Topoisomerase I-mediated DNA cleavage as a guide to the development of antitumor agents derived from the marine alkaloid lamellarin D: Triester derivatives incorporating amino acid residues

Article abstract of DOI:10.1016/j.bmc.2004.01.020

The marine alkaloid lamellarin D (LAM-D) has been recently characterized as a potent poison of human topoisomerase I endowed with remarkable cytotoxic activities against tumor cells. We report here the first structure-activity relationship study in the LAM-D series. Two groups of triester compounds incorporating various substituents on the three phenolic OH at positions 8, 14 and 20 of 6H-[1]benzopyrano[4′,3′:4,5]pyrrolo[2,1-a]isoquinolin-6- one pentacyclic planar chromophore typical of the parent alkaloid were tested as topoisomerase I inhibitors. The non-amino compounds in group A showed no activity against topoisomerase I and were essentially non cytotoxic. In sharp contrast, compounds in group B incorporating amino acid residues strongly promoted DNA cleavage by human topoisomerase I. LAM-D derivatives tri-substituted with leucine, valine, proline, phenylalanine or alanine residues, or a related amino side chain, stabilize topoisomerase I-DNA complexes. The DNA cleavage sites detected at T↓G or C↓G dinucleotides with these molecules were identical to that of LAM-D but slightly different from those seen with camptothecin which stimulates topoisomerase I-mediated cleavage at T↓G only. In the DNA relaxation and cleavage assays, the corresponding Boc-protected compounds and the analogues of the non-planar LAM-501 derivative lacking the 5-6 double bond in the quinoline B-ring showed no effect on topoisomerase I and were considerably less cytotoxic than the corresponding cationic compounds in the LAM-D series. The presence of positive charges on the molecules enhances DNA interaction but melting temperature studies indicate that DNA binding is not correlated with topoisomerase I inhibition or cytotoxicity. Cell growth inhibition by the 41 lamellarin derivatives was evaluated with a panel of tumor cells lines. With prostate (DU-145 and LN-CaP), ovarian (IGROV and IGROV-ET resistant to ecteinascidin-743) and colon (LoVo and LoVo-Dox cells resistant to doxorubicin) cancer cells (but not with HT29 colon carcinoma cells), the most cytotoxic compounds correspond to the most potent topoisomerase I poisons. The observed correlation between cytotoxicity and topoisomerase I inhibition strongly suggests that topoisomerase I-mediated DNA cleavage assays can be used as a guide to the development of superior analogues in this series. LAM-D is the lead compound of a new promising family of antitumor agents targeting topoisomerase I and the amino acid derivatives appear to be excellent candidates for a preclinical development.

Full text of DOI:10.1016/j.bmc.2004.01.020

Bioorganic & Medicinal Chemistry 12 (2004) 1697–1712
Topoisomerase I-mediated DNA cleavage as a guide to the
development of antitumor agents derived from the marine alkaloid
lamellarin D: triester derivatives incorporating amino acid residues
Christelle Tardy,a Michael Facompre,a William Laine,a Brigitte Baldeyrou,a
Dolores Garcıa-Gravalos,b Andres Francesch,b Cristina Mateo,b Alfredo Pastor,b
Jose A. Jimenez,b Ignacio Manzanares,b Carmen Cuevasb and Christian Baillya,*
aINSERM UR-524 and Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL, Place de Verdun,
59045 Lille, France
bPharmaMar, Avda. de losReyes1, Poligono Industrial La Mina-Norte, 28770 Colmenar Viejo, Madrid, Spain
Received 21 August 2003; accepted 13 January 2004
Abstract—The marine alkaloid lamellarin D (LAM-D) has been recently characterized as a potent poison of human topoisomerase
I endowed with remarkable cytotoxic activities against tumor cells. We report here the first structure–activity relationship study in
the LAM-D series. Two groups of triester compounds incorporating various substituents on the three phenolic OH at positions 8,
14 and 20 of 6H-[1]benzopyrano[40,30:4,5]pyrrolo[2,1-a]isoquinolin-6-one pentacyclic planar chromophore typical of the parent
alkaloid were tested as topoisomerase I inhibitors. The non-amino compounds in group A showed no activity against topoisome-
rase I and were essentially non cytotoxic. In sharp contrast, compounds in group B incorporating amino acid residues strongly
promoted DNA cleavage by human topoisomerase I. LAM-D derivatives tri-substituted with leucine, valine, proline, phenylalanine
or alanine residues, or a related amino side chain, stabilize topoisomerase I–DNA complexes. The DNA cleavage sites detected at
T#G or C#G dinucleotides with these molecules were identical to that of LAM-D but slightly different from those seen with
camptothecin which stimulates topoisomerase I-mediated cleavage at T#G only. In the DNA relaxation and cleavage assays, the
corresponding Boc-protected compounds and the analogues of the non-planar LAM-501 derivative lacking the 5–6 double bond in
the quinoline B-ring showed no effect on topoisomerase I and were considerably less cytotoxic than the corresponding cationic
compounds in the LAM-D series. The presence of positive charges on the molecules enhances DNA interaction but melting temp-
erature studies indicate that DNA binding is not correlated with topoisomerase I inhibition or cytotoxicity. Cell growth inhibition
by the 41 lamellarin derivatives was evaluated with a panel of tumor cells lines. With prostate (DU-145 and LN-CaP), ovarian
(IGROV and IGROV-ET resistant to ecteinascidin-743) and colon (LoVo and LoVo-Dox cells resistant to doxorubicin) cancer
cells (but not with HT29 colon carcinoma cells), the most cytotoxic compounds correspond to the most potent topoisomerase I
poisons. The observed correlation between cytotoxicity and topoisomerase I inhibition strongly suggests that topoisomerase I-
mediated DNA cleavage assays can be used as a guide to the development of superior analogues in this series. LAM-D is the lead
compound of a new promising family of antitumor agents targeting topoisomerase I and the amino acid derivatives appear to be
excellent candidates for a preclinical development.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
plants such as etoposide and taxol, or drugs derived
from plant alkaloids such as the camptothecin (CPT)
derivatives topotecan and irinotecan. Antibiotics are
equally well represented because several of the most
frequently used anticancer drugs, daunomycin and
bleomycin, for example, originate from Streptomyces.
The sea world is also a rich source of anticancer agents.
The marine environment has provided many lead com-
pounds, the bryostatins, aplidine and kahalalide F to
cite only a few of them.1ꢀ5 The most advanced anti-
Many natural products are used to combat cancer. The
chemotherapeutic arsenal includes drugs extracted from
Keywords: Lamellarin; Topoisomerase I; DNA interaction; Cytotoxi-
city; Anticancer drugs.
Abbreviations: CPT, Camptothecin; LAM-D, Lamellarin D.
* Corresponding author. Tel.: +33-320-16-92-18; fax: +33-320-16-92-
29; e-mail: bailly@lille.inserm.fr
0968-0896/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.01.020

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C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712
cancer drug issued from the sea is probably ET-743
(Yondelis1, trabectidin), a tetrahydroisoquinoline alka-
loid produced by the tunicate Ecteinascidia turbinata.6,7
considerably less cytotoxic than the parent natural
alkaloid. The 5–6 double bond seems to be an essential
element for the poisoning of topoisomerase I and the
antiproliferative activity. Here we have investigated
further the importance of this structural element by
comparing the activity of two series of lamellarin ana-
logues bearing a 6H-[1]benzopyrano[40,30:4,5]pyrrolo[2,1-
a]isoquinolin-6-one pentacyclic planar chromophore
typical of the parent alkaloid or a 5,6-dehydro non-pla-
nar chromophore as found in the analogue LAM-501.
The newly synthesized lamellarin derivatives are all tri-
esters and differ by the nature of the substituent intro-
duced on the three phenolic OH at positions 8, 14 and
20. Two sets of compounds were studied. Compounds
in group A (Chart 1) refer to the non-amino derivatives
and include various ester with aryl groups or diethyl-
phosphono and methylsulfone groups. Compounds in
group B (Chart 2) refer to cationic derivatives with
diverse amino acid substituents (such as Leu, Val, Ala,
Pro, Trp, Phe). The results of our first structure–activity
relationship study in the LAM-D series, aimed at iden-
tifying the structural elements implicated in the anti-
topoisomerase I activity, confirm that the lamellarins
represent a new and promising series of topoisomerase I
inhibitors. The correlation between the capacity of the
drugs to stimulate topoisomerase I-mediated DNA
cleavage and their cytotoxic potential is a good augur
for the development of antitumor agents.
PharmaMar, the spanish company which develops ET-
743, aplidine and kahalalide F, has characterized a large
number of marine compounds of potential interest for
the treatment of cancer. One of the novel drug candi-
date is the hexacyclic alkaloid lamellarin D (LAM-D)
initially isolated from a prosobranch mollusc of the
genus Lamellaria8 and subsequently found in ascidians.9
Over 30 lamellarins have been isolated and interestingly,
some of them including LAM-D, show equally potent
cytotoxic activities against both multi-drug resistant
(MDR) and their corresponding parental cell lines.10
Very recently, we have identified topoisomerase I as a
molecular target for LAM-D and this important dis-
covery has prompted us to exploit the LAM-D phar-
macophore for the development of topoisomerase I-
targeted anticancer agents. Molecular and cellular stud-
ies revealed that LAM-D potently stabilizes topoisome-
rase I–DNA covalent complexes so as to promote the
formation of DNA single strand breaks. In parallel,
camptothecin-resistant cell lines expressing a mutated
top1 gene were found to be cross-resistant to LAM-D.11
In sharp contrast, the synthetic analogue LAM-501
lacking the 5–6 double bond in the quinoline B-ring was
found to be totally inactive against topoisomerase I and
Chart 1.

C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712
1699
2. Results
(Scheme 2) by reaction of LAM-501 (2) with the
corresponding acid using EDC as a coupling reagent or
the acid chloride with triethylamine. Treatment of the
ester derivatives with DDQ afforded the derivatives of
LAM-D (1). Compounds in group B (Chart 2) were
obtained in a similar way (Scheme 3) by reaction of
LAM-501 (2) with N-protected aminoacid using EDC
as a coupling reagent, oxidation with DDQ to obtain
2.1. Chemistry
The synthesis of LAM-D (1) and LAM-501 (2) was
achieved by [3+2] cycloaddition reaction of the corres-
ponding dihydroisoquinoline 44 with ester 43 (Scheme
1) following the procedure described previously.12
Compounds in group A (Chart 1) were obtained
Chart 2.
Scheme 1. Reagents: (a) (i) ClCH2CH2Cl, 23 ꢁC; (ii) DlPEA, 85 ꢁC; (b) DDQ, CHCl3, reflux; (c) AlCl3, CH2Cl2, 23 ꢁC.

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C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712
the derivatives of LAM-D (1) and deprotection of the
amino group with trifluoroacetic acid or acetyl chloride
in MeOH to afford the cationic derivatives.
acetotetrafluorophenyl (11) group, show no effect on
DNA cleavage by the enzyme. The analogous com-
pounds with a 5–6 saturated bond (2,4,6,8,12) were also
inactive in this assay. Different ester groups, coumarin
(13), 1-aceto-(9H-fluoren-1-yl) (14), and diethylphos-
phate (15), were incorporated on the three phenoxy
positions of LAM-D but no topoisomerase I inhibitor
could be identified. The first design was unsuccessful
and therefore we adopted another strategy which con-
sisted to incorporate a cationic substituent with the
rational that a positively charged group might facilitate
interaction with DNA phosphate and possibly the tar-
get enzyme. Different cationic groups, mostly amino
acid residues, were incorporated at the three phenoxy
positions of LAM-D. Compounds of group B (Chart 2)
include Ala, Leu, Val, Pro and Phe derivatives and a
few analogous molecules such as the aminopentyl deriv-
ative 39. In all cases, we tested the uncharged Boc-pro-
tected intermediates and the final cationic products.
Here again, in most cases the compounds were prepared
in the LAM-D (C5–C6 double bond) and LAM-501
(C5–C6 single bond) series. A marked inhibition of
topoisomerase I was observed with the positively
charged molecules 18 (Ala), 22 (Leu), 26 (Val), 30 (Pro)
and 34 (Phe) but not with the corresponding NH-Boc
derivatives or the non-planar C5–C6 analogues (Fig.
2.2. Topoisomerase I inhibition
Two assays, based on DNA relaxation and DNA clea-
vage,13 were used evaluate the effects of the lamellarin
analogues on the catalytic activity of human topo-
isomerase I. In the first assay, a supercoiled plasmid
DNA was relaxed with topoisomerase I in the absence
or presence of the test compounds, each tested at 1 mM.
DNA relaxation products were then resolved by gel
electrophoresis on agarose gels containing ethidium
bromide to stain the DNA. The results are presented in
Figure 1. The alkaloid camptothecin, used as a positive
control, strongly stabilizes the cleaved complex with
topoisomerase I. Similarly, the intensity of the band
corresponding to nicked DNA is significantly amplified
in the presence of LAM-D indicating that this natural
product also stabilizes DNA–topoisomerase I covalent
complexes. This functional assay is useful to identify the
topoisomerase I poisons among the various analogues
synthesized. Compounds in group A are all inactive
against topoisomerase I. All the uncharged esters, be it a
small acetyl substituent (3) or with a bulkier 2,3,4,5-
ꢁ
.
Scheme 2. Reagents: (a) RCO2H, EDC HCl, DMAP, or RCOCl, Et3N, CH2Cl2, 23 C; (b) DDQ, CHCl3, reflux.
ꢁ
.
Scheme 3. Reagents: (a) BocHNaaCO2H, EDC HCl, DMAP, CH2Cl2, 23 C; (b) DDQ, CHCl3, reflux; (c) TFA, CH2Cl2, or ClCOCH3/MeOH,
EtOAc, 23 ꢁC.

