152.2,
151.0, 149.6, 144.8, 135.3, 132.1, 130.6, 130.0, 128.6-127.8 (11
C), 119.9, 98.4, 79.5, 75.2, 68.4, 67.9, 65.6, 33.0, 7.9, 7.2, 6.4.
HRMS m/z calcd for C42H44N4O8Si 760.293, m/z found 760.289.
13b: 1H NMR (CDCl3, 400 MHz) δ 8.85 (br s, 1 Η, Αr), 8.58 (br
s 1 H, Ar), 8.20 (br s, 1 H, Ar), 7.93 (br s, Ar), 7.81 (br t, 1 H, J
1
pale-yellow solid. 12a: H NMR (CDCl3, 400 MHz) δ 8.52 (s, 1
Η, Αr, H-7), 8.25 (d, 1 H, J ) 8.4 Hz, Ar), 7.96 (d, 1 H, J ) 8.4
Hz, Ar), 7.87 (t, 1 H, J ) 8.4 Hz, Ar), 7.69 (t, 1 H, J ) 8.4 Hz,

C-Ring-Modified Camptothecins
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1037
Ar), 7.59 (s, 1 H, H-14), 7.46 (d, 1 H, 1JHF ) 61.2 Hz, H-5), 5.69
(d, 1 H, J ) 16.8 Hz, H-17), 5.26 (d, 1 H, J ) 16.8 Hz, H-17),
3.87 (br s, 1 H, OH), 2.01-1.81 (m, 2 H, H-19), 1.05 (t, 3 H, J )
7.6 Hz, Me). 13C NMR (CDCl3, 100 MHz) δ 173.5, 157.6, 151.1,
151.0, 150.2, 144.1, 133.9, 131.9, 130.0, 129.0, 128.5, 127.8, 126.4,
NMR (CDCl3, 400 MHz) δ 8.71 (br s, 1 Η, Αr), 8.34 (br s 1 H,
Ar), 8.18 (br s, 1 H, Ar), 7.94 (br s, 1 H, Ar), 7.79 (br t, 1 H, J )
7.6 Hz, Ar), 7.70-6.70 (m, 12 H, Ar + H-14), 6.52 (br s, 1 H,
H-5), 5.53 (d, 1 H, J ) 16.4 Hz, H-17), 5.44-4.48 (m, 5 H), 3.87
(br s, 1 H, OH), 1.90-1.70 (m, 2 H, H-19), 0.99 (t, 3 H, J ) 7.6
Hz, Me). 13C NMR (CDCl3, 100 MHz) δ 173.4, 158.0, 156.3, 156.1,
153.0, 151.0, 150.9, 149.6, 145.3, 135.5, 132.3, 130.8, 129.8,
128.7-127.8 (11 C), 119.8, 98.4, 79.5, 72.7, 68.5, 67.8, 66.0, 31.6,
7.7.
1
121.7, 98.8, 93.8 (d, JCF ) 214.0 Hz, C-5), 72.5, 66.0, 31.5, 7.8.
HRMS m/z calcd for C20H15FN2O4 366.102, m/z found 366.109.
1
12b: H NMR (CDCl3, 400 MHz) δ 8.52 (s, 1 Η, Αr, H-7), 8.24
(d, 1 H, J ) 8.4 Hz, Ar), 7.96 (d, 1 H, J ) 8.4 Hz, Ar), 7.88 (t, 1
H, J ) 8.4 Hz, Ar), 7.69 (t, 1 H, J ) 8.4 Hz, Ar), 7.56 (s, 1 H,
H-14), 7.51 (d, 1 H, 1JHF ) 60.4 Hz, H-5), 5.69 (d, 1 H, J ) 16.4
Hz, H-17), 5.25 (d, 1 H, J ) 16.4 Hz, H-17), 3.87 (br s, 1 H, OH),
1.98-1.78 (m, 2 H, H-19), 1.04 (t, 3 H, J ) 7.6 Hz, Me). 13C
NMR (CDCl3, 100 MHz) δ 173.3, 157.7, 151.2, 151.2, 150.2, 144.2,
133.8, 131.9, 130.0, 129.0, 128.5, 127.8, 126.4, 121.6, 98.9, 93.7
20-OH-5-methylcamptothecin (7). Reaction of
5 with
Et3N·3HF afforded, after silica gel flash chromatography purifica-
tion (n-hexane/EtOAc, 1:3), 7 in 98% yield as a pale-yellow solid.
