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 Michaël Facompré,a William Laine,a Brigitte Baldeyrou,a
Dolores Garcı́a-Gravalos,b Andrés Francesch,b Cristina Mateo,b Alfredo Pastor,b
José A. Jiménez,b Ignacio Manzanares,b Carmen Cuevasb and Christian Baillya,*
a
INSERM UR-524 and Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL, Place de Verdun,
59045 Lille, France
b
PharmaMar, Avda. de los Reyes 1, 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)
Many natural products are used to combat cancer. The derivatives topotecan and irinotecan. Antibiotics are
chemotherapeutic arsenal includes drugs extracted from equally well represented because several of the most
frequently used anticancer drugs, daunomycin and
bleomycin, for example, originate from Streptomyces.
Keywords: Lamellarin; Topoisomerase I; DNA interaction; Cytotoxi- The sea world is also a rich source of anticancer agents.
city; Anticancer drugs.
Abbreviations: CPT, Camptothecin; LAM-D, Lamellarin D. The marine environment has provided many lead com-
* Corresponding author. Tel.: +33-320-16-92-18; fax: +33-320-16-92- pounds, the bryostatins, aplidine and kahalalide F to
29; e-mail: [email protected] cite only a few of them.15 The most advanced anti-

0968-0896/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.01.020
1698 C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712

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

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
2.1. Chemistry
the acid chloride with triethylamine. Treatment of the
The synthesis of LAM-D (1) and LAM-501 (2) was ester derivatives with DDQ afforded the derivatives of
achieved by [3+2] cycloaddition reaction of the corres- LAM-D (1). Compounds in group B (Chart 2) were
ponding dihydroisoquinoline 44 with ester 43 (Scheme obtained in a similar way (Scheme 3) by reaction of
1) following the procedure described previously.12 LAM-501 (2) with N-protected aminoacid using EDC
Compounds in group A (Chart 1) were obtained as a coupling reagent, oxidation with DDQ to obtain

Chart 2.

Scheme 1. Reagents: (a) (i) ClCH2CH2Cl, 23  C; (ii) DlPEA, 85  C; (b) DDQ, CHCl3, reflux; (c) AlCl3, CH2Cl2, 23  C.
1700 C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712

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

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 It is clear that the introduction of an amino acid func-
the other amino acid derivatives which are all more or tionality on the phenolic OH groups at positions 8, 14
less equally effective at inhibiting topoisomerase I. The and 20 of LAM-D is not detrimental to topoisomerase I
stereospecificity was investigated with the Val deriva- inhibition. The extent of topoisomerase I-mediated
tives for which we compared the activity of the (L) (25– DNA cleavage is fully maintained when a Leu, Val, Ala
28) and (D) (25r–28r) isomers but, as shown in Figure or Pro residue is incorporated on the LAM-D skeleton
1d, there was no difference between the two series. whereas a non charged group abolishes the anti-topoi-
Compound 26 and 26r both stimulated DNA cleavage somerase I activity. A phenylalanine residue is much less
by the enzyme. No effect was observed with the Boc- favorable than a proline or an alanine residue for
proteted analogues in the C5–C6 double stranded (25, example. The observations that the incorporation of a
25r) or C5–C6 single-stranded (27, 28, 27r, 28r) series. cationic group promoted topoisomerase I inhibition
The amino compound 40 was also found to inhibit suggested that the enhanced capacity of the drugs to bind
topoisomerase I (Fig. 1b). to DNA could be responsible for a better enzyme inhi-
bition. To verify this idea, melting temperature measure-
Concentration-dependent measurements were per- ments were carried out with the polymer poly(dAT)2
formed with each of the positive compounds identified and for each compound we calculated the
Tm values
and a few representative gels comparing the anti-topoi- (TmdrugDNA complexTmDNA alone). Poly(dAT)2 which
somerase I activity of LAM-D (1) with the three analo- melt at a low temperature (41  1  C in BPE buffer)
gues Val(D) (26r), Pro (30) and the amino compound 40 affords a sensitive determination of the DNA binding
are shown in Figure 2. This later compound is equally capacity of the studied molecules. As indicated in Table
efficient to 1 in terms of stimulation of DNA cleavage 1, the Ala (18) and Pro (30) derivatives markedly stabi-
by topoisomerase I and is also equally cytotoxic (see lize the polymer against heat denaturation with
Tm
below). In all cases, the dose–response analysis con- values > 10  C and this correlates with their potent anti-
firmed that the cationic LAM-D analogues potently topoisomerase I activity. In contrast, the Phe derivative
inhibit the enzyme. (34) showed no effect on DNA thermal stability and this
1702 C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712

may be the reason why this compound is more weakly that the cationic lamellarin derivatives do effectively
active against topoisomerase I compared to the other function as topoisomerase I poisons. A 117-bp DNA
amino acid derivatives. Enhanced DNA binding seems restriction fragment uniquely end-labeled at the 30 end
to facilitate topoisomerase I inhibition but tight binding was subjected to cleavage by topoisomerase I in the
to DNA is not required for enzyme inhibition since the presence of the different compounds and the resulting
natural product LAM-D shows little affinity for DNA DNA cleavage products were resolved on sequencing-
despite its potent topoisomerase I poisoning capacity. type polyacrylamide gels. A typical gel is presented in
Figure 3. The advantage of this assay is to detect the
A second assay, based on the cleavage of a radiolabeled cleavage sites and to locate their positions with nucleo-
DNA substrate by topoisomerase I, was used to confirm tide resolution, providing thus information on the site
selectivity of cleavage.

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).
Figure 2. Concentration-dependent effect for the inhibition of topoi- The amino compound 40 was also found to stimulate
somerase I by LAM-D (1) or the cationic derivatives 26r, 30 and 40. DNA cleavage by the enzyme and here also we found
All concentrations are given in mM. Other details as for Figure 1.
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-
Table 1. Topoisomerase I inhibition, DNA binding and cytotoxicity fore validate the conclusion that the cationic lamellarin
Nicked DNA (%)a
Tm ( C)b GI50 (nM)c derivatives potently inhibit topoisomerase I.

LAM-D (1) 25.7 2.9 10.9

LAM-501 (2) 12.3 0.0 2990 2.3. Cytotoxicity
18 29.7 13.3 16.2
19 7.0 6.8 3650 Seven human tumor cell lines were used to determine
22 31.1 4.5 18.0 the cytotoxicity of the lamellarin derivatives: HT29
23 9.2 1.0 4000 human colon carcinoma cells, LoVo human colon
26 33.6 8.0 10.8 lymph node metastasis cells and the corresponding
27 6.7 4.1 1600
30 24.6 12.0 18.6
LoVo-Dox cells resistant to doxorubicin, ovarian cells
31 13.4 5.3 2120 sensitive (IGROV) or resistant (IGROV-ET) to ectei-
34 31.4 0.0 25.2 nascidin-743, and the prostate tumor cells LN-CaP and
36 10.0 0.9 1630 DU-145. A colorimetric assay was used up to estimate
38 6.2 0.0 913 GI50 values, that is, the drug concentration which causes
40 27.6 4.1 15.1
50% cell growth inhibition after 72 h continuous expo-
a
Extent of topoisomerase I-mediated DNA cleavage measured with sure to the test molecules. We have shown recently that
the lamellarin derivatives (1 mM each). The% of nicked DNA (form LAM-D exhibits a pronounced cytotoxic effect toward
II) was determined by densitometry. Band intensities from three gels prostatic cells, in particular the DU-145 metastatic cells
such as those shown in Figure 1 were compiled for the quantitative
analysis. which are androgen-insensitive.11 This cell line was used
b
Variation of the melting temperature (
Tm) of helix-to-coil transi- as a reference to compare the different compound.
tion of poly(dAT)2 in the presence of the lamellarin derivatives. Tm Table 1 reports the GI50 values for each compound
measurements were performed in BPE buffer pH 7.1 (6 mM along with the effect on topoisomerase I and DNA
Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA) using 20 mM poly(dAT)2
(nucleotide concentration) and 20 mM drug.
binding activity. To quantify topoisomerase I poison-
c
Drug concentration that causes 50% growth inhibition of DU-145 ing, we measured the extent of DNA cleavage in the
metastatic cells. Values were calculated from dose–response curves. presence of the drug at 1 mM (% nicked DNA).