C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712
1701
Figure 1. Effect of the lamellarin derivatives on the stabilisation of covalent DNA–topoisomerase I complexes. Native supercoiled pLAZ3 DNA
(lane DNA) was incubated with topoisomerase I in the absence (lane TopoI) or presence of the indicated compound. Lanes marked CPT refer to
camptothecin (10 mM). LAM-D (1) and its derivatives were tested at 1 mM. DNA samples were separated by electrophoresis on agarose gels con-
taining ethidium bromide (1 mg/mL). Gel were photographed under UV light. Nck, nicked; Rel, relaxed; Sc, supercoiled.
1c). The Phe derivative is significantly less potent than
the other amino acid derivatives which are all more or
less equally effective at inhibiting topoisomerase I. The
stereospecificity was investigated with the Val deriva-
tives for which we compared the activity of the (L) (25–
28) and (D) (25r–28r) isomers but, as shown in Figure
1d, there was no difference between the two series.
Compound 26 and 26r both stimulated DNA cleavage
by the enzyme. No effect was observed with the Boc-
proteted analogues in the C5–C6 double stranded (25,
25r) or C5–C6 single-stranded (27, 28, 27r, 28r) series.
The amino compound 40 was also found to inhibit
topoisomerase I (Fig. 1b).
It is clear that the introduction of an amino acid func-
tionality on the phenolic OH groups at positions 8, 14
and 20 of LAM-D is not detrimental to topoisomerase I
inhibition. The extent of topoisomerase I-mediated
DNA cleavage is fully maintained when a Leu, Val, Ala
or Pro residue is incorporated on the LAM-D skeleton
whereas a non charged group abolishes the anti-topoi-
somerase I activity. A phenylalanine residue is much less
favorable than a proline or an alanine residue for
example. The observations that the incorporation of a
cationic group promoted topoisomerase I inhibition
suggested that the enhanced capacity of the drugs to bind
to DNA could be responsible for a better enzyme inhi-
bition. To verify this idea, melting temperature measure-
ments were carried out with the polymer poly(dAT)2
and for each compound we calculated the ÁTm values
(TmdrugꢀDNA complexꢀTmDNA alone). Poly(dAT)2 which
melt at a low temperature (41ꢂ1 ꢁC in BPE buffer)
affords a sensitive determination of the DNA binding
capacity of the studied molecules. As indicated in Table
1, the Ala (18) and Pro (30) derivatives markedly stabi-
lize the polymer against heat denaturation with ÁTm
values >10 ꢁC and this correlates with their potent anti-
topoisomerase I activity. In contrast, the Phe derivative
(34) showed no effect on DNA thermal stability and this
Concentration-dependent measurements were per-
formed with each of the positive compounds identified
and a few representative gels comparing the anti-topoi-
somerase I activity of LAM-D (1) with the three analo-
gues Val(D) (26r), Pro (30) and the amino compound 40
are shown in Figure 2. This later compound is equally
efficient to 1 in terms of stimulation of DNA cleavage
by topoisomerase I and is also equally cytotoxic (see
below). In all cases, the dose–response analysis con-
firmed that the cationic LAM-D analogues potently
inhibit the enzyme.

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C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712
may be the reason why this compound is more weakly
active against topoisomerase I compared to the other
amino acid derivatives. Enhanced DNA binding seems
to facilitate topoisomerase I inhibition but tight binding
to DNA is not required for enzyme inhibition since the
natural product LAM-D shows little affinity for DNA
despite its potent topoisomerase I poisoning capacity.
that the cationic lamellarin derivatives do effectively
function as topoisomerase I poisons. A 117-bp DNA
restriction fragment uniquely end-labeled at the 30 end
was subjected to cleavage by topoisomerase I in the
presence of the different compounds and the resulting
DNA cleavage products were resolved on sequencing-
type polyacrylamide gels. A typical gel is presented in
Figure 3. The advantage of this assay is to detect the
cleavage sites and to locate their positions with nucleo-
tide resolution, providing thus information on the site
selectivity of cleavage.
A second assay, based on the cleavage of a radiolabeled
DNA substrate by topoisomerase I, was used to confirm
From the two typical gels shown in Figure 3a and b, it
is easy to identify the compounds that induce DNA
cleavage by topoisomerase I. The reference drug CPT
produces three sites at nucleotide positions 26, 48 and
81 which all three correspond to T#G sites. Cleavage at
TG sites in the presence of CPT is believed to result
from the interaction of topoisomerase I with the T resi-
due combined with the stacking of the CPT molecule
with the adjacent G residue.14,15 A fourth weak site can
be detected at the top of the gels (T#G107). The
sequence selectivity profiles are slightly different with
the lamellarin analogues. LAM-D is less efficient than
CPT for topoisomerase I-mediated DNA cleavage at
sites T#G48 and T#G81 but it induces an additional
cleavage site at C#G73. This likely reflects a different
mode of interaction with the topoisomerase I–DNA
covalent complexes, as recently discussed.11 Cleavage
profiles identical to that of 1 were obtained with the
cationic derivatives such as 22 (Leu), 26 (Val), 30 (Pro)
and 34 (Phe) but not with the corresponding NH-Boc
derivatives or the non-planar C5–C6 analogue (Fig. 3a).
The amino compound 40 was also found to stimulate
DNA cleavage by the enzyme and here also we found
no difference between the (l) (26) and (d) (26d) Val
isomers (Fig. 3b). The results are thus entirely consistent
with those obtained by the relaxation assay and there-
fore validate the conclusion that the cationic lamellarin
derivatives potently inhibit topoisomerase I.
Figure 2. Concentration-dependent effect for the inhibition of topoi-
somerase I by LAM-D (1) or the cationic derivatives 26r, 30 and 40.
All concentrations are given in mM. Other details as for Figure 1.
Table 1. Topoisomerase I inhibition, DNA binding and cytotoxicity
Nicked DNA (%)a
ÁTm (ꢁC)b
GI50 (nM)c
LAM-D (1)
LAM-501 (2)
25.7
12.3
29.7
7.0
31.1
9.2
2.9
0.0
13.3
6.8
4.5
1.0
8.0
4.1
12.0
5.3
0.0
0.9
0.0
4.1
10.9
2990
16.2
3650
18.0
4000
10.8
1600
18.6
2120
25.2
1630
913
2.3. Cytotoxicity
18
19
22
23
26
27
30
31
34
36
38
40
Seven human tumor cell lines were used to determine
the cytotoxicity of the lamellarin derivatives: HT29
human colon carcinoma cells, LoVo human colon
lymph node metastasis cells and the corresponding
LoVo-Dox cells resistant to doxorubicin, ovarian cells
sensitive (IGROV) or resistant (IGROV-ET) to ectei-
nascidin-743, and the prostate tumor cells LN-CaP and
DU-145. A colorimetric assay was used up to estimate
GI50 values, that is, the drug concentration which causes
50% cell growth inhibition after 72 h continuous expo-
sure to the test molecules. We have shown recently that
LAM-D exhibits a pronounced cytotoxic effect toward
prostatic cells, in particular the DU-145 metastatic cells
which are androgen-insensitive.11 This cell line was used
as a reference to compare the different compound.
Table 1 reports the GI50 values for each compound
along with the effect on topoisomerase I and DNA
binding activity. To quantify topoisomerase I poison-
ing, we measured the extent of DNA cleavage in the
presence of the drug at 1 mM (% nicked DNA).
33.6
6.7
24.6
13.4
31.4
10.0
6.2
27.6
15.1
a Extent of topoisomerase I-mediated DNA cleavage measured with
the lamellarin derivatives (1 mM each). The% of nicked DNA (form
II) was determined by densitometry. Band intensities from three gels
such as those shown in Figure 1 were compiled for the quantitative
analysis.
b Variation of the melting temperature (ÁTm) of helix-to-coil transi-
tion of poly(dAT)2 in the presence of the lamellarin derivatives. Tm
measurements were performed in BPE buffer pH 7.1 (6 mM
Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA) using 20 mM poly(dAT)2
(nucleotide concentration) and 20 mM drug.
c Drug concentration that causes 50% growth inhibition of DU-145
metastatic cells. Values were calculated from dose–response curves.

C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712
1703
Figure 3. Cleavage of the 117 bp DNA fragment by topoisomerase I in the presence of the lamellarin derivatives. The 30-end labeled fragment was
incubated in the absence (lane TopoI) or presence of the test drug at 50 mM. CPT was used at 20 mM. Topoisomerase I cleavage reactions were
analyzed on a 8% denaturing polyacrylamide gel. Numbers at the right side of the gels show the nucleotide positions, determined with reference to
the guanine tracks labeled G. Arrows point to the five main cleavage sites.
Compounds 18, 22, 26, 30, 34 and 40 were found to be
equally efficient as 1 at stimulating DNA cleavage by
topoisomerase I. This effect is well correlated with the
GI50 data. Indeed, GI50 values are in the 10–25 nM
range with these compounds whereas the other mole-
cules inactive or weakly active against topoisomerase I
all give GI50 values in the 1–4 mM range. In contrast, the
DNA binding activity, measured through the extent of
stabilization of poly(dAT)2 against heat denaturation
(ÁTm values in Table 1), is not correlated with cyto-
toxicity or topoisomerase I inhibition. LAM-D and
compound 22 both give low ÁTm values, 2.9 and 4.5 ꢁC
respectively, and compounds 18 and 30 give high ÁTm
values, 13.3 and 12.0 ꢁC respectively, but these four
compounds are equally cytotoxic and represent potent
topoisomerase I poison. Nevertheless, if DNA binding
is not sufficient to confer activity against topoisomerase
I in this series, it may be useful because the compounds
which do not stabilize duplex DNA, such as 23, 34 and
38, are inactive. DNA binding is certainly not a good
criterion to predict cytotoxicity in this series. On the
opposite, topoisomerase I inhibition (i.e., enzyme-medi-
ated DNA cleavage) may represent a useful indicator of
the drug antiproliferative activity.
androgen-insensitive) and LN-CaP (hormonally respon-
sive but non metastatic). In both cases, the most cyto-
toxic compounds (low GI50 values) generally correspond
the most potent inhibitors of topoisomerase I. A similar
conclusion can be drawn when looking at the LoVo
human colon cells and IGROV ovarian cells. In most
cases (but not all) the compounds which strongly sti-
mulate DNA cleavage by topoisomerase I were the most
potent cytotoxic agents (Fig. 4b and c). The use of cells
resistant to doxorubicin (LOVO-DOX) or to ecteinasci-
din 743 (IGROV-ET) gave the same results. Here again,
we found that the less cytotoxic molecules were gen-
erally devoid of activity against topoisomerase I
whereas inhibition of the enzyme often coincided with a
marked cytotoxic potential. These data showing a
correlation between topoisomerase I inhibition and
cytotoxicity are extremely important as they strongly
suggest that the topoisomerase I-mediated DNA clea-
vage assay can be used as a guide to the development of
superior analogues in this series. This observation may
greatly facilitate the optimization of the LAM-D lead
compound. However, one should bear in mind that no
general rule can be drawn. This is illustrated with the
HT29 human colon carcinoma cell line which is poorly
sensitive to LAM-D11 and with which we could not
detect any correlation between cytotoxicity and topoi-
somerase I inhibition (Fig. 4d).
The relationship between topoisomerase I poisoning
and cytotoxicity was investigated further with the other
cell lines. GI50 values determined with each type of cells
were plotted against the extent of DNA cleavage mea-
sured with the different compounds. The graphics in
Figure 4a shows a satisfactory relation between the
enzyme inhibitory activity and the cytotoxicity toward
the two prostatic cell lines DU-145 (metastatic and
In conclusion, our SAR study reveals that LAM-D
derivatives represent a novel family of topoisomerase I-
targeted antitumor agents. The 5–6 double bond in the
quinoline B-ring of LAM-D is absolutely required to
maintain an activity against topoisomerase I and a