1H NMR (CDCl3) δ 8.25 (s, 1 H, Ar), 8.24 (d, 1 H, J ) 8.4 Hz,
Ar), 7.92 (d, 1 H, J ) 8.4 Hz, Ar), 7.82 (t, 1 H, J ) 8.4 Hz, Ar),
7.65 (t, 1 H, J ) 8.4 Hz, Ar), 7.63 (s, 1 H, Ar), 5.80-5.70 (m, 2
H, H-5 + H-17), 5.30 (d, 0.5 H, J ) 16.4 Hz, H-17), 5.28 (d, 0.5
H, J ) 16.4 Hz, H-17), 1.98-1.84 (m, 5 H, H-19 + Me), 1.06 (t,
1.5 H, J ) 7.6 Hz, Me), 1.05 (t, 1.5 H, J ) 7.6 Hz, Me).
1
(d, JCF ) 214.0 Hz, C-5), 72.5, 66.1, 31.6, 7.8. HRMS m/z calcd
for C20H15FN2O4 366.102, m/z found 366.110.
20-OH-5-azidocamptothecin (18a, 18b). Reaction of 17a (17b)
with Et3N·3HF afforded, after silica gel flash chromatography
purification (n-hexane/EtOAc, 1:1), 18a (18b) in 97% yield as a
20-OH-5-(3-hydroxypropyl)camptothecin (8). Reaction of 6
with Et3N·3HF afforded, after silica gel flash chromatography
purification (CH2Cl2-MeOH, 30:1), 8 in 98% yield as a as pale-
1
pale-yellow solid. 18a: H NMR (CDCl3, 400 MHz) δ 8.44 (s, 1
1
Η, Αr, H-7), 8.24 (d, 1 H, J ) 8.4 Hz, Ar), 7.93 (d, 1 H, J ) 8.4
Hz, Ar), 7.85 (t, 1 H, J ) 8.4 Hz, Ar), 7.67 (t, 1 H, J ) 8.4 Hz,
Ar), 7.63 (s, 1 H, H-14), 6.97 (s, 1 H, H-5), 5.70 (d, 1 H, J ) 16.8
Hz, H-17), 5.29 (d, 1 H, J ) 16.8 Hz, H-17), 3.99 (br s, 1 H, OH),
2.00-1.84 (m, 2 H, H-19), 1.04 (t, 3 H, J ) 7.6 Hz, Me). 13C
NMR (CDCl3, 100 MHz) δ 173.6, 158.3, 150.8, 150.7, 149.8, 144.4,
133.1, 131.5, 129.9, 128.6, 128.3, 128.3, 128.1, 120.6, 98.6, 75.4,
72.7, 66.0, 31.5, 7.8. HRMS m/z calcd for C20H15N5O4 389.112,
yellow solid. H NMR (CDCl3) δ 8.31 (s, 1 H, Ar), 8.22 (d, 1 H,
J ) 8.4 Hz, Ar), 7.92 (d, 1 H, J ) 8.4 Hz, Ar), 7.82 (t, 1 H, J )
8.4 Hz, Ar), 7.66 (t, 1 H, J ) 8.4 Hz, Ar), 7.65 (s, 0.5 H, Ar), 7.63
(s, 0.5 H, Ar), 5.88 (dd, 0.5 H, J1 ) 6.4 Hz, J2 ) 2.8 Hz, H-5),
5.82 (bt, 0.5 H, J ) 4.8 Hz, H-5), 5.71 (d, 0.5 H, J ) 16.0 Hz,
H-17), 5.70 (d, 0.5 H, J ) 16.0 Hz, H-17), 5.29 (d, 0.5 H, J )
16.0 Hz, H-17), 5.27 (d, 0.5 H, J ) 16.0 Hz, H-17), 3.76 (s, 1 H,
OH), 3.71-3.48 (m, 2 H), 2.68-2.48 (m, 2 H), 1.98-1.74 (m, 2
H, H-19), 1.48-1.30 (m, 2 H), 1.06 (t, 1.5 H, J ) 7.6 Hz, Me),
1.04 (t, 1.5 H, J ) 7.6 Hz, Me). 13C NMR (CDCl3) δ 173.9, 158.0,
150.1, 149.2, 148.8, 146.4, 133.2, 130.6, 130.3, 129.8, (128.3-128.2),
128.0, 119.4, 97.9, (72.7-72.7), (66.4-66.3), 62.4, 62.0, 31.6,
(27.9-27.4), (26.5-26.3), (7.9-7.8).