 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 androgen-insensitive) and LN-CaP (hormonally respon-
equally efficient as 1 at stimulating DNA cleavage by sive but non metastatic). In both cases, the most cyto-
topoisomerase I. This effect is well correlated with the toxic compounds (low GI50 values) generally correspond
GI50 data. Indeed, GI50 values are in the 10–25 nM the most potent inhibitors of topoisomerase I. A similar
range with these compounds whereas the other mole- conclusion can be drawn when looking at the LoVo
cules inactive or weakly active against topoisomerase I human colon cells and IGROV ovarian cells. In most
all give GI50 values in the 1–4 mM range. In contrast, the cases (but not all) the compounds which strongly sti-
DNA binding activity, measured through the extent of mulate DNA cleavage by topoisomerase I were the most
stabilization of poly(dAT)2 against heat denaturation potent cytotoxic agents (Fig. 4b and c). The use of cells
(
Tm values in Table 1), is not correlated with cyto- resistant to doxorubicin (LOVO-DOX) or to ecteinasci-
toxicity or topoisomerase I inhibition. LAM-D and din 743 (IGROV-ET) gave the same results. Here again,
compound 22 both give low
Tm values, 2.9 and 4.5  C we found that the less cytotoxic molecules were gen-
respectively, and compounds 18 and 30 give high
Tm erally devoid of activity against topoisomerase I
values, 13.3 and 12.0  C respectively, but these four whereas inhibition of the enzyme often coincided with a
compounds are equally cytotoxic and represent potent marked cytotoxic potential. These data showing a
topoisomerase I poison. Nevertheless, if DNA binding correlation between topoisomerase I inhibition and
is not sufficient to confer activity against topoisomerase cytotoxicity are extremely important as they strongly
I in this series, it may be useful because the compounds suggest that the topoisomerase I-mediated DNA clea-
which do not stabilize duplex DNA, such as 23, 34 and vage assay can be used as a guide to the development of
38, are inactive. DNA binding is certainly not a good superior analogues in this series. This observation may
criterion to predict cytotoxicity in this series. On the greatly facilitate the optimization of the LAM-D lead
opposite, topoisomerase I inhibition (i.e., enzyme-medi- compound. However, one should bear in mind that no
ated DNA cleavage) may represent a useful indicator of general rule can be drawn. This is illustrated with the
the drug antiproliferative activity. HT29 human colon carcinoma cell line which is poorly
sensitive to LAM-D11 and with which we could not
The relationship between topoisomerase I poisoning detect any correlation between cytotoxicity and topoi-
and cytotoxicity was investigated further with the other somerase I inhibition (Fig. 4d).
cell lines. GI50 values determined with each type of cells
were plotted against the extent of DNA cleavage mea- In conclusion, our SAR study reveals that LAM-D
sured with the different compounds. The graphics in derivatives represent a novel family of topoisomerase I-
Figure 4a shows a satisfactory relation between the targeted antitumor agents. The 5–6 double bond in the
enzyme inhibitory activity and the cytotoxicity toward quinoline B-ring of LAM-D is absolutely required to
the two prostatic cell lines DU-145 (metastatic and maintain an activity against topoisomerase I and a
1704 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- drous 1,2-dichloroethane (40 mL) under Argon atmos-
tions 8, 14 and 20 are also important structural elements phere. The reaction mixture was stirred for 8 h at 23  C
of the LAM-D structure but they can be substituted turning to an orange solution. At this time, diisopropyl-
with amino acid derivatives without loss of activity. The amine (DIPEA, 0.75 mL, 4.31 mmol) was added and the
cationic proline (30) and valine (26) derivatives have resulting brown solution stirred at 85  C for 32 h. The
been selected for a preclinial development to evaluate reaction mixture was cooled to 23  C, silica gel (6 g) was
their antitumor activity in vivo. 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
pale yellow solid (1.27 g, 47%). 1H NMR (300 MHz,
3. Experimental
CDCl3) d 7.08–7.04 (m, 3H), 6.92 (s, 1H), 6.76–6.74 (m,
3.1. Chemistry 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
3.1.1. General methods. 1H and 13C NMR spectra were Hz, 2H), 1.41–1.36 (m, 18H). 13C NMR (75 MHz,
recorded on a Varian AC300 instrument (300 MHz for CDCl3) d 155.5, 151.2, 148.5, 147.2, 146.9, 146.8, 146.4,
1
H, 75 MHz for 13C) using CDCl3, CD3OD or 145.8, 135.9, 128.5, 128.1, 126.3, 123.3, 120.1, 116.8,
(CD3)2SO. Chemical shifts were reported in ppm (d 114.8, 114.6, 114.5, 113.6, 110.3, 109.1, 104.8, 103.4,
scale) relative to Me4Si as an internal standard, and all J 71.7, 71.3, 71.2, 56.1, 55.4, 55.0, 42.3, 28.5, 22.0 (2C),
values were in Hz. Reagents obtained from commercial 21.8, 21.8, 21.7 (2C). MS (ESI) m/z: 628 (M+1)+. Rf:
suppliers were used without further purification unless 0.28 (hexane:CH2Cl2:Et2O, 5:5:2).
otherwise noted. All air- and water-sensitive reactions
were performed in flame-dried glassware under a posi- 3.1.3. 3,11 - Diisopropoxy - 14 - (4 - isopropoxy - 3 - methoxy-
tive pressure of argon. Thin-layer chromatography was phenyl)-2,12-dimethoxy-6H-[1]Benzopyrano[40 ,30 :4,5]pyr-
carried out on SDS precoated silica gel 60 F254 plates. rolo[2,1-a]isoquinolin-6-one (42). A suspension of 41
The spots were visualized with UV light (254 and 366 (301 mg, 0.480 mmol) and 2,3-dichloro-5,6-dicyano-1,4-
nm). Column chormatography was performed using the benzoquinone (DDQ, 139 mg, 0.611 mmol) in CHCl3
indicated solvents on silica gel 60 (0.040–0.063 mm, (10 mL) was refluxed for 2 h. The mixture was cooled at
SDS). Mass spectra (ESI and APCI) were recorded on a 23  C then filtered through Celite, and washed with
HP Series 1100. CH2Cl2. The filtrated was concentrated under reduced
pressure and the residue was purified by chromato-
3.1.2. 5,6-dihydro-3,11-Diisopropoxy-14-(4-isopropoxy-3- graphy on silica gel (hexane:EtOAc, 2:1) to give 42 (283
methoxyphenyl)-2,12-dimethoxy-6H-[1]Benzopyrano[40 ,30 : mg, 94%). 1H NMR (300 MHz, CDCl3) d 9.24 (d,
4,5]pyrrolo[2,1-a]isoquinolin-6-one (41). 6-Isopropoxy-7- J=7.3 Hz, 1H), 7.29–7.17 (m, 2H), 7.12–7.10 (m, 2H),
methoxy-3,4-dihydroisoquinoline 44 (1.06 g, 4.83 mmol) 7.04–7.02 (m, 2H), 6.98 (s, 1H), 6.76 (s, 1H), 4.70–4.56
was added to a solution of Iodo-acetic acid 5-iso- (m, 3H), 3.84 (s, 3H), 3.44 (s, 3H), 3.43 (s, 3H), 1.44–
propoxy-2-(4-isopropoxy-3-methoxy-phenylethynyl)-4- 1.39 (m, 18H). 13C NMR (75 MHz, CDCl3) d 155.3,
methoxy-phenyl ester 43 (2.30 g, 4.27 mmol) in anhy- 151.2, 150.0, 148.3, 147.7, 147.0, 146.4, 146.3, 134.2,
 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, 3.1.7. Compound 4. A solution of LAM-501 (2) (40 mg,
112.1, 110.8, 110.2, 109.8, 107.6, 105.5, 105.3, 103.2, 0.08 mmol) and Ac2O (0.5 mL, 5.289 mmol) in pyridine
71.6, 71.3, 71.0, 56.0, 55.3, 55.0, 21.8 (3C), 21.7, 21.7, (1 mL) was stirred at 23  C under Argon atmosphere for
21.6. MS (ESI) m/z: 648 (M+23)+, 626 (M+1)+. Rf: 4 h. The resulting pale yellow solution was washed with
0.35 (hexane:EtOAc, 2:1). HCl 1 N (210 mL). The organic phase was dried over
anhydrous Na2SO4, filtered, and the solvent removed
3.1.4. LAM-D (1). A suspension of 42 (271 mg, 0.433 under vacuum. The residue was purified by chromato-
mmol) and AlCl3 (462 mg, 3.465 mmol) in anhydrous graphy on silica gel (CH2Cl2:MeOH, from 50:1 to 40:1)
CH2Cl2 (4 mL) was stirred at 23  C for 5.5 h under to give 4 as a white solid (38 mg, 76%). 1H NMR
Argon atmosphere. H2O was added, then HCl 2 M until (300 MHz, CDCl3) d 7.22 (d, J=7.9 Hz, 1H), 7.15–7.09
pH 1–2, the resulting aqueous solution was extracted (m, 3H), 6.95 (s, 1H), 6.79 (s, 1H), 6.69 (s, 1H), 4.92–
with CH2Cl2 (3), dried over anhydrous Na2SO4, fil- 4.71 (m, 2H), 3.81 (s, 3H), 3.43 (s, 3H), 3.35 (s, 3H), 3.12
tered, and the solvent was evaporated under reduced (t, J=6.7 Hz, 2H), 2.35 (s, 3H), 2.31 (s, 3H), 2.30 (s,
pressure. The residue was purified by chromatography 3H). 13C NMR (75 MHz, CDCl3) d 169.0, 168.8, 168.6,
on silica gel (EtOAc, 100%) to afford LAM-D (1) as a 155.1, 152.2, 149.9, 147.7, 144.9, 140.0, 139.4, 138.9,
pale yellow solid (92 mg, 43%). 1H NMR (300 MHz, 135.0, 134.0, 127.1, 125.9, 125.7, 123.9, 123.2, 122.6,
DMSO-d6) d 9.92 (s, 1H), 9.81 (s, 1H), 9.32 (s, 1H), 8.98 116.0, 115.9, 114.9, 114.6, 112.0, 109.7, 105.4, 56.2, 55.7,
(d, J=7.3 Hz, 1H), 7.22–6.98 (m, 6H), 6.85 (s, 1H), 6.70 55.5, 42.5, 28.1, 20.6 (3C). MS (ESI) m/z: 628 (M+1)+.