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C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712
Figure 4. Correlation between cytotoxicity and topoisomerase I inhibition. In each graph the extent of topoisomerase I-mediated DNA cleavage
measured with each compound at 1 mM is plotted as a function of the GI50 values determined with the indicated cell lines (see text for details on the
origin of the tumor cells). In panels (a), (b) and (c), note that the most cytotoxic molecules are generally the most potent topoisomerase I poisons
(dashed box).
potent cytotoxic action. The three phenolic OH at posi-
tions 8, 14 and 20 are also important structural elements
of the LAM-D structure but they can be substituted
with amino acid derivatives without loss of activity. The
cationic proline (30) and valine (26) derivatives have
been selected for a preclinial development to evaluate
their antitumor activity in vivo.
drous 1,2-dichloroethane (40 mL) under Argon atmos-
phere. The reaction mixture was stirred for 8 h at 23 ꢁC
turning to an orange solution. At this time, diisopropyl-
amine (DIPEA, 0.75 mL, 4.31 mmol) was added and the
resulting brown solution stirred at 85 ꢁC for 32 h. The
reaction mixture was cooled to 23 ꢁC, silica gel (6 g) was
added and the solvent removed under reduced pressure.
The resulting crude was purified by chromatography on
silica gel (hexane:CH2Cl2:Et2O, 5:5:2) to afford 41 as a
1
pale yellow solid (1.27 g, 47%). H NMR (300 MHz,
3. Experimental
3.1. Chemistry
CDCl3) d 7.08–7.04 (m, 3H), 6.92 (s, 1H), 6.76–6.74 (m,
2H), 6.67 (s, 1H), 4.87–4.71 (m, 2H), 4.65–4.48 (m, 3H),
3.82 (s, 3H), 3.42 (s, 3H), 3.33 (s, 3H), 3.09 (t, J=6.6
Hz, 2H), 1.41–1.36 (m, 18H). 13C NMR (75 MHz,
CDCl3) d 155.5, 151.2, 148.5, 147.2, 146.9, 146.8, 146.4,
145.8, 135.9, 128.5, 128.1, 126.3, 123.3, 120.1, 116.8,
114.8, 114.6, 114.5, 113.6, 110.3, 109.1, 104.8, 103.4,
71.7, 71.3, 71.2, 56.1, 55.4, 55.0, 42.3, 28.5, 22.0 (2C),
21.8, 21.8, 21.7 (2C). MS (ESI) m/z: 628 (M+1)+. Rf:
0.28 (hexane:CH2Cl2:Et2O, 5:5:2).
1
3.1.1. General methods. H and 13C NMR spectra were
recorded on a Varian AC300 instrument (300 MHz for
1H, 75 MHz for 13C) using CDCl3, CD3OD or
(CD3)2SO. Chemical shifts were reported in ppm (d
scale) relative to Me4Si as an internal standard, and all J
values were in Hz. Reagents obtained from commercial
suppliers were used without further purification unless
otherwise noted. All air- and water-sensitive reactions
were performed in flame-dried glassware under a posi-
tive pressure of argon. Thin-layer chromatography was
carried out on SDS precoated silica gel 60 F254 plates.
The spots were visualized with UV light (254 and 366
nm). Column chormatography was performed using the
indicated solvents on silica gel 60 (0.040–0.063 mm,
SDS). Mass spectra (ESI and APCI) were recorded on a
HP Series 1100.
3.1.3. 3,11-Diisopropoxy-14-(4-isopropoxy-3-methoxy-
phenyl)-2,12-dimethoxy-6H-[1]Benzopyrano[40,30:4,5]pyr-
rolo[2,1-a]isoquinolin-6-one (42). A suspension of 41
(301 mg, 0.480 mmol) and 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ, 139 mg, 0.611 mmol) in CHCl3
(10 mL) was refluxed for 2 h. The mixture was cooled at
23 ꢁC then filtered through Celite, and washed with
CH2Cl2. The filtrated was concentrated under reduced
pressure and the residue was purified by chromato-
graphy on silica gel (hexane:EtOAc, 2:1) to give 42 (283
mg, 94%). 1H NMR (300 MHz, CDCl3) d 9.24 (d,
J=7.3 Hz, 1H), 7.29–7.17 (m, 2H), 7.12–7.10 (m, 2H),
7.04–7.02 (m, 2H), 6.98 (s, 1H), 6.76 (s, 1H), 4.70–4.56
(m, 3H), 3.84 (s, 3H), 3.44 (s, 3H), 3.43 (s, 3H), 1.44–
1.39 (m, 18H). 13C NMR (75 MHz, CDCl3) d 155.3,
151.2, 150.0, 148.3, 147.7, 147.0, 146.4, 146.3, 134.2,
3.1.2. 5,6-dihydro-3,11-Diisopropoxy-14-(4-isopropoxy-3-
methoxyphenyl)-2,12-dimethoxy-6H-[1]Benzopyrano[40,30:
4,5]pyrrolo[2,1-a]isoquinolin-6-one (41). 6-Isopropoxy-7-
methoxy-3,4-dihydroisoquinoline 44 (1.06 g, 4.83 mmol)
was added to a solution of Iodo-acetic acid 5-iso-
propoxy-2-(4-isopropoxy-3-methoxy-phenylethynyl)-4-
methoxy-phenyl ester 43 (2.30 g, 4.27 mmol) in anhy-

C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712
1705
129.2, 128.6, 124.6, 123.8, 122.9, 118.8, 116.7, 114.9,
112.1, 110.8, 110.2, 109.8, 107.6, 105.5, 105.3, 103.2,
71.6, 71.3, 71.0, 56.0, 55.3, 55.0, 21.8 (3C), 21.7, 21.7,
21.6. MS (ESI) m/z: 648 (M+23)+, 626 (M+1)+. Rf:
0.35 (hexane:EtOAc, 2:1).
3.1.7. Compound 4. A solution of LAM-501 (2) (40 mg,
0.08 mmol) and Ac2O (0.5 mL, 5.289 mmol) in pyridine
(1 mL) was stirred at 23 ꢁC under Argon atmosphere for
4 h. The resulting pale yellow solution was washed with
HCl 1 N (2ꢃ10 mL). The organic phase was dried over
anhydrous Na2SO4, filtered, and the solvent removed
under vacuum. The residue was purified by chromato-
graphy on silica gel (CH2Cl2:MeOH, from 50:1 to 40:1)
to give 4 as a white solid (38 mg, 76%). 1H NMR
(300 MHz, CDCl3) d 7.22 (d, J=7.9 Hz, 1H), 7.15–7.09
(m, 3H), 6.95 (s, 1H), 6.79 (s, 1H), 6.69 (s, 1H), 4.92–
4.71 (m, 2H), 3.81 (s, 3H), 3.43 (s, 3H), 3.35 (s, 3H), 3.12
(t, J=6.7 Hz, 2H), 2.35 (s, 3H), 2.31 (s, 3H), 2.30 (s,
3H). 13C NMR (75 MHz, CDCl3) d 169.0, 168.8, 168.6,
155.1, 152.2, 149.9, 147.7, 144.9, 140.0, 139.4, 138.9,
135.0, 134.0, 127.1, 125.9, 125.7, 123.9, 123.2, 122.6,
116.0, 115.9, 114.9, 114.6, 112.0, 109.7, 105.4, 56.2, 55.7,
55.5, 42.5, 28.1, 20.6 (3C). MS (ESI) m/z: 628 (M+1)+.
Rf: 0.32 (CH2Cl2:MeOH, 100:1).
3.1.4. LAM-D (1). A suspension of 42 (271 mg, 0.433
mmol) and AlCl3 (462 mg, 3.465 mmol) in anhydrous
CH2Cl2 (4 mL) was stirred at 23 ꢁC for 5.5 h under
Argon atmosphere. H2O was added, then HCl 2 M until
pH 1–2, the resulting aqueous solution was extracted
with CH2Cl2 (3ꢃ), dried over anhydrous Na2SO4, fil-
tered, and the solvent was evaporated under reduced
pressure. The residue was purified by chromatography
on silica gel (EtOAc, 100%) to afford LAM-D (1) as a
1
pale yellow solid (92 mg, 43%). H NMR (300 MHz,
DMSO-d6) d 9.92 (s, 1H), 9.81 (s, 1H), 9.32 (s, 1H), 8.98
(d, J=7.3 Hz, 1H), 7.22–6.98 (m, 6H), 6.85 (s, 1H), 6.70
(s, 1H), 3.75 (s, 3H), 3.36 (s, 6H). 13C NMR (75 MHz,
DMSO-d6) d 154.3, 148.7, 148.5, 148.3, 147.8, 146.8,
146.3, 144.6, 134.1, 129.2, 128.9, 125.5, 124.7, 123.9,
117.6, 116.4, 115.1, 113.9, 112.3, 111.5, 110.8, 106.4,
105.7, 105.4, 103.7, 56.0, 55.1, 54.5. MS (APCI) m/z:
500 (M+1)+. Rf: 0.60 (EtOAc).
3.1.8. Compound 5. A suspension of 6 (25 mg, 0.034
mmol) and DDQ (15 mg, 0.068 mmol) in CHCl3 (2 mL)
was refluxed for 77 h. The mixture was cooled to 23 ꢁC
then filtered through Celite, and washed with CH2Cl2
(50 mL). The filtrated was concentrated under reduced
pressure and the residue was purified by chromato-
graphy on silica gel (CH2Cl2:MeOH, 80:1) to give 5 (17
mg, 68%). 1H NMR (300 MHz, CDCl3) d 9.16 (d,
J=7.5 Hz, 1H), 7.66 (s, 1H), 7.60 (d, J=8.6 Hz, 1H),
7.33–7.30 (m, 3H), 7.18 (s, 1H), 7.06 (d, J=7.7 Hz, 1H),
6.76 (s, 1H), 3.96 (s, 3H), 3.49 (s, 6H), 3.37 (s, 3H), 3.24
(s, 3H), 3.21 (s, 3H). MS (APCI) m/z: 734 (M+1)+. Rf:
0.33 (CH2Cl2:MeOH, 80:1).
3.1.5. LAM-501 (2). A suspension of 41 (1.422 g, 2.265
mmol) and AlCl3 (1.208 g, 9.061 mmol) in anhydrous
CH2Cl2 (43 mL) was stirred at 23 ꢁC for 2.5 h under
Argon atmosphere. MeOH (20 mL) was added and the
solvent evaporated under reduced pressure. The brown
residue was purified by chromatography on silica gel
(CH2Cl2:MeOH, from 20:1 to 10:1 to 5:1) to afford
1
LAM-501 (2) as a pale brown solid (1.11 g, 97%). H
NMR (300 MHz, DMSO-d6) d 9.64 (s, 1H), 9.41 (s, 1H),
9.24 (s, 1H), 7.01 (s, 1H), 6.99 (d, J=8.1 Hz, 1H), 6.88
(d, J=8.4 Hz, 1H), 6.78 (s, 1H), 6.73 (s, 1H), 6.67 (s,
1H), 6.59 (s, 1H), 4.58 (t, J=6.5 Hz, 2H), 3.73 (s, 3H),
3.34 (s, 3H), 3.25 (s, 3H), 2.99 (t, J=6.4 Hz, 2H). 13C
NMR (75 MHz, DMSO-d6) d 154.3, 148.5, 147.1, 146.9,
146.5, 146.0, 145.7, 144.4, 135.9, 127.7, 127.1, 125.5,
123.4, 118.1, 116.3, 115.3, 114.7, 114.3, 112.2, 109.2,
108.8, 105.1, 103.6, 56.0, 55.0, 54.7, 42.0, 27.5. MS
(ESI) m/z: 524 (M+23)+. Rf: 0.55 (CH2Cl2:MeOH
10:1).
3.1.9. Compound 6. To a suspension of LAM-501 (2) (50
mg, 0.0997 mmol) in anhydrous CH2Cl2 (2 mL) under
Argon at 0 ꢁC, Et3N (83 mL, 0.5982 mmol) and metha-
nesulfonyl chloride (47 mL, 0.5982 mmol) were added.
The resulting mixture was stirred at 23 ꢁC for 6 h, then
quenched with H2O and extracted with CH2Cl2 (3ꢃ20
mL). The combined organic layers were washed with
saturated aqueous solution of NaHCO3, dried over
anhydrous Na2SO4, filtered, and evaporated under
vacuum. The resulting residue was purified on silica gel
(CH2Cl2:MeOH, 80:1) to afford 6 as a pale yellow solid
1
3.1.6. Compound 3. A suspension of 4 (20 mg, 0.032
mmol) and DDQ (15 mg, 0.064 mmol) in CHCl3 (2 mL)
was refluxed for 24 h. The mixture was cooled to 23 ꢁC
then filtered through Celite, and washed with CH2Cl2
(50 mL). The filtrated was concentrated under reduced
pressure and the residue was purified by chromato-
graphy on silica gel (CH2Cl2:MeOH, 40:1) to give 3 (12
mg, 60%). 1H NMR (300 MHz, CDCl3) d 9.22 (d,
J=7.3 Hz, 1H), 7.39 (s, 1H), 7.30 (d, J=7.7 Hz, 1H),
7.25–7.22 (m, 3H), 7.14 (s, 1H), 7.05 (d, J=7.5 Hz, 1H),
6.81 (s, 1H), 3.84 (s, 3H), 3.45 (s, 6H), 2.37 (s, 3H), 2.34
(s, 3H), 2.32 (s, 3H). 13C NMR (75 MHz, CDCl3) d
168.9, 168.7, 168.7, 155.0, 152.4, 151.0, 147.8, 145.4,
140.9, 140.3, 139.7, 134.2, 133.5, 128.2, 124.1, 123.8,
123.7, 123.6, 123.1, 120.7, 115.7, 115.0, 112.8, 112.3,
112.2, 109.1, 106.4, 106.1, 56.2, 55.7, 55.6, 20.6 (3C).
MS (ESI) m/z: 626 (M+1)+. Rf: 0.40 (CH2Cl2:MeOH,
100:1).
(47 mg, 64%). H NMR (300 MHz, CDCl3) d 7.52 (d,
J=8.1 Hz, 1H), 7.30 (s, 1H), 7.23–7.20 (m, 2H), 7.17 (d,
J=1.6 Hz, 1H), 6.75 (s, 1H), 6.65 (s, 1H), 4.99–4.90 (m,
1H), 4.71–4.61 (m, 1H), 3.92 (s, 3H), 3.46 (s, 3H), 3.38
(s, 3H), 3.34 (s, 3H), 3.19 (s, 3H), 3.18 (s, 3H), 3.14 (t,
J=6.0 Hz, 2H). 13C NMR (75 MHz, CDCl3) d 154.5,
152.8, 150.1, 148.0, 144.6, 138.0, 137.7, 136.9, 135.4,
134.4, 126.5, 126.4, 126.3, 125.6, 124.4, 123.3, 117.0,
115.8, 115.4, 115.2, 113.5, 109.9, 105.6. MS (ESI) m/z:
736 (M+1)+. Rf: 0.33 (CH2Cl2:MeOH, 80:1).
3.1.10. Compound 7. A suspension of 8 (23 mg, 0.026
mmol) and DDQ (12 mg, 0.051 mmol) in CHCl3 (2 mL)
was refluxed for 21 h. The mixture was cooled to 23 ꢁC
then filtered through Celite and washed with CH2Cl2
(50 mL). The filtrated was concentrated under reduced
pressure and the residue was purified by chromato-
graphy on silica gel (CH2Cl2:MeOH, 100:1) to give 7 (16