1
m/z found: 389.108. 18b: H NMR (CDCl3, 400 MHz) δ 8.45 (s,
1 Η, Αr, H-7), 8.23 (d, 1 H, J ) 8.4 Hz, Ar), 7.95 (d, 1 H, J ) 8.4
Hz, Ar), 7.85 (t, 1 H, J ) 8.4 Hz, Ar), 7.68 (t, 1 H, J ) 8.4 Hz,
Ar), 7.60 (s, 1 H, H-14), 7.00 (s, 1 H, H-5), 5.74 (d, 1 H, J ) 16.8
Hz, H-17), 5.28 (d, 1 H, J ) 16.8 Hz, H-17), 3.86 (br s, 1 H, OH),
1.98-1.82 (m, 2 H, H-19), 1.04 (t, 3 H, J ) 7.6 Hz, Me). 13C
NMR (CDCl3, 100 MHz) δ 173.4, 158.4, 150.9, 150.7, 149.8, 144.5,
133.0, 131.5, 129.9, 128.6, 128.4, 128.3, 128.1, 120.6, 98.6, 75.3,
72.6, 66.1, 31.6, 7.8. HRMS m/z calcd for C20H15N5O4 389.112,
m/z found 389.109.
Synthesis of 20. To a solution of 20-OTES-5-DTBAC-CPT (14)
(225 mg, 0.32 mmol) in dry dichloroethane (8 mL), TFA (0.9 mL,
11.7 mmol) was added. The mixture was stirred at room temperature
for 20 h and then refluxed for 4 h. The excess of solvents was
removed under reduced pressure, and the residue was purified by
silica gel flash chromatography (CH2Cl2-MeOH, 30:1). The
product was further purified by a second chromatography (tolu-
ene-AcOEt, 1:1) affording 20 as a yellow solid (84 mg, 55%). 1H
NMR (CDCl3, 400 MHz) δ 10.61 (br s, 0.5 Η, Ν5-NHdC16a),
10.39 (br s, 0.5 Η, Ν5-NHdC16a), 8.67 (s, 1 H, Ar, H-7),
8.22-8.15 (m, 1 H, Ar), 7.96-7.92 (m, 1 H, Ar), 7.88-7.78 (m,
1 H, Ar), 7.69-7.60 (m, 2 H, Ar), 6.38-6.36 (m, 1 H, Ar, H-5),
5.72-5.62 (m, 1 H, Ar, H-17), 5.32-5.20 (m, 2 H, Ar, H-17 +
N5H), 4.08-3.86 (br s, 1 H, OH), 1.96-1.74 (m, 2 H, H-19),
1.05-0.98 (t, 3 H, J ) 7.6 Hz, Me). 13C NMR (CDCl3, 100 MHz)
δ 173.4 (0.5 C), 173.8 (0.5 C), 159.1, 159.0, 156.7 (q CF3COOH),
156.5 (q CF3COOH) 151.5, 151.3, 150.7, 150.5, 150.1, 149.9, 144.8,
144.7, 134.0, 133.8, 131.6, 131.5, 129.9, 129.8, 128.7, 128.7, 128.4,
128.4, 128.2, 128.2, 127.1, 126.9, 120.5, 120.3, 99.1(2 C), 78.9,
78.6, 72.7, 72.7, 66.0 (2 C), 31.7 (2 C), 7.7, 7.7. HRMS m/z calcd
for C22H17F3N4O5 474.115, m/z found 474.109.
Synthesis of 21. DDQ (25 mg, 0.110 mmol) was added to a
solution of 20 (20 mg, 0042 mmol) in dry THF (4 mL). The reaction
mixture was warmed to 60 °C and reacted at this temperature for
3 h. The solvent was removed under reduced pressure, and water
was added. The aqueous layers were extracted with CH2Cl2 (15
mL × 3), and the combined organic extracts were dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
crude material was purified by silica gel flash chromatography (n-
hexane-OEtAc, 1:1) to afford 21 as a yellow solid (14 mg, 94%).