(s, 1H), 3.75 (s, 3H), 3.36 (s, 6H). 13C NMR (75 MHz, Rf: 0.32 (CH2Cl2:MeOH, 100:1).
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, 3.1.8. Compound 5. A suspension of 6 (25 mg, 0.034
117.6, 116.4, 115.1, 113.9, 112.3, 111.5, 110.8, 106.4, mmol) and DDQ (15 mg, 0.068 mmol) in CHCl3 (2 mL)
105.7, 105.4, 103.7, 56.0, 55.1, 54.5. MS (APCI) m/z: was refluxed for 77 h. The mixture was cooled to 23  C
500 (M+1)+. Rf: 0.60 (EtOAc). then filtered through Celite, and washed with CH2Cl2
(50 mL). The filtrated was concentrated under reduced
3.1.5. LAM-501 (2). A suspension of 41 (1.422 g, 2.265 pressure and the residue was purified by chromato-
mmol) and AlCl3 (1.208 g, 9.061 mmol) in anhydrous graphy on silica gel (CH2Cl2:MeOH, 80:1) to give 5 (17
CH2Cl2 (43 mL) was stirred at 23  C for 2.5 h under mg, 68%). 1H NMR (300 MHz, CDCl3) d 9.16 (d,
Argon atmosphere. MeOH (20 mL) was added and the J=7.5 Hz, 1H), 7.66 (s, 1H), 7.60 (d, J=8.6 Hz, 1H),
solvent evaporated under reduced pressure. The brown 7.33–7.30 (m, 3H), 7.18 (s, 1H), 7.06 (d, J=7.7 Hz, 1H),
residue was purified by chromatography on silica gel 6.76 (s, 1H), 3.96 (s, 3H), 3.49 (s, 6H), 3.37 (s, 3H), 3.24
(CH2Cl2:MeOH, from 20:1 to 10:1 to 5:1) to afford (s, 3H), 3.21 (s, 3H). MS (APCI) m/z: 734 (M+1)+. Rf:
LAM-501 (2) as a pale brown solid (1.11 g, 97%). 1H 0.33 (CH2Cl2:MeOH, 80:1).
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 3.1.9. Compound 6. To a suspension of LAM-501 (2) (50
(d, J=8.4 Hz, 1H), 6.78 (s, 1H), 6.73 (s, 1H), 6.67 (s, mg, 0.0997 mmol) in anhydrous CH2Cl2 (2 mL) under
1H), 6.59 (s, 1H), 4.58 (t, J=6.5 Hz, 2H), 3.73 (s, 3H), Argon at 0  C, Et3N (83 mL, 0.5982 mmol) and metha-
3.34 (s, 3H), 3.25 (s, 3H), 2.99 (t, J=6.4 Hz, 2H). 13C nesulfonyl chloride (47 mL, 0.5982 mmol) were added.
NMR (75 MHz, DMSO-d6) d 154.3, 148.5, 147.1, 146.9, The resulting mixture was stirred at 23  C for 6 h, then
146.5, 146.0, 145.7, 144.4, 135.9, 127.7, 127.1, 125.5, quenched with H2O and extracted with CH2Cl2 (320
123.4, 118.1, 116.3, 115.3, 114.7, 114.3, 112.2, 109.2, mL). The combined organic layers were washed with
108.8, 105.1, 103.6, 56.0, 55.0, 54.7, 42.0, 27.5. MS saturated aqueous solution of NaHCO3, dried over
(ESI) m/z: 524 (M+23)+. Rf: 0.55 (CH2Cl2:MeOH anhydrous Na2SO4, filtered, and evaporated under
10:1). vacuum. The resulting residue was purified on silica gel
(CH2Cl2:MeOH, 80:1) to afford 6 as a pale yellow solid
3.1.6. Compound 3. A suspension of 4 (20 mg, 0.032 (47 mg, 64%). 1H NMR (300 MHz, CDCl3) d 7.52 (d,
mmol) and DDQ (15 mg, 0.064 mmol) in CHCl3 (2 mL) J=8.1 Hz, 1H), 7.30 (s, 1H), 7.23–7.20 (m, 2H), 7.17 (d,
was refluxed for 24 h. The mixture was cooled to 23  C J=1.6 Hz, 1H), 6.75 (s, 1H), 6.65 (s, 1H), 4.99–4.90 (m,
then filtered through Celite, and washed with CH2Cl2 1H), 4.71–4.61 (m, 1H), 3.92 (s, 3H), 3.46 (s, 3H), 3.38
(50 mL). The filtrated was concentrated under reduced (s, 3H), 3.34 (s, 3H), 3.19 (s, 3H), 3.18 (s, 3H), 3.14 (t,
pressure and the residue was purified by chromato- J=6.0 Hz, 2H). 13C NMR (75 MHz, CDCl3) d 154.5,
graphy on silica gel (CH2Cl2:MeOH, 40:1) to give 3 (12 152.8, 150.1, 148.0, 144.6, 138.0, 137.7, 136.9, 135.4,
mg, 60%). 1H NMR (300 MHz, CDCl3) d 9.22 (d, 134.4, 126.5, 126.4, 126.3, 125.6, 124.4, 123.3, 117.0,
J=7.3 Hz, 1H), 7.39 (s, 1H), 7.30 (d, J=7.7 Hz, 1H), 115.8, 115.4, 115.2, 113.5, 109.9, 105.6. MS (ESI) m/z:
7.25–7.22 (m, 3H), 7.14 (s, 1H), 7.05 (d, J=7.5 Hz, 1H), 736 (M+1)+. Rf: 0.33 (CH2Cl2:MeOH, 80:1).