1706
C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712
mg, 70%). 1H NMR (300 MHz, CDCl3) d 9.23 (d,
J=7.3 Hz, 1H), 7.39–7.20 (m, 20H), 7.08–7.03 (m, 2H),
6.80 (s, 1H), 3.79 (s, 3H), 3.42 (s, 6H), 3.16–3.06 (m,
6H), 3.00–2.90 (m, 6H). 13C NMR (75 MHz, CDCl3) d
170.8, 170.7, 170.6, 155.1, 152.4, 151.0, 147.7, 145.4,
140.9, 140.2, 140.1 (2C), 139.7, 134.2, 133.5, 128.5 (6C),
128.4 (2C), 128.4 (4C), 128.2, 126.4, 126.4 (2C), 124.0,
123.8, 123.6, 123.6, 123.1, 120.7, 115.6, 115.0, 112.8,
112.3, 112.1, 109.0, 106.4, 106.1, 56.2, 55.7, 55.6, 35.4
(3C), 30.9, 30.8 (2C). MS (APCI) m/z: 896 (M+1)+. Rf:
0.25 (CH2Cl2:MeOH, 200:1).
mL) was stirred under Argon atmosphere at 23 ꢁC for 2
h. The resulting pale yellow solution was washed with
H2O (10 mL), the aqueous phase was extracted
with CH2Cl2 (10 mL) and the combined organic layers
were dried over anhydrous Na2SO4, filtered, and the
solvent removed under vacuum. The residue was pur-
ified by chromatography on silica gel (hexane:EtOAc,
1
60:40) to give 10 as a white solid (47 mg, 94%). H
NMR (300 MHz, CDCl3) d 7.57–7.49 (m, 6H), 7.16–
7.00 (m, 10H), 6.86 (s, 1H), 6.74 (s, 1H), 6.64 (s, 1H),
4.89–4.85 (m, 1H), 4.75–4.70 (m, 1H), 3.85 (s, 2H), 3.79
(s, 4H), 3.75 (s, 3H), 3.34 (s, 3H), 3.28 (s, 3H), 3.09 (t,
J=6.6 Hz, 2H). 13C NMR (75 MHz, CDCl3) d 167.7,
167.5, 167.4, 164.2, 160.9, 154.9, 152.1, 149.7, 147.5,
144.8, 139.7, 139.2, 138.6, 134.9, 134.3, 133.8, 133.7,
133.6, 133.6, 129.3, 129.3, 126.9, 125.9, 125.8, 123.6,
123.1, 122.3, 116.4 (6C), 116.2, 116.1 (6C), 115.8, 115.0,
114.7, 111.7, 109.7, 105.5, 56.1, 55.6, 55.4, 42.4, 37.5
(3C), 27.8. MS (ESI) m/z: 1006 (M+1)+. Rf: 0.40
(hexane:EtOAc, 60:40).
3.1.11. Compound 8. To a suspension of LAM-501 (2)
(25 mg, 0.050 mmol) in anhydrous CH2Cl2 (2 mL), 4-
(dimethylamino)pyridine (DMAP, 4 mg, 0.030 mmol),
pyridine (24 mL, 0.300 mmol) and hydrocinnamoyl
chloride (45 mL, 0.300 mmol) were added. The reaction
mixture was stirred under Argon atmosphere at 23 ꢁC
for 22 h, then diluted with EtOAc (50 mL) and washed
with H2O (2ꢃ20 mL) and saturated aqueous solution of
NaHCO3 (2ꢃ20 mL). The combined organic layers were
dried over anhydrous Na2SO4, filtered, and con-
centrated under reduced pressure. The resulting residue
was purified by chromatography on silica gel
(CH2Cl2:MeOH, from 200:1 to 100:1) to give 8 as a
3.1.14. Compound 11. A suspension of 12 (20 mg, 0.019
mmol) and DDQ (9 mg, 0.039 mmol) in CCl4 (2 mL)
was refluxed for 7 h. The mixture was cooled at 23 ꢁC
then filtered through Celite, and washed with CH2Cl2
(50 mL). The filtrated was concentrated under reduced
pressure and the residue was purified by chromato-
graphy on silica gel (CH2Cl2:MeOH, 100:1) to give 11
(15 mg, 75%) as a white solid. 1H NMR (300 MHz,
CDCl3) d 9.24 (d, J=7.5 Hz, 1H), 7.84–7.75 (m, 3H),
7.54 (s, 1H), 7.45 (d, J=8.4 Hz, 1H), 7.37–7.35 (m, 2H),
7.30 (s, 1H), 7.26 (s, 1H), 7.08 (d, J=7.3 Hz, 1H), 6.90
(s, 1H), 3.87 (s, 3H), 3.52 (s, 3H), 3.52 (s, 3H). 13C
NMR (75 MHz, CDCl3) d 159.7 (3C), 154.8, 152.3,
150.8, 148.2, 147.6, 145.4, 140.3, 139.8, 139.2, 135.0,
133.4, 128.0, 124.0, 123.9, 123.8, 123.7, 123.2, 120.7,
116.2, 115.3, 113.8, 113.6, 112.8, 112.4, 112.1, 109.2,
106.6, 106.3, 56.4, 55.9, 55.8. MS (ESI) m/z: 1050
(M+23)+, 1028 (M+1)+. Rf: 0.63 (CH2Cl2).
1
white solid (31 mg, 69%). H NMR (300 MHz, CDCl3)
d 7.37–7.21 (m, 15H), 7.13–7.02 (m, 4H), 6.87 (s, 1H),
6.77 (s, 1H), 6.68 (s, 1H), 4.92–4.83 (m, 1H), 4.79–4.70
(m, 1H), 3.76 (s, 3H), 3.39 (s, 3H), 3.32 (s, 3H), 3.13–
3.04 (m, 8H), 2.97–2.87 (m, 6H). 13C NMR (75 MHz,
CDCl3) d 171.0, 170.7, 170.6, 155.1, 152.2, 149.8, 147.7,
144.9, 140.1 (3C), 140.0, 139.4, 138.9, 135.0, 133.9, 128.5
(6C), 128.4 (6C), 127.1, 126.4, 126.4, 126.4, 125.9, 125.6,
123.8, 123.1, 122.6, 116.0, 115.9, 114.9, 114.6, 111.9,
109.7, 105.4, 56.1, 55.7, 55.5, 42.4, 35.5 (3C), 30.9, 30.9,
30.8, 28.0. MS (ESI) m/z: 898 (M+1)+. Rf: 0.25
(CH2Cl2:MeOH, 200:1).
3.1.12. Compound 9. A suspension of 10 (26 mg, 0.026
mmol) and DDQ (9 mg, 0.039 mmol) in CHCl3 (1.5
mL) was refluxed for 40 h. The mixture was cooled at
23 ꢁC, filtered through Celite, and washed with CH2Cl2
(50 mL). The filtrated was concentrated under reduced
pressure and the residue was purified by chromato-
graphy on silica gel (CH2Cl2:MeOH, from 200:1 to
100:1) to give 9 (17 mg, 65%) as a white solid. 1H NMR
(300 MHz, CDCl3) d 9.21 (d, J=7.3 Hz, 1H), 7.58–7.50
(m, 6H), 7.31 (s, 1H), 7.21–7.16 (m, 4H), 7.10–7.01 (m,
8H), 6.75 (s, 1H), 3.87 (s, 2H), 3.84 (s, 2H), 3.81 (s, 2H),
3.78 (s, 3H), 3.37 (s, 6H). 13C NMR (75 MHz, CDCl3) d
167.6, 167.5 (2C), 162.6 (d, JC-F=248.3, 3C), 154.9,
152.3, 150.8, 147.6, 145.4, 140.6, 140.0, 139.5, 134.6,
133.9, 133.8, 133.8, 133.7, 133.6, 133.4, 129.5, 129.3,
128.1, 123.8, 123.6, 123.2, 120.5, 116.4 (6C), 116.1 (6C),
115.9, 115.1, 112.8, 112.3, 112.0, 109.1, 106.4, 106.2. MS
(ESI) m/z: 1026 (M+23)+, 1004 (M+1)+. Rf: 0.35
(CH2Cl2).
3.1.15. Compound 12. A suspension of LAM-501 (2) (25
mg, 0.050 mmol), 2,3,4,5-tetrafluorobenzoic acid (58
.
mg, 0.30 mmol), EDC HCl (58 mg, 0.30 mmol) and
DMAP (4 mg, 0.03 mmol) in anhydrous CH2Cl2 (5 mL)
was stirred under Argon atmosphere at 23 ꢁC for 6 h.
The resulting pale yellow solution was washed with H2O
(10 mL), the aqueous phase was extracted with CH2Cl2
(10mL), the combined organic phases were dried over
anhydrous Na2SO4, filtered, and the solvent removed
under vacuum. The residue was purified by chroma-
tography on silica gel (CH2Cl2:MeOH, 200:1) to give 12
as
a
white solid (33 mg, 64%). 1H NMR
(300 MHz, CDCl3) d 7.81–7.74 (m, 3H), 7.36 (d, J=8.1
Hz, 1H), 7.25 (s, 1H), 7.23 (s, 1H), 7.19 (s, 1H), 7.10 (s,
1H), 6.86 (s, 1H), 6.78 (s, 1H), 4.97–4.91 (m, 1H), 4.82–
4.77 (m, 1H), 3.83 (s, 3H), 3.48 (s, 3H), 3.41 (s, 3H),
3.17 (t, J=6.5 Hz, 2H). 13C NMR (75 MHz, CDCl3) d
159.6 (3C), 154.9, 152.1, 149.7, 147.5, 144.9, 139.5,
138.9, 138.3, 134.9, 134.7, 126.9, 126.2, 126.1, 123.7,
123.3, 122.5, 116.5, 115.9, 115.2, 114.9, 113.8, 113.5,
111.9, 109.9, 105.6, 56.3, 55.9, 55.7, 42.5, 28.1. MS
(APCI) m/z: 1030 (M+1)+. Rf: 0.50 (CH2Cl2:MeOH,
200:1).
3.1.13. Compound 10. A suspension of LAM-501 (2) (25
mg, 0.05 mmol), (4-Fluorophenyl)thioacetic acid (56
mg, 0.30 mmol), 1-(3-dimethylaminopropyl)-3-ethylcar-
.
bodiimide hydrochloride (EDC HCl, 58 mg, 0.30 mmol)
and DMAP (4 mg, 0.03 mmol) in anhydrous CH2Cl2 (5