1H NMR (CDCl3, 400 MHz) δ 8.89 (s, 1 Η, Αr, H-7), 8.20 (d, 1
H, J ) 8.4 Hz, Ar), 8.00 (d, 1 H, J ) 8.4 Hz, Ar), 7.88 (t, 1 H, J
) 8.4 Hz, Ar), 7.79 (s, 1 H, Ar H-14), 7.69 (t, 1 H, J ) 8.4 Hz,
Ar), 5.70 (d, 1 H, J ) 17.2 Hz, H-17), 5.28 (d, 1 H, J ) 17.2 Hz,
H-17), 3.83 (br s, 1 H, OH), 2.00-1.74 (m, 2 H, H-19), 1.08 (t, 3
H, J ) 7.6 Hz, Me). 13C NMR (CDCl3, 100 MHz) δ 172.6, 157.4,
152.5, 150.8, 148.9, 143.7, 134.9, 132.5, 132.4, 130.0, 129.5, 128.7,
20-OH-5-DTBAC-camptothecin (16a, 16b). Reaction of 14a
(14b) with Et3N·3HF afforded, after silica gel flash chromatography
purification (n-hexane/EtOAc, 3:2), 16a (16b) in 97% yield as a
1
pale-yellow solid. 16a: H NMR (CDCl3, 400 MHz) δ 8.77 (br s,
1 Η, Αr), 8.16 (br d, 1 H, J ) 8.0 Hz, Ar), 7.97 (br s, 1 H, Ar),
7.86-7.50 (m, 4 H, Ar), 6.51 (br s, 1 H, H-5), 5.66 (d, 1 H, J )
16.4 Hz, H-17), 5.24 (d, 1 H, J ) 16.4 Hz, H-17), 3.86 (br s, 1 H,
OH), 2.00-1.80 (m, 2 H, H-19), 1.79-1.13 (br s, 18 H), 1.03 (t,
3 H, J ) 7.6 Hz, Me). 13C NMR (CDCl3, 100 MHz) δ 173.7, 157.9,
155.5, 155.5, 152.1, 151.3, 150.7, 149.6, 145.7, 132.3, 130.7, 129.9,
128.7, 127.9, 127.6, 120.0, 97.9, 82.8, 81.6, 79.7, 72.7, 66.1, 31.8,
1
28.3, 27.7, 7.7. 16b: H NMR (CDCl3, 400 MHz) δ 8.79 (br s, 1
Η, Αr), 8.22 (br d, 1 H, J ) 8.4 Hz, Ar), 7.99 (br s, 1 H, Ar),
7.88-7.50 (m, 4 H, Ar), 6.53 (br s, 1 H, H-5), 5.65 (d, 1 H, J )
16.4 Hz, H-17), 5.26 (d, 1 H, J ) 16.4 Hz, H-17), 3.80 (br s, 1 H,
OH), 2.00-1.80 (m, 2 H, H-19), 1.79-1.13 (br s, 18 H), 1.03 (t,
3 H, J ) 7.2 Hz, Me). 13C NMR (CDCl3, 100 MHz) δ 173.6, 157.9,
155.4, 155.4, 152.1, 151.3, 150.8, 149.5, 145.6, 132.3, 130.8, 129.8,
128.7, 127.9, 127.8, 119.8, 98.0, 83.0, 81.5, 79.7, 72.7, 66.3, 31.8,
28.3, 27.7, 7.8.
20-OH-5-DBAC-camptothecin (15a, 15b). Reaction of 13a
(13b) with Et3N·3HF afforded, after silica gel flash chromatography
purification (n-hexane/EtOAc, 1:1 followed by 2:3), 15a (15b) in
98% yield as a pale-yellow solid. 15a: 1H NMR (CDCl3, 400 MHz)
δ 8.67 (br s, 1 Η, Αr), 8.39 (br s 1 H, Ar), 8.12 (br d, 1 H, J ) 7.6
Hz, Ar), 7.95 (br s, 1 H, Ar), 7.74 (br t, 1 H, J ) 7.6 Hz, Ar),
7.65-6.66 (m, 12 H, Ar + H-14), 6.48 (br s, 1 H, H-5), 5.55 (d,
1 H, J ) 16.0 Hz, H-17), 5.42-4.44 (m, 5 H), 3.86 (br s, 1 H,
OH), 1.92-1.72 (m, 2 H, H-19), 0.95 (t, 3 H, J ) 7.6 Hz, Me).
13C NMR (CDCl3, 100 MHz) δ 173.5, 158.0, 156.2, 156.0, 153.0,
150.9, 150.9, 149.5, 145.3, 135.4, 132.2, 130.7, 129.8, 128.7-127.8
(11 C), 119.9, 98.2, 79.6, 72.7, 68.5, 68.0, 65.9, 31.6, 7.8. 15b: 1H

1038 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Samor`ı et al.
127.5, 122.6, 121.4, 101.2, 72.4, 66.0, 31.6, 7.7. HRMS m/z calcd
for C20H14N4O3 358.107, m/z found 358.101.
contributions to ligand-protein interaction; hydrogen-bonding, lipo-
philic contact, and metal binding (if present) terms are also taken into
account. In the second step, a mixed quantum mechanical/molecular
mechanics (QM/MM) method is used to compute the ligand charge
distribution. For QM/MM calculations the QSITE program is used.