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 3.1.10. Compound 7. A suspension of 8 (23 mg, 0.026
168.9, 168.7, 168.7, 155.0, 152.4, 151.0, 147.8, 145.4, mmol) and DDQ (12 mg, 0.051 mmol) in CHCl3 (2 mL)
140.9, 140.3, 139.7, 134.2, 133.5, 128.2, 124.1, 123.8, was refluxed for 21 h. The mixture was cooled to 23  C
123.7, 123.6, 123.1, 120.7, 115.7, 115.0, 112.8, 112.3, then filtered through Celite and washed with CH2Cl2
112.2, 109.1, 106.4, 106.1, 56.2, 55.7, 55.6, 20.6 (3C). (50 mL). The filtrated was concentrated under reduced
MS (ESI) m/z: 626 (M+1)+. Rf: 0.40 (CH2Cl2:MeOH, pressure and the residue was purified by chromato-
100:1). 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, mL) was stirred under Argon atmosphere at 23  C for 2
J=7.3 Hz, 1H), 7.39–7.20 (m, 20H), 7.08–7.03 (m, 2H), h. The resulting pale yellow solution was washed with
6.80 (s, 1H), 3.79 (s, 3H), 3.42 (s, 6H), 3.16–3.06 (m, H2O (10 mL), the aqueous phase was extracted
6H), 3.00–2.90 (m, 6H). 13C NMR (75 MHz, CDCl3) d with CH2Cl2 (10 mL) and the combined organic layers
170.8, 170.7, 170.6, 155.1, 152.4, 151.0, 147.7, 145.4, were dried over anhydrous Na2SO4, filtered, and the
140.9, 140.2, 140.1 (2C), 139.7, 134.2, 133.5, 128.5 (6C), solvent removed under vacuum. The residue was pur-
128.4 (2C), 128.4 (4C), 128.2, 126.4, 126.4 (2C), 124.0, ified by chromatography on silica gel (hexane:EtOAc,
123.8, 123.6, 123.6, 123.1, 120.7, 115.6, 115.0, 112.8, 60:40) to give 10 as a white solid (47 mg, 94%). 1H
112.3, 112.1, 109.0, 106.4, 106.1, 56.2, 55.7, 55.6, 35.4 NMR (300 MHz, CDCl3) d 7.57–7.49 (m, 6H), 7.16–
(3C), 30.9, 30.8 (2C). MS (APCI) m/z: 896 (M+1)+. Rf: 7.00 (m, 10H), 6.86 (s, 1H), 6.74 (s, 1H), 6.64 (s, 1H),
0.25 (CH2Cl2:MeOH, 200:1). 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,
3.1.11. Compound 8. To a suspension of LAM-501 (2) J=6.6 Hz, 2H). 13C NMR (75 MHz, CDCl3) d 167.7,
(25 mg, 0.050 mmol) in anhydrous CH2Cl2 (2 mL), 4- 167.5, 167.4, 164.2, 160.9, 154.9, 152.1, 149.7, 147.5,
(dimethylamino)pyridine (DMAP, 4 mg, 0.030 mmol), 144.8, 139.7, 139.2, 138.6, 134.9, 134.3, 133.8, 133.7,
pyridine (24 mL, 0.300 mmol) and hydrocinnamoyl 133.6, 133.6, 129.3, 129.3, 126.9, 125.9, 125.8, 123.6,
chloride (45 mL, 0.300 mmol) were added. The reaction 123.1, 122.3, 116.4 (6C), 116.2, 116.1 (6C), 115.8, 115.0,
mixture was stirred under Argon atmosphere at 23  C 114.7, 111.7, 109.7, 105.5, 56.1, 55.6, 55.4, 42.4, 37.5
for 22 h, then diluted with EtOAc (50 mL) and washed (3C), 27.8. MS (ESI) m/z: 1006 (M+1)+. Rf: 0.40
with H2O (220 mL) and saturated aqueous solution of (hexane:EtOAc, 60:40).
NaHCO3 (220 mL). The combined organic layers were
dried over anhydrous Na2SO4, filtered, and con- 3.1.14. Compound 11. A suspension of 12 (20 mg, 0.019
centrated under reduced pressure. The resulting residue mmol) and DDQ (9 mg, 0.039 mmol) in CCl4 (2 mL)
was purified by chromatography on silica gel was refluxed for 7 h. The mixture was cooled at 23  C
(CH2Cl2:MeOH, from 200:1 to 100:1) to give 8 as a then filtered through Celite, and washed with CH2Cl2
white solid (31 mg, 69%). 1H NMR (300 MHz, CDCl3) (50 mL). The filtrated was concentrated under reduced
d 7.37–7.21 (m, 15H), 7.13–7.02 (m, 4H), 6.87 (s, 1H), pressure and the residue was purified by chromato-
6.77 (s, 1H), 6.68 (s, 1H), 4.92–4.83 (m, 1H), 4.79–4.70 graphy on silica gel (CH2Cl2:MeOH, 100:1) to give 11
(m, 1H), 3.76 (s, 3H), 3.39 (s, 3H), 3.32 (s, 3H), 3.13– (15 mg, 75%) as a white solid. 1H NMR (300 MHz,
3.04 (m, 8H), 2.97–2.87 (m, 6H). 13C NMR (75 MHz, CDCl3) d 9.24 (d, J=7.5 Hz, 1H), 7.84–7.75 (m, 3H),
CDCl3) d 171.0, 170.7, 170.6, 155.1, 152.2, 149.8, 147.7, 7.54 (s, 1H), 7.45 (d, J=8.4 Hz, 1H), 7.37–7.35 (m, 2H),
144.9, 140.1 (3C), 140.0, 139.4, 138.9, 135.0, 133.9, 128.5 7.30 (s, 1H), 7.26 (s, 1H), 7.08 (d, J=7.3 Hz, 1H), 6.90
(6C), 128.4 (6C), 127.1, 126.4, 126.4, 126.4, 125.9, 125.6, (s, 1H), 3.87 (s, 3H), 3.52 (s, 3H), 3.52 (s, 3H). 13C
123.8, 123.1, 122.6, 116.0, 115.9, 114.9, 114.6, 111.9, NMR (75 MHz, CDCl3) d 159.7 (3C), 154.8, 152.3,
109.7, 105.4, 56.1, 55.7, 55.5, 42.4, 35.5 (3C), 30.9, 30.9, 150.8, 148.2, 147.6, 145.4, 140.3, 139.8, 139.2, 135.0,
30.8, 28.0. MS (ESI) m/z: 898 (M+1)+. Rf: 0.25 133.4, 128.0, 124.0, 123.9, 123.8, 123.7, 123.2, 120.7,
(CH2Cl2:MeOH, 200:1). 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
3.1.12. Compound 9. A suspension of 10 (26 mg, 0.026 (M+23)+, 1028 (M+1)+. Rf: 0.63 (CH2Cl2).
mmol) and DDQ (9 mg, 0.039 mmol) in CHCl3 (1.5
mL) was refluxed for 40 h. The mixture was cooled at 3.1.15. Compound 12. A suspension of LAM-501 (2) (25
23  C, filtered through Celite, and washed with CH2Cl2 mg, 0.050 mmol), 2,3,4,5-tetrafluorobenzoic acid (58
(50 mL). The filtrated was concentrated under reduced mg, 0.30 mmol), EDC.HCl (58 mg, 0.30 mmol) and
pressure and the residue was purified by chromato- DMAP (4 mg, 0.03 mmol) in anhydrous CH2Cl2 (5 mL)
graphy on silica gel (CH2Cl2:MeOH, from 200:1 to was stirred under Argon atmosphere at 23  C for 6 h.
100:1) to give 9 (17 mg, 65%) as a white solid. 1H NMR The resulting pale yellow solution was washed with H2O
(300 MHz, CDCl3) d 9.21 (d, J=7.3 Hz, 1H), 7.58–7.50 (10 mL), the aqueous phase was extracted with CH2Cl2
(m, 6H), 7.31 (s, 1H), 7.21–7.16 (m, 4H), 7.10–7.01 (m, (10mL), the combined organic phases were dried over
8H), 6.75 (s, 1H), 3.87 (s, 2H), 3.84 (s, 2H), 3.81 (s, 2H), anhydrous Na2SO4, filtered, and the solvent removed
3.78 (s, 3H), 3.37 (s, 6H). 13C NMR (75 MHz, CDCl3) d under vacuum. The residue was purified by chroma-
167.6, 167.5 (2C), 162.6 (d, JC-F=248.3, 3C), 154.9, tography on silica gel (CH2Cl2:MeOH, 200:1) to give 12
152.3, 150.8, 147.6, 145.4, 140.6, 140.0, 139.5, 134.6, as a white solid (33 mg, 64%). 1H NMR
133.9, 133.8, 133.8, 133.7, 133.6, 133.4, 129.5, 129.3, (300 MHz, CDCl3) d 7.81–7.74 (m, 3H), 7.36 (d, J=8.1
128.1, 123.8, 123.6, 123.2, 120.5, 116.4 (6C), 116.1 (6C), Hz, 1H), 7.25 (s, 1H), 7.23 (s, 1H), 7.19 (s, 1H), 7.10 (s,
115.9, 115.1, 112.8, 112.3, 112.0, 109.1, 106.4, 106.2. MS 1H), 6.86 (s, 1H), 6.78 (s, 1H), 4.97–4.91 (m, 1H), 4.82–
(ESI) m/z: 1026 (M+23)+, 1004 (M+1)+. Rf: 0.35 4.77 (m, 1H), 3.83 (s, 3H), 3.48 (s, 3H), 3.41 (s, 3H),
(CH2Cl2). 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,
3.1.13. Compound 10. A suspension of LAM-501 (2) (25 138.9, 138.3, 134.9, 134.7, 126.9, 126.2, 126.1, 123.7,
mg, 0.05 mmol), (4-Fluorophenyl)thioacetic acid (56 123.3, 122.5, 116.5, 115.9, 115.2, 114.9, 113.8, 113.5,
mg, 0.30 mmol), 1-(3-dimethylaminopropyl)-3-ethylcar- 111.9, 109.9, 105.6, 56.3, 55.9, 55.7, 42.5, 28.1. MS
bodiimide hydrochloride (EDC.HCl, 58 mg, 0.30 mmol) (APCI) m/z: 1030 (M+1)+. Rf: 0.50 (CH2Cl2:MeOH,
and DMAP (4 mg, 0.03 mmol) in anhydrous CH2Cl2 (5 200:1).