C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712
1707
3.1.16. Compound 13. A suspension of LAM-501 (2) (25
mg, 0.050 mmol), coumarin 3-carboxylic acid (38 mg,
(s, 3H), 3.09 (br t, 2H), 1.44–1.31 (m, 18H). 13C NMR
(75 MHz, CDCl3) d 155.1, 151.7 (d, JC-P=4.5 Hz), 149.3
(d, JC-P=5.5 Hz), 147.4 (d, JC-P=5.0 Hz), 144.9, 139.9
(d, JC-P=7.1 Hz), 139.6 (d, JC-P=6.5 Hz), 139.1 (d,
JC-P=7.6 Hz), 135.0, 133.0, 127.0, 126.2, 124.4, 123.2,
122.6, 122.6, 121.1, 115.5, 114.9, 114.8, 110.5, 109.8,
105.6, 64.7, 64.7, 64.7 (3C), 64.6, 56.2, 55.8, 55.5, 42.4,
28.1, 16.2, 16.1 (3C), 16.0, 16.0. MS (ESI) m/z: 910
(M+1)+. Rf: 0.23 (CH2Cl2:MeOH, 30:1).
.
0.200 mmol), EDC HCl (38 mg, 0.200 mmol) and
DMAP (7 mg, 0.0598 mmol) in anhydrous CH2Cl2 (4
mL) was stirred under Argon atmosphere at 23 ꢁC for
3.5 h. The resulting pale yellow solution was washed
with H2O (10 mL) and saturated aqueous solution of
NaHCO3 (10 mL). Both aqueous phases were extracted
with CH2Cl2 (10 mL). The combined organic layers
were dried over anhydrous Na2SO4, filtered, and the
solvent removed under vacuum. The residue was pur-
ified by chromatography on silica gel (CH2Cl2:MeOH,
from 50:1 to 40:1) to give 13 as a white solid (41 mg,
3.1.19. Compound 17. A suspension of 19 (63 mg, 0.062
mmol) and DDQ (21 mg, 0.093 mmol) in CHCl3 (5 mL)
was refluxed for 2 h. The mixture was cooled at 23 ꢁC
then filtered through Celite, and washed with CH2Cl2
(50 mL). The filtrated was concentrated under reduced
pressure and the residue was purified by chromato-
graphy on silica gel (hexane:EtOAc, 50:50) to give 17
1
80%). H NMR (300 MHz, CDCl3) d 8.80 (s, 1H), 8.79
(s, 1H), 8.75 (s, 1H), 7.72–7.65 (m, 6H), 7.42–7.34 (m,
7H), 7.26–7.21 (m, 3H), 7.23 (s, 1H), 6.85 (s, 1H), 6.81
(s, 1H), 4.93–4.86 (m, 1H), 4.78–4.69 (m, 1H), 3.84 (s,
3H), 3.50 (s, 3H), 3.44 (s, 3H), 3.15 (br t, 2H). MS (ESI)
m/z: 1040 (M+23)+. Rf: 0.24 (CH2Cl2:MeOH, 50:1).
1
(58 mg, 92%). H NMR (300 MHz, CDCl3) d 9.24 (d,
J=7.3 Hz, 1H), 7.44–7.32 (m, 2H), 7.25–7.18 (m, 4H),
7.07 (d, J=7.5 Hz, 1H), 6.79 (d, J=7.5 Hz, 1H), 5.11–
5.09 (m, 3H), 4.64–4.60 (m, 3H), 3.81 (s, 3H), 3.44 (s,
6H), 1.63–1.55 (m, 9H), 1.49 (s, 9H), 1.47 (s, 18H). 13C
NMR (75 MHz, CDCl3) d 171.5, 171.3, 171.1, 155.0,
154.8, 152.2, 150.8, 147.6, 145.3, 140.6, 140.0, 139.4,
134.4, 133.3, 128.0, 127.9, 123.9, 123.7, 123.7, 123.7,
123.6, 123.0, 120.6, 115.7, 115.1, 112.7, 112.2, 112.0,
108.9, 106.3, 106.1, 80.0 (3C), 56.2 (2C), 55.8, 55.7, 55.7,
55.5, 28.3 (9C), 18.6 (3C). MS (ESI) m/z: 1035
(M+23)+, 1013 (M+1)+. Rf: 0.43 (hexane:EtOAc,
50:50).
3.1.17. Compound 14. A suspension of LAM-501 (2) (25
mg, 0.05 mmol), 9H-fluorene-4-carboxylic acid (63 mg,
.
0.30 mmol), EDC HCl (58 mg, 0.30 mmol) and DMAP
(4 mg, 0.03 mmol) in anhydrous CH2Cl2 (5 mL) was
stirred under Argon atmosphere at 23 ꢁC for 2 h. The
resulting pale yellow solution was washed with H2O (10
mL), the aqueous phase was extracted with CH2Cl2 (10
mL), the combined organic phases were dried over
anhydrous Na2SO4 and the solvent removed under
vacuum. The residue was purified by chromatography
on silica gel (CH2Cl2:MeOH, 200:1) to give 14 as a
1
white solid (26 mg, 48%). H NMR (300 MHz, CDCl3)
3.1.20. Compound 18. TFA (1 mL) was added to a
solution of 17 (15 mg, 0.015 mmol) in anhydrous
CH2Cl2 (3 mL) at 0 ꢁC under Argon atmosphere. The
reaction mixture was stirred at 23 ꢁC for 5 h. The sol-
vent was evaporated under reduced pressure and, in
order to eliminate the remaining TFA, the mixture was
treated with CH2Cl2 (3ꢃ15 mL) and evaporated to
d 8.56–8.52 (m, 2H), 8.46–8.43 (m, 1H), 8.18–8.11 (m,
3H), 7.77–7.74 (m, 3H), 7.58–7.56 (m, 3H), 7.50–7.30
(m, 13H), 7.21 (s, 1H), 7.04 (s, 1H), 6.93 (s, 1H), 5.04–
4.95 (m, 1H), 4.92–4.83 (m, 1H), 3.96 (s, 6H), 3.93 (s,
3H), 3.62 (s, 3H), 3.54 (s, 3H), 3.25 (t, J=6.5 Hz, 2H).
13C NMR (75 MHz, CDCl3) d 166.1, 166.0, 166.0,
155.2, 152.6, 150.2, 148.0, 145.2, 145.2, 145.1, 144.2,
144.2, 144.2, 141.5, 141.3, 140.4, 139.9, 139.8, 139.2,
135.2, 134.3, 129.5, 129.3, 129.1, 129.0, 127.7, 127.3,
126.9, 126.8, 126.1, 126.1, 126.0, 125.9, 125.4, 125.1,
125.1, 125.0, 124.7, 124.6, 124.1, 123.4, 122.8, 116.3,
116.1, 115.1, 114.8, 112.2, 109.9, 105.7, 56.3, 55.9, 55.7,
42.6, 37.0 (3C), 28.2. MS (ESI) m/z: 1100 (M+23)+. Rf:
0.47 (CH2Cl2).
1
dryness to give 18 as a white solid (16 mg, quant.). H
NMR (300 MHz, CD3OD) d 9.03–8.99 (m, 1H), 7.63–
7.60 (m, 2H), 7.52 (dd, J=8.0, 1.6 Hz, 1H), 7.39–7.27
(m, 2H), 7.18–7.15 (m, 2H), 6.85 (d, J=9.2 Hz, 1H),
4.53–4.36 (m, 3H), 3.93 (s, 3H), 3.48 (s, 6H), 1.80 (d,
J=7.1 Hz, 3H), 1.74 (d, J=7.3 Hz, 3H), 1.71–1.68 (m,
3H). MS (ESI) m/z: 713 (M+1)+.
3.1.21. Compound 19. A suspension of LAM-501 (2) (50
mg, 0.0997 mmol), Boc-l-Ala-OH (75 mg, 0.3988
3.1.18. Compound 15. To a suspension of LAM-501 (2)
(15 mg, 0.030 mmol) in anhydrous CH2Cl2 under Argon
atmosphere, Et3N (17 mL, 0.120 mmol) and diethyl
chlorophosphate (18 mL, 0.120 mmol) were added and
the mixture was stirred at 23 ꢁC. After 4.5 h, two more
equivalents of Et3N (9 mL, 0.060 mmol) and diethyl
chlorophosphate (9 mL, 0.060 mmol) were added and
the mixture stirred at 23 ꢁC overnight. The mixture was
concentrated under reduced pressure and the residue
.
mmol), EDC HCl (76 mg, 0.3988 mmol) and DMAP (7
mg, 0.0598 mmol) in anhydrous CH2Cl2 (4.3 mL) was
stirred under Argon atmosphere at 23 ꢁC for 2 h. The
resulting pale yellow solution was washed with H2O (10
mL) and HCl 0.1 N (10 mL) and the aqueous phases
were extracted with CH2Cl2 (10 mL). The organic pha-
ses were dried over anhydrous Na2SO4, filtered, and the
solvent removed under vacuum. The residue was pur-
ified by chromatography on silica gel (hexane:EtOAc,
from 2:1 to 1:1) to give 19 as a white solid (81 mg, 80%).
1H NMR (300 MHz, CDCl3) d 7.25–7.09 (m, 4H), 6.97
(s, 1H), 6.75 (d, J=7.7 Hz, 1H), 6.67 (d, J=10.1 Hz,
1H), 5.12 (br s, 2H), 4.89–4.85 (m, 1H), 4.70–4.55 (m,
3H), 3.78 (s, 3H), 3.40 (s, 3H), 3.33 (s, 3H), 2.03 (br t,
2H), 1.58 (d, J=7.1 Hz, 3H), 1.52 (d, J=7.1 Hz, 6H),
purified
by
chromatography
on
silica
gel
(CH2Cl2:MeOH, from 30:1 to 15:1) to give 15 as a white
solid (20 mg, 74%). 1H NMR (300 MHz, CDCl3) d 7.45
(dd, J=8.1, 1.5 Hz, 1H), 7.29 (d, J=1.5 Hz, 1H), 7.18
(s, 1H), 7.10 (dd, J=8.1, 1.6 Hz, 1H), 7.06 (s, 1H), 6.71
(s, 1H), 6.64 (s, 1H), 4.94–4.86 (m, 1H), 4.72–4.63 (m,
1H), 4.34–4.18 (m, 12H), 3.84 (s, 3H), 3.44 (s, 3H), 3.36