The protein is defined as the MM region (force field, OPLS-AA), and
the ligand is defined as the QM region. To represent the QM region,
ab initio density functional theory (DFT) methods were used (6-31G*
basis set and hybrid DFT functional B3LYP). At each configuration,
a single point energy calculation was performed and the output was
used for further computation. The optimization of the QM wave
function was performed incorporating coupling of the surrounding MM
point charges. It is the effect of these point charges that leads to
polarization of the ligand charge distribution. After performance of
the DFT calculation for the ligand, atomic charges are assigned
employing electrostatic potential (ESP) fitting. In the third step, the
ligands are submitted to another GLIDE docking run where the ligand
charges are substituted with the new charge sets calculated in the
second step. Finally, GLIDE returns the most energetically favorable
poses.
20-OH-5-NH2-CPT (22). To a solution of 20-OH-5-azido-
camptothecin (18) (50 mg, 0.129 mmol) in a mixture of dry THF
(1.5 mL) and dry MeOH (6 mL) under nitrogen, 10% palladium
on activated carbon (14 mg) was added. The oxygen dissolved in
the reaction mixture was removed under vacuum. Then a hydrogen
balloon was mounted and the mixture was stirred for 3 h at room
temperature. The catalyst was removed by filtration through a Celite
pad, and the filtrate was concentrated under reduced pressure to
remove solvents. The crude product was purified by silica gel flash
chromatography CH2Cl2-MeOH (step gradient elution 35:1 and
1
25:1) to afford 22 as a pale-yellow solid (46 mg, 98%). H NMR
(CDCl3, 400 MHz) δ 8.48 (s, 1 Η, Αr, H-7), 8.22-8.17 (m, 1 H,
Ar), 7.95-7.90 (m, 1 H, Ar), 7.85-7.78 (m, 1 H, Ar), 7.68-7.60
(m, 1 H, Ar), 7.58 (s, 0.5 H, H-14), 7.54 (s, 0.5 H, H-14) 6.50 (s,
0.5 H, H-5), 6.47 (s, 0.5 H, H-5), 5.74-5.64 (m, 1 H, H-17),
5.28-5.22 (m, 1 H, H-17), 4.00-2.40 (br s, 3 H, OH + NH2),
1.98-1.82 (m, 2 H, H-19), 1.07-1.01 (m, 3 H, Me). 13C NMR
(CDCl3, 100 MHz) δ 173.8 (2 C), 158.5 (2 C), 151.2 (2 C), 150.4
(2 C), 149.7 (2 C), 144.5 (2 C), 132.7 (2 C), 131.0 (2 C), 129.8 (2
C), 128.5 (2 C), 128.3 (2 C), 128.0 (2 C), 127.8 (2 C), 120.2 (2 C),
113.8 (2 C), 97.7 (2 C), 72.7 (2 C), 66.3, 66.0 31.5 (2 C), 7.8, 7.8.
Growth Inhibition Assay. Human non-small-cell lung cancer
NCI-H460 cells26 were cultured in RPMI 1640 containing 10%
FCS. Cell sensitivity was assessed by growth inhibition assay.
Briefly, cells in the logarithmic phase of growth were seeded in
duplicate into six-well plates. Twenty-four hours later, cells were
exposed for 1 h to the drug and counted with a Coulter counter
72 h later. IC50 is defined as the inhibitory drug concentration
causing a 50% decrease of cell growth over that of untreated control.
Topoisomerase I Dependent DNA Cleavage Assay. A gel-
purified 751-bp BamHI-EcoRI fragment of SV40 DNA was used
for the cleavage assay.27 DNA fragments were uniquely 3′-end
labeled. Topoisomerase I-DNA cleavage reactions (20 000 cpm per
sample) were done in 20 µL of 10 mM Tris-HCl (pH 7.6), 150
mM KCl, 5 mM MgCl2, 15 µg/mL bovine serum albumin, 0.1 mM
DTT, and the human recombinant enzyme (full-length topo-
isomerase I) for 30 min at 37 °C. Reactions were stopped using
0.5% SDS and 0.3 mg/mL of proteinase K for 45 min at 42 °C.
DNA was resuspended in denaturing buffer (80% formamide, 10
mM NaOH, 0.01 mM EDTA, and 1 mg/mL dyes) before loading
on a denaturing 8% polyacrylamide gel in Tris-borate EDTA buffer.
Overall, DNA cleavage levels were measured with a PhosphoImager
425 model (Molecular Dynamics, Sunnyvale, CA).28
Supporting Information Available: Elemental analysis data for
all new and enantiomeric pure compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
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