 C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712 1707

3.1.16. Compound 13. A suspension of LAM-501 (2) (25 (s, 3H), 3.09 (br t, 2H), 1.44–1.31 (m, 18H). 13C NMR
mg, 0.050 mmol), coumarin 3-carboxylic acid (38 mg, (75 MHz, CDCl3) d 155.1, 151.7 (d, JC-P=4.5 Hz), 149.3
0.200 mmol), EDC.HCl (38 mg, 0.200 mmol) and (d, JC-P=5.5 Hz), 147.4 (d, JC-P=5.0 Hz), 144.9, 139.9
DMAP (7 mg, 0.0598 mmol) in anhydrous CH2Cl2 (4 (d, JC-P=7.1 Hz), 139.6 (d, JC-P=6.5 Hz), 139.1 (d,
mL) was stirred under Argon atmosphere at 23  C for JC-P=7.6 Hz), 135.0, 133.0, 127.0, 126.2, 124.4, 123.2,
3.5 h. The resulting pale yellow solution was washed 122.6, 122.6, 121.1, 115.5, 114.9, 114.8, 110.5, 109.8,
with H2O (10 mL) and saturated aqueous solution of 105.6, 64.7, 64.7, 64.7 (3C), 64.6, 56.2, 55.8, 55.5, 42.4,
NaHCO3 (10 mL). Both aqueous phases were extracted 28.1, 16.2, 16.1 (3C), 16.0, 16.0. MS (ESI) m/z: 910
with CH2Cl2 (10 mL). The combined organic layers (M+1)+. Rf: 0.23 (CH2Cl2:MeOH, 30:1).
were dried over anhydrous Na2SO4, filtered, and the
solvent removed under vacuum. The residue was pur- 3.1.19. Compound 17. A suspension of 19 (63 mg, 0.062
ified by chromatography on silica gel (CH2Cl2:MeOH, mmol) and DDQ (21 mg, 0.093 mmol) in CHCl3 (5 mL)
from 50:1 to 40:1) to give 13 as a white solid (41 mg, was refluxed for 2 h. The mixture was cooled at 23  C
80%). 1H NMR (300 MHz, CDCl3) d 8.80 (s, 1H), 8.79 then filtered through Celite, and washed with CH2Cl2
(s, 1H), 8.75 (s, 1H), 7.72–7.65 (m, 6H), 7.42–7.34 (m, (50 mL). The filtrated was concentrated under reduced
7H), 7.26–7.21 (m, 3H), 7.23 (s, 1H), 6.85 (s, 1H), 6.81 pressure and the residue was purified by chromato-
(s, 1H), 4.93–4.86 (m, 1H), 4.78–4.69 (m, 1H), 3.84 (s, graphy on silica gel (hexane:EtOAc, 50:50) to give 17
3H), 3.50 (s, 3H), 3.44 (s, 3H), 3.15 (br t, 2H). MS (ESI) (58 mg, 92%). 1H NMR (300 MHz, CDCl3) d 9.24 (d,
m/z: 1040 (M+23)+. Rf: 0.24 (CH2Cl2:MeOH, 50:1). 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–
3.1.17. Compound 14. A suspension of LAM-501 (2) (25 5.09 (m, 3H), 4.64–4.60 (m, 3H), 3.81 (s, 3H), 3.44 (s,
mg, 0.05 mmol), 9H-fluorene-4-carboxylic acid (63 mg, 6H), 1.63–1.55 (m, 9H), 1.49 (s, 9H), 1.47 (s, 18H). 13C
0.30 mmol), EDC.HCl (58 mg, 0.30 mmol) and DMAP NMR (75 MHz, CDCl3) d 171.5, 171.3, 171.1, 155.0,
(4 mg, 0.03 mmol) in anhydrous CH2Cl2 (5 mL) was 154.8, 152.2, 150.8, 147.6, 145.3, 140.6, 140.0, 139.4,
stirred under Argon atmosphere at 23  C for 2 h. The 134.4, 133.3, 128.0, 127.9, 123.9, 123.7, 123.7, 123.7,
resulting pale yellow solution was washed with H2O (10 123.6, 123.0, 120.6, 115.7, 115.1, 112.7, 112.2, 112.0,
mL), the aqueous phase was extracted with CH2Cl2 (10 108.9, 106.3, 106.1, 80.0 (3C), 56.2 (2C), 55.8, 55.7, 55.7,
mL), the combined organic phases were dried over 55.5, 28.3 (9C), 18.6 (3C). MS (ESI) m/z: 1035
anhydrous Na2SO4 and the solvent removed under (M+23)+, 1013 (M+1)+. Rf: 0.43 (hexane:EtOAc,
vacuum. The residue was purified by chromatography 50:50).
on silica gel (CH2Cl2:MeOH, 200:1) to give 14 as a
white solid (26 mg, 48%). 1H NMR (300 MHz, CDCl3) 3.1.20. Compound 18. TFA (1 mL) was added to a
d 8.56–8.52 (m, 2H), 8.46–8.43 (m, 1H), 8.18–8.11 (m, solution of 17 (15 mg, 0.015 mmol) in anhydrous
3H), 7.77–7.74 (m, 3H), 7.58–7.56 (m, 3H), 7.50–7.30 CH2Cl2 (3 mL) at 0  C under Argon atmosphere. The
(m, 13H), 7.21 (s, 1H), 7.04 (s, 1H), 6.93 (s, 1H), 5.04– reaction mixture was stirred at 23  C for 5 h. The sol-
4.95 (m, 1H), 4.92–4.83 (m, 1H), 3.96 (s, 6H), 3.93 (s, vent was evaporated under reduced pressure and, in
3H), 3.62 (s, 3H), 3.54 (s, 3H), 3.25 (t, J=6.5 Hz, 2H). order to eliminate the remaining TFA, the mixture was
13
C NMR (75 MHz, CDCl3) d 166.1, 166.0, 166.0, treated with CH2Cl2 (315 mL) and evaporated to
155.2, 152.6, 150.2, 148.0, 145.2, 145.2, 145.1, 144.2, dryness to give 18 as a white solid (16 mg, quant.). 1H
144.2, 144.2, 141.5, 141.3, 140.4, 139.9, 139.8, 139.2, NMR (300 MHz, CD3OD) d 9.03–8.99 (m, 1H), 7.63–
135.2, 134.3, 129.5, 129.3, 129.1, 129.0, 127.7, 127.3, 7.60 (m, 2H), 7.52 (dd, J=8.0, 1.6 Hz, 1H), 7.39–7.27
126.9, 126.8, 126.1, 126.1, 126.0, 125.9, 125.4, 125.1, (m, 2H), 7.18–7.15 (m, 2H), 6.85 (d, J=9.2 Hz, 1H),
125.1, 125.0, 124.7, 124.6, 124.1, 123.4, 122.8, 116.3, 4.53–4.36 (m, 3H), 3.93 (s, 3H), 3.48 (s, 6H), 1.80 (d,
116.1, 115.1, 114.8, 112.2, 109.9, 105.7, 56.3, 55.9, 55.7, J=7.1 Hz, 3H), 1.74 (d, J=7.3 Hz, 3H), 1.71–1.68 (m,
42.6, 37.0 (3C), 28.2. MS (ESI) m/z: 1100 (M+23)+. Rf: 3H). MS (ESI) m/z: 713 (M+1)+.
0.47 (CH2Cl2).