1708
C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712
1.47 (s, 9H), 1.45 (s, 18H). 13C NMR (75 MHz, CDCl3)
d 171.4, 155.0, 152.0, 149.7, 147.4, 144.8, 139.7, 139.1,
138.6, 135.0, 134.1, 126.9, 126.8, 126.0, 125.9, 125.7,
123.7, 123.1, 122.4, 116.1, 115.8, 114.9, 114.7, 111.7, 109.6,
105.4, 79.9, 60.3, 56.1, 55.7, 55.7, 55.5, 55.4, 49.2, 42.3,
28.2, 27.9, 21.0, 18.5, 14.1. MS (ESI) m/z: 1037 (M+23)+,
1015 (M+1)+. Rf: 0.44 (hexane:EtOAc, 50:50).
mg, 0.0598 mmol) in anhydrous CH2Cl2 (4.3 mL) was
stirred under Argon atmosphere at 23 ꢁC for 3 h. The
resulting pale yellow solution was washed with H2O (10
mL) and HCl 0.1 N (10 mL) and the aqueous phases
were extracted with CH2Cl2 (10 mL). The combined
organic layers were dried over anhydrous Na2SO4, fil-
tered, and the solvent removed under vacuum. The
residue was purified by chromatography on silica gel
(hexane:EtOAc, 2:1) to give 23 as a white solid (77 mg,
68%). 1H NMR (300 MHz, CDCl3) d 7.24–7.08 (m,
4H),6.99 (s, 1H), 6.76 (d, J=7.0 Hz, 1H), 6.69 (d,
J=8.4 Hz, 1H), 4.94–4.86 (m, 3H), 4.78–4.68 (m, 1H),
4.63–4.50 (m, 2H), 4.37–4.26 (m, 2H), 3.78 (s, 3H), 3.41
(s, 3H), 3.34 (s, 3H), 3.12 (br t, 2H), 1.90–1.60 (m, 9H),
1.45 (s, 27H), 1.05–0.95 (m, 18H). 13C NMR (75 MHz,
CDCl3) d 177.6 (2C), 171.4, 155.6, 155.4, 155.1, 152.1,
149.7, 147.6, 144.8, 139.8, 139.2, 138.7, 135.0, 134.1,
127.0, 127.0, 126.0, 125.7, 123.8, 123.1, 122.5, 116.1,
115.8, 114.9, 114.7, 111.9, 109.7, 105.5, 80.0 (3C), 56.1,
55.7, 55.5, 53.1, 52.2 (2C), 42.4, 41.5 (3C), 28.3 (9C),
28.0, 24.7 (2C), 22.9, 22.8 (4C), 21.8 (2C). MS (ESI) m/z:
1163 (M+23)+, 1141 (M+1)+. Rf: 0.26 (hexane:EtOAc,
2:1).
3.1.22. Compound 20. TFA (1 mL) was added to a
solution of 19 (15 mg, 0.0148 mmol) in anhydrous
CH2Cl2 (3 mL) at 0 ꢁC under Argon atmosphere. The
reaction mixture was stirred at 23 ꢁC for 2 h. The sol-
vent was evaporated under reduced pressure and, in
order to eliminate the remaining TFA, the mixture was
treated with CH2Cl2 (3ꢃ15 mL) and evaporated to
1
dryness to give 20 as a white solid (17 mg, quant.) H
NMR (300 MHz, CD3OD) d 7.46–7.44 (m, 2H), 7.28–
7.27 (m, 1H), 7.19 (s, 1H), 7.16 (s, 1H), 6.87 (d, J=8.8
Hz, 1H), 6.78 (d, J=10.0 Hz, 1H), 4.75 (t, J=6.2 Hz,
2H), 4.77–4.37 (m, 3H), 3.87 (s, 3H), 3.45 (s, 3H), 3.38
(s, 3H), 3.16 (t, J=6.2 Hz, 2H), 1.77 (d, J=6.9 Hz, 3H),
1.71–1.67 (m, 6H). MS (ESI) m/z: 715 (M+1)+.
3.1.23. Compound 21. A suspension of 23 (52 mg, 0.046
mmol) and DDQ (16 mg, 0.068 mmol) in CHCl3 (5 mL)
was refluxed for 35 h. The mixture was cooled at 23 ꢁC
then filtered through Celite, and washed with CH2Cl2
(50 mL). The filtrated was concentrated under reduced
pressure and the residue was purified by chromato-
graphy on silica gel (CH2Cl2:MeOH, 100:1) to give 21
(38 mg, 73%) as a white solid. 1H NMR (300 MHz,
CDCl3) d 9.22 (d, J=7.3 Hz, 1H), 7.42 (s, 1H), 7.32 (d,
J=7.9 Hz, 1H), 7.25–7.17 (m, 4H), 7.05 (d, J=7.7 Hz,
1H), 6.79 (d, J=5.9 Hz, 1H), 4.98–4.96 (m, 3H), 4.62–
4.56 (m, 3H), 3.80 (s, 3H), 3.44 (s, 3H), 3.43 (s, 3H),
1.87–1.64 (m, 9H), 1.49 (s, 9H), 1.46 (s, 18H), 1.06–0.99
(m, 18H). 13C NMR (75 MHz, CDCl3) d 171.4 (4C),
155.3, 155.0, 152.3, 150.9, 147.7, 145.4, 140.7, 140.1,
139.6, 134.4, 128.1, 124.0, 123.8, 123.6, 123.2, 120.7,
115.8, 115.1, 112.8, 112.3, 112.2, 109.1, 106.4, 106.2,
80.0 (3C), 56.2 (2C), 55.8 (2C), 55.7, 52.2, 41.7 (2C),
41.5, 28.3 (9C), 24.8 (3C), 23.0, 22.9 (3C), 21.9. MS
(ESI) m/z: 1161 (M+23)+, 1139 (M+1)+. Rf: 0.45
(CH2Cl2:MeOH, 50:1).
3.1.26. Compound 24. TFA (1 mL) was added to a
solution of 23 (15 mg, 0.013 mmol) in CH2Cl2 (3 mL) at
0 ꢁC under Argon atmosphere. The reaction mixture
was stirred at 23 ꢁC for 5 h. The solvent was evaporated
under reduced pressure and, in order to eliminate the
remaining TFA, the mixture was treated with CH2Cl2
(3ꢃ15 mL) and evaporated to dryness to give 24 as a
white solid (14 mg, 88%). 1H NMR (300 MHz,
CD3OD) d 7.47–7.41 (m, 2H), 7.29–7.24 (m, 2H), 7.16
(s, 1H), 6.87 (d, J=8.2 Hz, 1H), 6.80 (d, J=10.1 Hz,
1H), 4.42–4.29 (m, 3H), 3.92–3.87 (m, 2H), 3.85 (s, 3H),
3.45 (s, 3H), 3.37 (s, 3H), 3.18 (t, J=6.2 Hz, 2H), 2.14–
1.61 (m, 9H), 1.12–0.98 (m, 18H). MS (ESI) m/z: 841
(M+1)+.
3.1.27. Compound 25. A suspension of 27 (49 mg, 0.045
mmol) and DDQ (20 mg, 0.089 mmol) in CCl4 (2 mL)
was refluxed for 5 h. The mixture was cooled to 23 ꢁC
then filtered through Celite and washed with CH2Cl2
(50 mL). The filtrated was concentrated under reduced
pressure and the residue was purified by chromato-
graphy on silica gel (CH2Cl2:MeOH, from 100:1 to
50:1) to give 25 (43 mg, 88%). 1H NMR (300 MHz,
CDCl3) d 9.20 (d, J=7.3 Hz, 1H), 7.40 (s, 1H), 7.30–
7.13 (m, 5H), 7.04 (d, J=7.3 Hz, 1H), 6.79 (d, J=7.5
Hz, 1H), 5.10–5.06 (m, 3H), 4.56–4.48 (m, 3H), 3.81 (s,
3H), 3.43 (s, 6H), 2.45–2.32 (m, 3H), 1.49 (s, 9H), 1.47
(s, 18H), 1.14–1.00 (m, 18H). 13C NMR (75 MHz,
CDCl3) d 170.4 (3C), 155.7, 155.0, 152.3, 150.9, 147.6,
145.4, 140.6, 140.0, 139.4, 134.5, 133.5, 130.9, 128.8,
128.2, 128.1, 124.1, 123.8, 123.6, 123.2, 120.8, 115.9,
115.1, 112.8, 112.3, 112.2, 109.1, 106.4, 106.2, 80.0 (3C),
58.5, 56.0, 55.6, 55.6, 55.5, 55.5, 31.3 (2C), 31.2, 28.3
(9C), 19.2, 19.1, 17.2 (2C), 17.1 (2C). MS (ESI) m/z:
3.1.24. Compound 22. TFA (1 mL) was added to a
solution of 21 (15 mg, 0.013 mmol) in anhydrous
CH2Cl2 (3 mL) at 0 ꢁC under Argon atmosphere. The
reaction mixture was stirred at 23 ꢁC for 5 h. The sol-
vent was evaporated under reduced pressure and, in
order to eliminate the remaining TFA, the mixture was
treated with CH2Cl2 (3ꢃ15 mL) and evaporated to
1
dryness to give 22 as a white solid (19 mg, quant.). H
NMR (300 MHz, CD3OD) d 9.15–9.12 (m, 1H), 7.64 (d,
J=2.7 Hz, 1H), 7.58–7.52 (m, 2H), 7.40–7.29 (m, 2H),
7.26–7.22 (m, 2H), 6.89 (d, J=7.3 Hz, 1H), 4.45–4.31
(m, 3H), 3.90 (s, 3H), 3.48 (s, 3H), 3.48 (s, 3H), 2.11–
1.79 (m, 9H), 1.13–1.06 (m, 18H). MS (ESI) m/z: 839
(M+1)+.
1119
(CH2Cl2:MeOH, 100:1).
(M+23)+,
1097
(M+1)+.
Rf:
0.33
3.1.25. Compound 23. A suspension of LAM-501 (2) (50
.
.
mmol), EDC HCl (76 mg, 0.3988 mmol) and DMAP (7
mg, 0.0997 mmol), Boc-l-Leu-OH H2O (99 mg, 0.3988
3.1.28. Compound 26. TFA (1 mL) was added to a
solution of 25 (15 mg, 0.014 mmol) in CH2Cl2 (3 mL) at

C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712
1709
0 ꢁC under Argon atmosphere. The reaction mixture
was stirred at 23 ꢁC for 4 h. The solvent was evaporated
under reduced pressure and, in order to eliminate the
remaining TFA, the mixture was treated with CH2Cl2
(3ꢃ15 mL) and evaporated to dryness to give 26 as a
white solid (21 mg, quant.). 1H NMR (300 MHz,
CD3OD) d 9.09–9.04 (m, 1H), 7.62–7.51 (m, 3H), 7.41–
7.32 (m, 2H), 7.23–7.18 (m, 2H), 6.88 (d, J=9.0 Hz,
1H), 4.36 (d, J=4.4 Hz, 1H), 4.31 (d, J=4.4 Hz, 1H),
4.24 (t, J=4.4 Hz, 1H), 3.92 (s, 3H), 3.48 (s, 6H), 2.62–
2.43 (m, 3H), 1.29–1.19 (m, 18H). MS (APCI) m/z: 797
(M+1)+.
d 9.27–9.23 (m, 1H), 7.47–7.36 (m, 1H), 7.26–7.08 (m,
6H), 6.84–6.78 (m, 1H), 4.56–4.49 (m, 3H), 3.80 (s, 3H),
3.66–3.47 (m, 6H), 3.43 (s, 6H), 2.40–2.29 (m, 6H),
2.04–1.98 (m, 6H), 1.49 (s, 27H). MS (ESI) m/z: 1091
(M+1)+. Rf: 0.31 (CH2Cl2:MeOH 30:1).
3.1.32. Compound 30. TFA (1 mL) was added to a
solution of 29 (15 mg, 0.014 mmol) in anhydrous
CH2Cl2 (3 mL) at 0 ꢁC under Argon atmosphere. The
reaction mixture was stirred at 23 ꢁC for 2 h. The sol-
vent was evaporated under reduced pressure and, in
order to eliminate the remaining TFA, the mixture was
treated with CH2Cl2 (3ꢃ15 mL) and evaporated to
1
3.1.29. Compound 27. A suspension of LAM-501 (2) (50
mg, 0.0997 mmol), Boc-l-Val-OH (87 mg, 0.3988
dryness to give 30 as a white solid (19 mg, quant.). H
NMR (300 MHz, CD3OD) d 8.96–8.90 (m, 1H), 7.67–
7.52 (m, 3H), 7.40–7.24 (m, 2H), 7.13–7.08 (m, 2H),
6.84–6.80 (m, 1H), 4.86–4.67 (m, 3H), 3.95 (s, 3H),
3.55–3.43 (m, 12H), 2.66–2.35 (m, 6H), 2.27–2.14 (m,
6H). MS (ESI) m/z: 791 (M+1)+.
.
mmol), EDC HCl (76 mg, 0.3988 mmol) and DMAP (7
mg, 0.0598 mmol) in anhydrous CH2Cl2 (4.3 mL) was
stirred under Argon atmosphere at 23 ꢁC for 4 h. The
resulting pale yellow solution was washed with H2O
(10 mL) and saturated aqueous solution of NaHCO3
(10 mL). The aqueous phase was extracted with
CH2Cl2 (10 mL). The combined organic phases were
dried over anhydrous Na2SO4 and the solvent removed
under vacuum. The residue was purified by chromato-
graphy on silica gel (CH2Cl2:MeOH, from 100:1 to
50:1) to give 27 as a white solid (87 mg, 79%). 1H NMR
(300 MHz, CDCl3) d 7.26–7.13 (m, 2H), 7.09 (s, 2H),
6.96 (s, 1H), 6.76 (d, J=7.5 Hz, 1H), 6.68 (d, J=9.5 Hz,
1H), 5.08–5.05 (m, 3H), 4.91–4.69 (m, 2H), 4.52–4.46
(m, 3H), 3.77 (s, 3H), 3.40 (s, 3H), 3.32 (s, 3H), 3.18 (br
t, 2H), 2.42–2.38 (m, 3H), 1.48 (s, 9H), 1.45 (s, 18H),
1.11–0.98 (m, 18H). 13C NMR (75 MHz, CDCl3) d
170.3 (3C), 155.6, 154.9, 152.1, 149.7, 147.5, 144.8,
139.7, 139.1, 138.5, 134.9, 134.2, 126.9, 126.0, 125.7,
123.8, 123.1, 122.5, 116.1, 115.8, 115.0, 114.6, 111.9,
109.6, 105.4, 79.9 (3C), 58.5, 56.0, 55.6, 55.3, 55.3, 53.4,
42.4, 31.2 (3C), 28.3 (9C), 28.0, 19.1 (2C), 17.1 (4C). MS
(ESI) m/z: 1121 (M+23)+, 1099 (M+1)+. Rf: 0.35
(CH2Cl2:MeOH, 50:1).
3.1.33. Compound 31. A suspension of LAM-501 (2) (50
mg, 0.0997 mmol), Boc-l-Pro-OH (86 mg, 0.3988
.
mmol), EDC HCl (76 mg, 0.3988 mmol) and DMAP (7
mg, 0.0598 mmol) in anhydrous CH2Cl2 (4.3 mL) was
stirred under Argon atmosphere at 23 ꢁC for 4 h. The
resulting pale yellow solution was washed with H2O
(10 mL) and saturated aqueous solution of NaHCO3
(10 mL). The aqueous phase was extracted with
CH2Cl2 (10 mL). The combined organic layers were
dried over anhydrous Na2SO4 and the solvent removed
under vacuum. The residue was purified by chromato-
graphy on silica gel (CH2Cl2:MeOH, from 100:1 to
50:1) to give 31 as a white solid (74 mg, 68%). 1H NMR
(300 MHz, CDCl3) d 7.16–7.13 (m, 2H), 7.06–7.03 (m,
2H), 6.90 (s, 1H), 6.78–6.63 (m, 2H), 4.95–4.62 (m, 2H),
4.56–4.46 (m, 3H), 3.78 (s, 3H), 3.69–3.43 (m, 6H), 3.40
(s, 3H), 3.33 (s, 3H), 3.11 (br t, 2H), 2.40–2.26 (m, 6H),
2.08–1.90 (m, 6H), 1.47 (s, 27H). 13C NMR (75 MHz,
CDCl3) d 171.0, 170.8, 170.7, 155.0, 154.3, 153.7, 153.7
(2C), 152.1, 149.7, 147.6, 144.8, 139.8, 139.3, 138.7,
134.1, 125.9, 125.6, 124.0, 123.4, 123.1 (2C), 122.7,
122.2, 116.0, 114.7, 111.6, 109.6, 105.3, 80.1, 80.0, 79.8,
58.9 (2C), 56.1, 55.7, 55.6, 55.5, 46.5, 46.3 (2C), 42.4,
31.5, 30.9, 29.9, 28.3 (9C), 28.0, 24.2, 23.4, 22.5. MS
(ESI) m/z: 1115 (M+23)+, 1093 (M+1)+. Rf: 0.18
(CH2Cl2:MeOH, 50:1).
3.1.30. Compound 28. TFA (1 mL) was added to a
solution of 27 (15 mg, 0.0136 mmol) in anhydrous
CH2Cl2 (3 mL) at 0 ꢁC under Argon atmosphere. The
reaction mixture was stirred at 23 ꢁC for 4 h. The sol-
vent was evaporated under reduced pressure and, in
order to eliminate the remaining TFA, the mixture was
treated with CH2Cl2 (3ꢃ15 mL) and evaporated to
1
dryness to give 28 as a white solid (16 mg, quant.). H
3.1.34. Compound 32. TFA (1 mL) was added to a
solution of 31 (15 mg, 0.014 mmol) in anhydrous
CH2Cl2 (3 mL) at 0 ꢁC under Argon atmosphere. The
reaction mixture was stirred at 23 ꢁC for 3 h. The sol-
vent was evaporated under reduced pressure and, in
order to eliminate the remaining TFA, the mixture was
treated with CH2Cl2 (3ꢃ15 mL) and evaporated to
NMR (300 MHz, CD3OD) d 7.47–7.44 (m, 2H), 7.28 (d,
J=8.1 Hz, 1H), 7.19 (s, 1H), 7.15 (s, 1H), 6.90–6.78 (m,
2H), 4.77 (br t, 2H), 4.32 (s, 1H), 4.24 (s, 2H), 3.87 (s,
3H), 3.45 (s, 3H), 3.37 (s, 3H), 3.17 (br t, 2H), 2.54–2.46
(m, 3H), 1.26–1.17 (m, 18H). MS (ESI) m/z: 799
(M+1)+.
1
dryness to give 32 as a white solid (17 mg, quant.). H
3.1.31. Compound 29. A suspension of 31 (45 mg, 0.041
mmol) and DDQ (19 mg, 0.082 mmol) in CCl4 (2 mL)
was refluxed for 17 h. The mixture was cooled at 23 ꢁC
then filtered through Celite, and washed with CH2Cl2
(50 mL). The filtrated was concentrated under reduced
pressure and the residue was purified by chromato-
graphy on silica gel (CH2Cl2:MeOH, 30:1) to give 29 (30
mg, 67%) as a white solid. 1H NMR (300 MHz, CDCl3)
NMR (300 MHz, CD3OD) d 7.49–7.44 (m, 2H), 7.29–
7.17 (m, 3H), 6.88–6.75 (m, 2H), 4.79–4.68 (m, 3H), 3.89
(s, 3H), 3.52–3.38 (m, 12H), 3.15 (br t, 2H), 2.63–2.35
(m, 6H), 2.25–2.15 (m, 6H). MS (ESI) m/z: 793
(M+1)+.
3.1.35. Compound 33. A suspension of 35 (50 mg, 0.040
mmol) and DDQ (18 mg, 0.080 mmol) in CHCl3 (2 mL)

1710
C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712
was refluxed for 22 h. The mixture was cooled to 23 ꢁC
then filtered through Celite, and washed with CH2Cl2
(50 mL). The filtrated was concentrated under reduced
pressure and the residue was purified by chromato-
graphy on silica gel (CH2Cl2:MeOH, 80:1) to give 33 (43
mg, 86%) as a white solid. 1H NMR (300 MHz, CDCl3)
d 9.22 (d, J=7.3 Hz, 1H), 7.37–7.20 (m, 20H), 7.08–7.03
(m, 2H), 6.80 (d, J=2.2 Hz, 1H), 5.29–5.02 (m, 3H),
4.90–4.88 (m, 3H), 3.85 (s, 3H), 3.44 (s, 6H), 3.41–3.23
(m, 6H), 1.46 (s, 9H), 1.43 (s, 18H). 13C NMR (75 MHz,
CDCl3) d 170.1, 170.0, 169.9, 155.1 (2C), 154.9, 152.3,
150.9, 147.6, 145.3, 140.5, 139.9, 139.3, 135.8 (2C),
134.5, 133.4, 125.0 (9C), 128.6 (6C), 128.1, 128.0, 127.1
(2C), 124.0, 123.7, 123.6, 123.1, 120.7, 115.8, 115.1,
112.8, 112.3, 112.1, 109.1, 106.4, 106.1, 80.1 (3C), 56.2
(2C), 55.7, 55.6, 55.5, 54.4, 38.1 (3C), 28.2 (9C). MS
(ESI) m/z: 1263 (M+23)+, 1241 (M+1)+. Rf: 0.56
(CH2Cl2:MeOH, 50:1).
CH2Cl2 (3 mL) at 0 ꢁC under Argon atmosphere. The
reaction mixture was stirred to 23 ꢁC for 2.5 h. The sol-
vent was evaporated under reduced pressure and, in
order to eliminate the remaining TFA, the mixture was
treated with CH2Cl2 (3ꢃ15 mL) and evaporated to
1
dryness to give 36 as a white solid (17 mg, quant.). H
NMR (300 MHz, CD3OD) d 7.44–7.35 (m, 17H), 7.26
(d, J=8.1 Hz, 1H), 7.10 (d, J=1.6 Hz, 1H), 7.06 (s,
1H), 6.88 (d, J=3.5 Hz, 1H), 6.78 (d, J=3.5 Hz, 1H),
4.79–4.70 (m, 3H), 4.63 (t, J=6.7 Hz, 2H), 3.89 (s, 3H),
3.53–3.26 (m, 6H), 3.44 (s, 3H), 3.36 (s, 3H), 3.16 (t,
J=6.7 Hz, 2H). MS (ESI) m/z: 965 (M+23)+, 943
(M+1)+.
3.1.39. Compound 37. A suspension of LAM-501 (2) (50
mg, 0.0997 mmol), Boc-l-Trp-OH (121 mg, 0.3988
.
mmol), EDC HCl (76 mg, 0.3988 mmol) and DMAP (7
mg, 0.0598 mmol) in anhydrous CH2Cl2 (4.3 mL) was
stirred under Argon atmosphere at 23 ꢁC for 4 h. The
resulting pale yellow solution was washed with H2O
(10 mL) and aqueous saturated solution of NaHCO3
(10 mL). The combined aqueous phases were extracted
with CH2Cl2 (10 mL). The combined organic layers
were dried over anhydrous Na2SO4, filtered, and the
solvent removed under vacuum. The residue was pur-
ified by chromatography on silica gel (CH2Cl2:MeOH,
from 30:1 to 15:1) to give 37 as a white solid (115 mg,
3.1.36. Compound 34. TFA (1 mL) was added to a
solution of 33 (15 mg, 0.012 mmol) in CH2Cl2 (3 mL) at
0 ꢁC under Argon atmosphere. The reaction mixture
was stirred to 23 ꢁC for 1 h. The solvent was evaporated
under reduced pressure and, in order to eliminate the
remaining TFA, the mixture was treated with CH2Cl2
(3ꢃ15 mL) and evaporated to dryness to give 34 as a
white solid (20 mg, quant.). 1H NMR (300 MHz,
CD3OD) d 9.08 (d, J=7.5 Hz, 1H), 7.60 (d, J=2.2 Hz,
1H), 7.53 (s, 1H), 7.49–7.36 (m, 17H), 7.30 (d, J=3.5
Hz, 1H), 7.20 (d, J=7.5 Hz, 1H), 7.08 (d, J=4.8 Hz,
1H), 6.87 (d, J=4.2 Hz, 1H), 4.76 (t, J=7.0 Hz, 1H),
4.70 (t, J=6.9 Hz, 1H), 4.63 (t, J=6.2 Hz, 1H), 3.95 (s,
3H), 3.47 (s, 6H), 3.61–3.36 (m, 6H). MS (ESI) m/z: 941
(M+1)+.
1
85%). H NMR (300 MHz, CDCl3) d 8.35 (s, 1H), 8.28
(s, 2H), 7.68–7.62 (m, 3H), 7.39–7.36 (m, 3H), 7.26–7.07
(m, 12H), 6.90 (s, 1H), 6.72 (s, 1H), 6.65 (br s, 2H),
5.15–5.12 (m, 2H), 5.00–4.59 (m, 6H), 3.75 (s, 3H),
3.52–3.28 (m, 12H), 3.00 (br t, 2H), 1.43 (s, 27H). 13C
NMR (75 MHz, CDCl3) d 170.7, 170.4, 170.4, 155.3
(2C), 154.9, 152.0, 149.6, 147.5, 144.6, 139.6, 139.0,
138.4, 136.1 (3C), 134.9, 134.0, 127.7 (3C), 126.8, 125.9,
125.5, 123.8, 123.1 (3C), 122.5, 122.0 (3C), 119.5 (3C),
118.6 (3C), 116.0, 115.8, 114.7, 111.7, 111.3 (3C), 109.5
(3C), 105.3, 80.0 (3C), 56.0 (2C), 55.6, 55.4, 54.4 (2C),
42.3, 28.2 (12C), 27.7. MS (ESI) m/z: 1382 (M+23)+.
Rf: 0.13 (CH2Cl2:MeOH, 30:1).
3.1.37. Compound 35. A suspension of LAM-501 (2) (50
mg, 0.0997 mmol), Boc-l-Phe-OH (106 mg, 0.3988
.
mmol), EDC HCl (76 mg, 0.3988 mmol) and DMAP (7
mg, 0.0598 mmol) in anhydrous CH2Cl2 (4.3 mL) was
stirred under Argon atmosphere at 23 ꢁC for 6 h. The
resulting pale yellow solution was washed with H2O
(10 mL) and saturated aqueous solution of NaHCO3
(10 mL) and both aqueous phases were extracted with
CH2Cl2 (10 mL). The combined organic layers were
dried over anhydrous Na2SO4, filtered, and the solvent
removed under vacuum. The residue was purified by
chromatography on silica gel (CH2Cl2:MeOH, 100:1) to
give 35 as a white solid (87 mg, 68%). 1H NMR
(300 MHz, CDCl3) d 7.34–7.26 (m, 15H), 7.16 (br s,
2H), 7.10 (s, 1H), 7.05 (s, 1H), 6.91 (s, 1H), 6.78–6.68
(m, 2H), 4.99 (t, J=8.6 Hz, 2H), 4.88–4.72 (m, 6H), 3.81
(s, 3H), 3.41 (s, 3H), 3.34 (s, 3H), 3.30–3.12 (m, 8H),
1.44 (s, 9H), 1.43 (s, 18H). 13C NMR (75 MHz, CDCl3)
d 170.1, 169.9 (2C), 155.0, 154.9, 152.0, 149.7, 147.5,
144.7, 139.6, 139.0, 138.4, 135.8 (2C), 134.8, 134.2, 129.4
(9C), 129.2, 128.5 (6C), 127.1 (2C), 126.9, 125.9, 125.7,
123.8, 123.1, 122.5, 116.1, 115.8, 114.9, 114.7, 111.8,
109.7, 105.4, 80.0 (3C), 56.1, 55.6, 55.4, 54.3 (3C), 42.3,
38.0 (3C), 28.2 (9C), 27.9. MS (ESI) m/z: 1265
(M+23)+. Rf: 0.65 (CH2Cl2:MeOH, 30:1).
3.1.40. Compound 38. TFA (1 mL) was added to a
solution of 37 (25 mg, 0.018 mmol) in CH2Cl2 (3 mL) at
0 ꢁC under Argon atmosphere. The reaction mixture
was stirred to 23 ꢁC for 1.5 h. The solvent was evapo-
rated under reduced pressure and, in order to eliminate
the remaining TFA, the mixture was treated with
CH2Cl2 (3ꢃ15 mL) and evaporated to dryness to give
38 as a white solid (27 mg, quant.) 1H NMR (300 MHz,
CD3OD) d 7.68 (d, J=8.1 Hz, 1H), 7.62 (s, 1H), 7.60 (s,
1H), 7.44 (s, 1H), 7.42 (s, 3H), 7.34 (s, 1H), 7.30 (s, 1H),
7.29 (s, 1H), 7.21–7.16 (m, 5H), 7.12–7.09 (m, 3H), 6.98
(s, 1H), 6.85 (d, J=2.0 Hz, 1H), 6.77–6.76 (m, 2H),
4.73–4.69 (m, 3H), 4.60 (br t, 2H), 3.87 (s, 3H), 3.77–
3.34 (m, 6H), 3.43 (s, 3H), 3.34 (s, 3H). MS (ESI) m/z:
1060 (M+1)+.
3.1.41. Compound 39. A suspension of LAM-D (1) (300
mg, 0.601 mmol), 6-tert-Butoxycarbonylamino-hex-
.
anoic acid (834 mg, 3.604 mmol), EDC HCl (691 mg,
0.3.604 mmol) and DMAP (44 mg, 0.360 mmol) in
CH2Cl2 anh. (50 mL) was stirred under Argon atmos-
phere at 23 ꢁC for 3 h. The resulting pale yellow solution
3.1.38. Compound 36. TFA (1 mL) was added to a
solution of 35 (15 mg, 0.012 mmol) in anhydrous