3.1.21. Compound 19. A suspension of LAM-501 (2) (50
3.1.18. Compound 15. To a suspension of LAM-501 (2) mg, 0.0997 mmol), Boc-l-Ala-OH (75 mg, 0.3988
(15 mg, 0.030 mmol) in anhydrous CH2Cl2 under Argon mmol), EDC.HCl (76 mg, 0.3988 mmol) and DMAP (7
atmosphere, Et3N (17 mL, 0.120 mmol) and diethyl mg, 0.0598 mmol) in anhydrous CH2Cl2 (4.3 mL) was
chlorophosphate (18 mL, 0.120 mmol) were added and stirred under Argon atmosphere at 23  C for 2 h. The
the mixture was stirred at 23  C. After 4.5 h, two more resulting pale yellow solution was washed with H2O (10
equivalents of Et3N (9 mL, 0.060 mmol) and diethyl mL) and HCl 0.1 N (10 mL) and the aqueous phases
chlorophosphate (9 mL, 0.060 mmol) were added and were extracted with CH2Cl2 (10 mL). The organic pha-
the mixture stirred at 23  C overnight. The mixture was ses were dried over anhydrous Na2SO4, filtered, and the
concentrated under reduced pressure and the residue solvent removed under vacuum. The residue was pur-
purified by chromatography on silica gel ified by chromatography on silica gel (hexane:EtOAc,
(CH2Cl2:MeOH, from 30:1 to 15:1) to give 15 as a white from 2:1 to 1:1) to give 19 as a white solid (81 mg, 80%).
solid (20 mg, 74%). 1H NMR (300 MHz, CDCl3) d 7.45 1
H NMR (300 MHz, CDCl3) d 7.25–7.09 (m, 4H), 6.97
(dd, J=8.1, 1.5 Hz, 1H), 7.29 (d, J=1.5 Hz, 1H), 7.18 (s, 1H), 6.75 (d, J=7.7 Hz, 1H), 6.67 (d, J=10.1 Hz,
(s, 1H), 7.10 (dd, J=8.1, 1.6 Hz, 1H), 7.06 (s, 1H), 6.71 1H), 5.12 (br s, 2H), 4.89–4.85 (m, 1H), 4.70–4.55 (m,
(s, 1H), 6.64 (s, 1H), 4.94–4.86 (m, 1H), 4.72–4.63 (m, 3H), 3.78 (s, 3H), 3.40 (s, 3H), 3.33 (s, 3H), 2.03 (br t,
1H), 4.34–4.18 (m, 12H), 3.84 (s, 3H), 3.44 (s, 3H), 3.36 2H), 1.58 (d, J=7.1 Hz, 3H), 1.52 (d, J=7.1 Hz, 6H),
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) mg, 0.0598 mmol) in anhydrous CH2Cl2 (4.3 mL) was
d 171.4, 155.0, 152.0, 149.7, 147.4, 144.8, 139.7, 139.1, stirred under Argon atmosphere at 23  C for 3 h. The
138.6, 135.0, 134.1, 126.9, 126.8, 126.0, 125.9, 125.7, resulting pale yellow solution was washed with H2O (10
123.7, 123.1, 122.4, 116.1, 115.8, 114.9, 114.7, 111.7, 109.6, mL) and HCl 0.1 N (10 mL) and the aqueous phases
105.4, 79.9, 60.3, 56.1, 55.7, 55.7, 55.5, 55.4, 49.2, 42.3, were extracted with CH2Cl2 (10 mL). The combined
28.2, 27.9, 21.0, 18.5, 14.1. MS (ESI) m/z: 1037 (M+23)+, organic layers were dried over anhydrous Na2SO4, fil-
1015 (M+1)+. Rf: 0.44 (hexane:EtOAc, 50:50). tered, and the solvent removed under vacuum. The
residue was purified by chromatography on silica gel
3.1.22. Compound 20. TFA (1 mL) was added to a (hexane:EtOAc, 2:1) to give 23 as a white solid (77 mg,
solution of 19 (15 mg, 0.0148 mmol) in anhydrous 68%). 1H NMR (300 MHz, CDCl3) d 7.24–7.08 (m,
CH2Cl2 (3 mL) at 0  C under Argon atmosphere. The 4H),6.99 (s, 1H), 6.76 (d, J=7.0 Hz, 1H), 6.69 (d,
reaction mixture was stirred at 23  C for 2 h. The sol- J=8.4 Hz, 1H), 4.94–4.86 (m, 3H), 4.78–4.68 (m, 1H),
vent was evaporated under reduced pressure and, in 4.63–4.50 (m, 2H), 4.37–4.26 (m, 2H), 3.78 (s, 3H), 3.41
order to eliminate the remaining TFA, the mixture was (s, 3H), 3.34 (s, 3H), 3.12 (br t, 2H), 1.90–1.60 (m, 9H),
treated with CH2Cl2 (315 mL) and evaporated to 1.45 (s, 27H), 1.05–0.95 (m, 18H). 13C NMR (75 MHz,
dryness to give 20 as a white solid (17 mg, quant.) 1H CDCl3) d 177.6 (2C), 171.4, 155.6, 155.4, 155.1, 152.1,
NMR (300 MHz, CD3OD) d 7.46–7.44 (m, 2H), 7.28– 149.7, 147.6, 144.8, 139.8, 139.2, 138.7, 135.0, 134.1,
7.27 (m, 1H), 7.19 (s, 1H), 7.16 (s, 1H), 6.87 (d, J=8.8 127.0, 127.0, 126.0, 125.7, 123.8, 123.1, 122.5, 116.1,
Hz, 1H), 6.78 (d, J=10.0 Hz, 1H), 4.75 (t, J=6.2 Hz, 115.8, 114.9, 114.7, 111.9, 109.7, 105.5, 80.0 (3C), 56.1,
2H), 4.77–4.37 (m, 3H), 3.87 (s, 3H), 3.45 (s, 3H), 3.38 55.7, 55.5, 53.1, 52.2 (2C), 42.4, 41.5 (3C), 28.3 (9C),
(s, 3H), 3.16 (t, J=6.2 Hz, 2H), 1.77 (d, J=6.9 Hz, 3H), 28.0, 24.7 (2C), 22.9, 22.8 (4C), 21.8 (2C). MS (ESI) m/z:
1.71–1.67 (m, 6H). MS (ESI) m/z: 715 (M+1)+. 1163 (M+23)+, 1141 (M+1)+. Rf: 0.26 (hexane:EtOAc,
2:1).