C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712
1711
was diluted with CH2Cl2 (50 mL), washed with H2O
(100 mL) and aqueous saturated solution of NaHCO3
(100 mL). The combined organic layers were dried over
anhydrous Na2SO4, filtered, and the solvent removed
under vacuum. The residue was purified by chromato-
graphy on silica gel (CH2Cl2:MeOH, 40:1) to give 39 as
a white solid (608 mg, 89%). 1H NMR (300 MHz,
CDCl3) d 9.24 (d, J=7.3 Hz, 1H), 7.38 (s, 1H), 7.29–
7.13 (m, 5H), 7.07 (d, J=7.5 Hz, 1H), 6.80 (s, 1H), 4.56
(bs, 3H), 3.82 (s, 3H), 3.44 (s, 6H), 3.20–3.13 (m, 6H),
2.66–2.56 (m, 6H), 1.84–1.75 (m, 6H), 1.60–1.44 (m,
39H). 13C NMR (75 MHz, CDCl3) d 171.4, 171.2, 155.9,
155.0, 152.4, 151.0, 147.7, 145.4, 140.9, 140.3, 139.8,
134.1, 133.5, 128.2, 124.0, 123.8, 123.6, 123.5, 123.0,
120.6, 115.5, 115.0, 112.7, 112.2, 112.1, 108.9, 106.3,
106.1, 79.0(3C), 56.2, 55.7, 55.6, 40.3(3C), 33.8(2C),
33.7, 29.7(3C), 28.4(9C), 26.2, 26.1(2C), 24.5(2C), 24.5.
MS (ESI) m/z: 1161.8 (M+23)+. Rf: 0.30
(CH2Cl2:MeOH, 40:1).
Supercoiled pLAZ3 DNA (0.25 mg) was incubated with
3 units human topoisomerase I at 37 ꢁC for 1 h in
relaxation buffer (50 mM Tris pH 7.8, 50 mM KCl, 10
mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA) in the
presence of varying concentrations of the drug under
study. Reactions were terminated by adding SDS to
0.25% and proteinase K to 250 mg/mL. DNA samples
were then added to the electrophoresis dye mixture (3
mL) and electrophoresed at room temperature for 2 h at
120V in 1% agarose gels containing ethidium bromide
(1 mg/mL). After electrophoresis, gels were washed and
photographed under UV light.13
3.5. Purification of the DNA restriction fragment and
radiolabeling
The 117-bp DNA fragment was prepared by 30-[32P]-end
labeling of the EcoRI-PvuII double digest of the pBS
plasmid (Stratagene) using a-[32P]-dATP (Amersham,
3000 Ci/mmol) and AMV reverse transcriptase (Roche).
The labeled digestion products were separated on a 6%
polyacrylamide gel under non-denaturing conditions in
TBE buffer (89 mM Tris–borate pH 8.3, 1 mM EDTA).
After autoradiography, the requisite band of DNA was
excised, crushed and soaked in water overnight at 37 ꢁC.
This suspension was filtered through a Millipore 0.22
mm filter and the DNA was precipitated with ethanol.
Following washing with 70% ethanol and vacuum dry-
ing of the precipitate, the labeled DNA was re-sus-
pended in 10 mM Tris adjusted to pH 7.0 containing 10
mM NaCl.
3.1.42. Compound 40. A solution of 39 (503 mg, 0.442
mmol) in a 3.0 M solution of HCl in EtOAc was stirred
at 23 ꢁC for 30 min. The resulting suspension was fil-
tered and the solid washed with EtOAc and n-hexane to
give 40 as a white solid (389 mg, 93%). 1H NMR
(300 MHz, CD3OD) d 9.07 (d, J=7.3 Hz, 1H), 7.49 (s,
2H), 7.39 (d, J=8.1 Hz, 1H), 7.31–7.28 (m, 2H), 7.16–
7.11 (m, 2H), 6.86 (s, 1H), 3.88 (s, 3H), 3.46 (s, 6H),
3.02–2.94 (m, 6H), 2.73–2.60 (m, 6H), 1.88–1.75 (m,
12H), 1.54–1.52 (m, 6H). MS (ESI) m/z: 839 (M+1)+.
3.2. Drugs and chemicals
3.6. Sequencing of topoisomerase I-mediated DNA clea-
vage sites
CPT was purchased from Sigma Chemical Co. All drug
solutions were prepared in DMSO at 5 mM and then
further diluted with water. The final DMA concen-
tration never exceeded 0.3% (v/v) in the cleavage reac-
tions. Under these conditions DMSO which is also used
in the controls, does not affect the topoisomerase activ-
ity. The stock solutions of drugs were kept at ꢀ20 ꢁC
and freshly diluted to the desired concentration imme-
diately prior to use. All other chemicals were analytical
grade reagents.
Each reaction mixture contained 2 mL of 30-end [32P]
labeled DNA (ꢄ1 mM), 5 mL of water, 2 mL of 10X
topoisomerase I buffer, 10 mL of drug solution at the
desired concentration (50 mM final concentration). After
10 min incubation to ensure equilibration, the reaction
was initiated by addition of 2 mL (20 units) topoisome-
rase I. Samples were incubated for 45 min at 37 ꢁC prior
to adding SDS to 0.25% and proteinase K to 250 mg/
mL to dissociate the drug-DNA–topoisomerase I clea-
vable complexes. The DNA was precipitated with etha-
nol and then resuspended in 5 mL of formamide-TBE
loading buffer, denatured at 90 ꢁC for 4 min then chilled
in ice for 4 min prior to loading on to the sequencing
gel. DNA cleavage products were resolved by poly-
acrylamide gel electrophoresis under denaturing condi-
tions (0.3 mm thick, 8% acrylamide containing 8 M
urea). After electrophoresis (about 2.5 h at 60 Watts,
1600 V in TBE buffer, BRL sequencer model S2), gels
were soaked in 10% acetic acid for 10 min, transferred
to Whatman 3MM paper, and dried under vacuum at
80 ꢁC. A Molecular Dynamics 425E PhosphorImager
was used to collect data from the storage screens
exposed to dried gels overnight at room temperature.
Base line-corrected scans were analyzed by integrating
all the densities between two selected boundaries using
ImageQuant version 3.3 software. Each resolved band
was assigned to a particular bond within the DNA
fragment by comparison of its position relative to
3.3. Melting temperature studies
Melting curves were measured using an Uvikon 943
spectrophotometer coupled to a Neslab RTE111 cryo-
stat. For each series of Tm measurements, 12 samples
were placed in a thermostatically controlled cell-holder,
and the quartz cuvettes (10 mm pathlength) were heated
by circulating water. The measurements were performed
in BPE buffer pH 7.1 (6 mM Na2HPO4, 2 mM
NaH2PO4, 1 mM EDTA). The temperature inside the
cuvette was measured with a platinum probe; it was
increased over the range 20–100 ꢁC with a heating rate
of 1 ꢁC/min. The ‘melting’ temperature Tm was taken as
the mid-point of the hyperchromic transition.
3.4. DNA relaxation experiments
Recombinant topoisomerase I protein was produced
and purified from baculovirus infected Sf9 cells.16

1712
C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712
sequencing standards generated by treatment of the
DNA with dimethylsulphate followed by piperidine-
induced cleavage at the modified guanine residues.
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a
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4 ꢁC. Plates are washed with deionized water and dried;
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Acknowledgements
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This work was done under the support of research
grants (to C.B.) from the Ligue Nationale Franc¸ aise
Contre le Cancer (Comite du Nord).