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) 3.1.26. Compound 24. TFA (1 mL) was added to a
was refluxed for 35 h. The mixture was cooled at 23  C solution of 23 (15 mg, 0.013 mmol) in CH2Cl2 (3 mL) at
then filtered through Celite, and washed with CH2Cl2 0  C under Argon atmosphere. The reaction mixture
(50 mL). The filtrated was concentrated under reduced was stirred at 23  C for 5 h. The solvent was evaporated
pressure and the residue was purified by chromato- under reduced pressure and, in order to eliminate the
graphy on silica gel (CH2Cl2:MeOH, 100:1) to give 21 remaining TFA, the mixture was treated with CH2Cl2
(38 mg, 73%) as a white solid. 1H NMR (300 MHz, (315 mL) and evaporated to dryness to give 24 as a
CDCl3) d 9.22 (d, J=7.3 Hz, 1H), 7.42 (s, 1H), 7.32 (d, white solid (14 mg, 88%). 1H NMR (300 MHz,
J=7.9 Hz, 1H), 7.25–7.17 (m, 4H), 7.05 (d, J=7.7 Hz, CD3OD) d 7.47–7.41 (m, 2H), 7.29–7.24 (m, 2H), 7.16
1H), 6.79 (d, J=5.9 Hz, 1H), 4.98–4.96 (m, 3H), 4.62– (s, 1H), 6.87 (d, J=8.2 Hz, 1H), 6.80 (d, J=10.1 Hz,
4.56 (m, 3H), 3.80 (s, 3H), 3.44 (s, 3H), 3.43 (s, 3H), 1H), 4.42–4.29 (m, 3H), 3.92–3.87 (m, 2H), 3.85 (s, 3H),
1.87–1.64 (m, 9H), 1.49 (s, 9H), 1.46 (s, 18H), 1.06–0.99 3.45 (s, 3H), 3.37 (s, 3H), 3.18 (t, J=6.2 Hz, 2H), 2.14–
(m, 18H). 13C NMR (75 MHz, CDCl3) d 171.4 (4C), 1.61 (m, 9H), 1.12–0.98 (m, 18H). MS (ESI) m/z: 841
155.3, 155.0, 152.3, 150.9, 147.7, 145.4, 140.7, 140.1, (M+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, 3.1.27. Compound 25. A suspension of 27 (49 mg, 0.045
80.0 (3C), 56.2 (2C), 55.8 (2C), 55.7, 52.2, 41.7 (2C), mmol) and DDQ (20 mg, 0.089 mmol) in CCl4 (2 mL)
41.5, 28.3 (9C), 24.8 (3C), 23.0, 22.9 (3C), 21.9. MS was refluxed for 5 h. The mixture was cooled to 23  C
(ESI) m/z: 1161 (M+23)+, 1139 (M+1)+. Rf: 0.45 then filtered through Celite and washed with CH2Cl2
(CH2Cl2:MeOH, 50:1). (50 mL). The filtrated was concentrated under reduced
pressure and the residue was purified by chromato-
3.1.24. Compound 22. TFA (1 mL) was added to a graphy on silica gel (CH2Cl2:MeOH, from 100:1 to
solution of 21 (15 mg, 0.013 mmol) in anhydrous 50:1) to give 25 (43 mg, 88%). 1H NMR (300 MHz,
CH2Cl2 (3 mL) at 0  C under Argon atmosphere. The CDCl3) d 9.20 (d, J=7.3 Hz, 1H), 7.40 (s, 1H), 7.30–
reaction mixture was stirred at 23  C for 5 h. The sol- 7.13 (m, 5H), 7.04 (d, J=7.3 Hz, 1H), 6.79 (d, J=7.5
vent was evaporated under reduced pressure and, in Hz, 1H), 5.10–5.06 (m, 3H), 4.56–4.48 (m, 3H), 3.81 (s,
order to eliminate the remaining TFA, the mixture was 3H), 3.43 (s, 6H), 2.45–2.32 (m, 3H), 1.49 (s, 9H), 1.47
treated with CH2Cl2 (315 mL) and evaporated to (s, 18H), 1.14–1.00 (m, 18H). 13C NMR (75 MHz,
dryness to give 22 as a white solid (19 mg, quant.). 1H CDCl3) d 170.4 (3C), 155.7, 155.0, 152.3, 150.9, 147.6,
NMR (300 MHz, CD3OD) d 9.15–9.12 (m, 1H), 7.64 (d, 145.4, 140.6, 140.0, 139.4, 134.5, 133.5, 130.9, 128.8,
J=2.7 Hz, 1H), 7.58–7.52 (m, 2H), 7.40–7.29 (m, 2H), 128.2, 128.1, 124.1, 123.8, 123.6, 123.2, 120.8, 115.9,
7.26–7.22 (m, 2H), 6.89 (d, J=7.3 Hz, 1H), 4.45–4.31 115.1, 112.8, 112.3, 112.2, 109.1, 106.4, 106.2, 80.0 (3C),
(m, 3H), 3.90 (s, 3H), 3.48 (s, 3H), 3.48 (s, 3H), 2.11– 58.5, 56.0, 55.6, 55.6, 55.5, 55.5, 31.3 (2C), 31.2, 28.3
1.79 (m, 9H), 1.13–1.06 (m, 18H). MS (ESI) m/z: 839 (9C), 19.2, 19.1, 17.2 (2C), 17.1 (2C). MS (ESI) m/z:
(M+1)+. 1119 (M+23)+, 1097 (M+1)+. Rf: 0.33
(CH2Cl2:MeOH, 100:1).
3.1.25. Compound 23. A suspension of LAM-501 (2) (50
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
mmol), EDC.HCl (76 mg, 0.3988 mmol) and DMAP (7 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 d 9.27–9.23 (m, 1H), 7.47–7.36 (m, 1H), 7.26–7.08 (m,
was stirred at 23  C for 4 h. The solvent was evaporated 6H), 6.84–6.78 (m, 1H), 4.56–4.49 (m, 3H), 3.80 (s, 3H),
under reduced pressure and, in order to eliminate the 3.66–3.47 (m, 6H), 3.43 (s, 6H), 2.40–2.29 (m, 6H),
remaining TFA, the mixture was treated with CH2Cl2 2.04–1.98 (m, 6H), 1.49 (s, 27H). MS (ESI) m/z: 1091
(315 mL) and evaporated to dryness to give 26 as a (M+1)+. Rf: 0.31 (CH2Cl2:MeOH 30:1).
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– 3.1.32. Compound 30. TFA (1 mL) was added to a
7.32 (m, 2H), 7.23–7.18 (m, 2H), 6.88 (d, J=9.0 Hz, solution of 29 (15 mg, 0.014 mmol) in anhydrous
1H), 4.36 (d, J=4.4 Hz, 1H), 4.31 (d, J=4.4 Hz, 1H), CH2Cl2 (3 mL) at 0  C under Argon atmosphere. The
4.24 (t, J=4.4 Hz, 1H), 3.92 (s, 3H), 3.48 (s, 6H), 2.62– reaction mixture was stirred at 23  C for 2 h. The sol-
2.43 (m, 3H), 1.29–1.19 (m, 18H). MS (APCI) m/z: 797 vent was evaporated under reduced pressure and, in
(M+1)+. order to eliminate the remaining TFA, the mixture was
treated with CH2Cl2 (315 mL) and evaporated to
3.1.29. Compound 27. A suspension of LAM-501 (2) (50 dryness to give 30 as a white solid (19 mg, quant.). 1H
mg, 0.0997 mmol), Boc-l-Val-OH (87 mg, 0.3988 NMR (300 MHz, CD3OD) d 8.96–8.90 (m, 1H), 7.67–
mmol), EDC.HCl (76 mg, 0.3988 mmol) and DMAP (7 7.52 (m, 3H), 7.40–7.24 (m, 2H), 7.13–7.08 (m, 2H),
mg, 0.0598 mmol) in anhydrous CH2Cl2 (4.3 mL) was 6.84–6.80 (m, 1H), 4.86–4.67 (m, 3H), 3.95 (s, 3H),
stirred under Argon atmosphere at 23  C for 4 h. The 3.55–3.43 (m, 12H), 2.66–2.35 (m, 6H), 2.27–2.14 (m,
resulting pale yellow solution was washed with H2O 6H). MS (ESI) m/z: 791 (M+1)+.
(10 mL) and saturated aqueous solution of NaHCO3
(10 mL). The aqueous phase was extracted with 3.1.33. Compound 31. A suspension of LAM-501 (2) (50
CH2Cl2 (10 mL). The combined organic phases were mg, 0.0997 mmol), Boc-l-Pro-OH (86 mg, 0.3988
dried over anhydrous Na2SO4 and the solvent removed mmol), EDC.HCl (76 mg, 0.3988 mmol) and DMAP (7
under vacuum. The residue was purified by chromato- mg, 0.0598 mmol) in anhydrous CH2Cl2 (4.3 mL) was
graphy on silica gel (CH2Cl2:MeOH, from 100:1 to stirred under Argon atmosphere at 23  C for 4 h. The
50:1) to give 27 as a white solid (87 mg, 79%). 1H NMR resulting pale yellow solution was washed with H2O
(300 MHz, CDCl3) d 7.26–7.13 (m, 2H), 7.09 (s, 2H), (10 mL) and saturated aqueous solution of NaHCO3
6.96 (s, 1H), 6.76 (d, J=7.5 Hz, 1H), 6.68 (d, J=9.5 Hz, (10 mL). The aqueous phase was extracted with
1H), 5.08–5.05 (m, 3H), 4.91–4.69 (m, 2H), 4.52–4.46 CH2Cl2 (10 mL). The combined organic layers were
(m, 3H), 3.77 (s, 3H), 3.40 (s, 3H), 3.32 (s, 3H), 3.18 (br dried over anhydrous Na2SO4 and the solvent removed
t, 2H), 2.42–2.38 (m, 3H), 1.48 (s, 9H), 1.45 (s, 18H), under vacuum. The residue was purified by chromato-
1.11–0.98 (m, 18H). 13C NMR (75 MHz, CDCl3) d graphy on silica gel (CH2Cl2:MeOH, from 100:1 to
170.3 (3C), 155.6, 154.9, 152.1, 149.7, 147.5, 144.8, 50:1) to give 31 as a white solid (74 mg, 68%). 1H NMR
139.7, 139.1, 138.5, 134.9, 134.2, 126.9, 126.0, 125.7, (300 MHz, CDCl3) d 7.16–7.13 (m, 2H), 7.06–7.03 (m,
123.8, 123.1, 122.5, 116.1, 115.8, 115.0, 114.6, 111.9, 2H), 6.90 (s, 1H), 6.78–6.63 (m, 2H), 4.95–4.62 (m, 2H),
109.6, 105.4, 79.9 (3C), 58.5, 56.0, 55.6, 55.3, 55.3, 53.4, 4.56–4.46 (m, 3H), 3.78 (s, 3H), 3.69–3.43 (m, 6H), 3.40
42.4, 31.2 (3C), 28.3 (9C), 28.0, 19.1 (2C), 17.1 (4C). MS (s, 3H), 3.33 (s, 3H), 3.11 (br t, 2H), 2.40–2.26 (m, 6H),
(ESI) m/z: 1121 (M+23)+, 1099 (M+1)+. Rf: 0.35 2.08–1.90 (m, 6H), 1.47 (s, 27H). 13C NMR (75 MHz,
(CH2Cl2:MeOH, 50:1). 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,
3.1.30. Compound 28. TFA (1 mL) was added to a 134.1, 125.9, 125.6, 124.0, 123.4, 123.1 (2C), 122.7,
solution of 27 (15 mg, 0.0136 mmol) in anhydrous 122.2, 116.0, 114.7, 111.6, 109.6, 105.3, 80.1, 80.0, 79.8,
CH2Cl2 (3 mL) at 0  C under Argon atmosphere. The 58.9 (2C), 56.1, 55.7, 55.6, 55.5, 46.5, 46.3 (2C), 42.4,
reaction mixture was stirred at 23  C for 4 h. The sol- 31.5, 30.9, 29.9, 28.3 (9C), 28.0, 24.2, 23.4, 22.5. MS
vent was evaporated under reduced pressure and, in (ESI) m/z: 1115 (M+23)+, 1093 (M+1)+. Rf: 0.18
order to eliminate the remaining TFA, the mixture was (CH2Cl2:MeOH, 50:1).
treated with CH2Cl2 (315 mL) and evaporated to
dryness to give 28 as a white solid (16 mg, quant.). 1H 3.1.34. Compound 32. TFA (1 mL) was added to a
NMR (300 MHz, CD3OD) d 7.47–7.44 (m, 2H), 7.28 (d, solution of 31 (15 mg, 0.014 mmol) in anhydrous
J=8.1 Hz, 1H), 7.19 (s, 1H), 7.15 (s, 1H), 6.90–6.78 (m, CH2Cl2 (3 mL) at 0  C under Argon atmosphere. The
2H), 4.77 (br t, 2H), 4.32 (s, 1H), 4.24 (s, 2H), 3.87 (s, reaction mixture was stirred at 23  C for 3 h. The sol-
3H), 3.45 (s, 3H), 3.37 (s, 3H), 3.17 (br t, 2H), 2.54–2.46 vent was evaporated under reduced pressure and, in
(m, 3H), 1.26–1.17 (m, 18H). MS (ESI) m/z: 799 order to eliminate the remaining TFA, the mixture was
(M+1)+. treated with CH2Cl2 (315 mL) and evaporated to
dryness to give 32 as a white solid (17 mg, quant.). 1H
3.1.31. Compound 29. A suspension of 31 (45 mg, 0.041 NMR (300 MHz, CD3OD) d 7.49–7.44 (m, 2H), 7.29–
mmol) and DDQ (19 mg, 0.082 mmol) in CCl4 (2 mL) 7.17 (m, 3H), 6.88–6.75 (m, 2H), 4.79–4.68 (m, 3H), 3.89
was refluxed for 17 h. The mixture was cooled at 23  C (s, 3H), 3.52–3.38 (m, 12H), 3.15 (br t, 2H), 2.63–2.35
then filtered through Celite, and washed with CH2Cl2 (m, 6H), 2.25–2.15 (m, 6H). MS (ESI) m/z: 793
(50 mL). The filtrated was concentrated under reduced (M+1)+.
pressure and the residue was purified by chromato-
graphy on silica gel (CH2Cl2:MeOH, 30:1) to give 29 (30 3.1.35. Compound 33. A suspension of 35 (50 mg, 0.040
mg, 67%) as a white solid. 1H NMR (300 MHz, CDCl3) 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 CH2Cl2 (3 mL) at 0  C under Argon atmosphere. The
then filtered through Celite, and washed with CH2Cl2 reaction mixture was stirred to 23  C for 2.5 h. The sol-
(50 mL). The filtrated was concentrated under reduced vent was evaporated under reduced pressure and, in
pressure and the residue was purified by chromato- order to eliminate the remaining TFA, the mixture was
graphy on silica gel (CH2Cl2:MeOH, 80:1) to give 33 (43 treated with CH2Cl2 (315 mL) and evaporated to
mg, 86%) as a white solid. 1H NMR (300 MHz, CDCl3) dryness to give 36 as a white solid (17 mg, quant.). 1H
d 9.22 (d, J=7.3 Hz, 1H), 7.37–7.20 (m, 20H), 7.08–7.03 NMR (300 MHz, CD3OD) d 7.44–7.35 (m, 17H), 7.26
(m, 2H), 6.80 (d, J=2.2 Hz, 1H), 5.29–5.02 (m, 3H), (d, J=8.1 Hz, 1H), 7.10 (d, J=1.6 Hz, 1H), 7.06 (s,
4.90–4.88 (m, 3H), 3.85 (s, 3H), 3.44 (s, 6H), 3.41–3.23 1H), 6.88 (d, J=3.5 Hz, 1H), 6.78 (d, J=3.5 Hz, 1H),
(m, 6H), 1.46 (s, 9H), 1.43 (s, 18H). 13C NMR (75 MHz, 4.79–4.70 (m, 3H), 4.63 (t, J=6.7 Hz, 2H), 3.89 (s, 3H),
CDCl3) d 170.1, 170.0, 169.9, 155.1 (2C), 154.9, 152.3, 3.53–3.26 (m, 6H), 3.44 (s, 3H), 3.36 (s, 3H), 3.16 (t,
150.9, 147.6, 145.3, 140.5, 139.9, 139.3, 135.8 (2C), J=6.7 Hz, 2H). MS (ESI) m/z: 965 (M+23)+, 943
134.5, 133.4, 125.0 (9C), 128.6 (6C), 128.1, 128.0, 127.1 (M+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 3.1.39. Compound 37. A suspension of LAM-501 (2) (50
(2C), 55.7, 55.6, 55.5, 54.4, 38.1 (3C), 28.2 (9C). MS mg, 0.0997 mmol), Boc-l-Trp-OH (121 mg, 0.3988
(ESI) m/z: 1263 (M+23)+, 1241 (M+1)+. Rf: 0.56 mmol), EDC.HCl (76 mg, 0.3988 mmol) and DMAP (7
(CH2Cl2:MeOH, 50:1). mg, 0.0598 mmol) in anhydrous CH2Cl2 (4.3 mL) was
stirred under Argon atmosphere at 23  C for 4 h. The
3.1.36. Compound 34. TFA (1 mL) was added to a resulting pale yellow solution was washed with H2O
solution of 33 (15 mg, 0.012 mmol) in CH2Cl2 (3 mL) at (10 mL) and aqueous saturated solution of NaHCO3
0  C under Argon atmosphere. The reaction mixture (10 mL). The combined aqueous phases were extracted
was stirred to 23  C for 1 h. The solvent was evaporated with CH2Cl2 (10 mL). The combined organic layers
under reduced pressure and, in order to eliminate the were dried over anhydrous Na2SO4, filtered, and the
remaining TFA, the mixture was treated with CH2Cl2 solvent removed under vacuum. The residue was pur-
(315 mL) and evaporated to dryness to give 34 as a ified by chromatography on silica gel (CH2Cl2:MeOH,
white solid (20 mg, quant.). 1H NMR (300 MHz, from 30:1 to 15:1) to give 37 as a white solid (115 mg,
CD3OD) d 9.08 (d, J=7.5 Hz, 1H), 7.60 (d, J=2.2 Hz, 85%). 1H NMR (300 MHz, CDCl3) d 8.35 (s, 1H), 8.28
1H), 7.53 (s, 1H), 7.49–7.36 (m, 17H), 7.30 (d, J=3.5 (s, 2H), 7.68–7.62 (m, 3H), 7.39–7.36 (m, 3H), 7.26–7.07
Hz, 1H), 7.20 (d, J=7.5 Hz, 1H), 7.08 (d, J=4.8 Hz, (m, 12H), 6.90 (s, 1H), 6.72 (s, 1H), 6.65 (br s, 2H),
1H), 6.87 (d, J=4.2 Hz, 1H), 4.76 (t, J=7.0 Hz, 1H), 5.15–5.12 (m, 2H), 5.00–4.59 (m, 6H), 3.75 (s, 3H),
4.70 (t, J=6.9 Hz, 1H), 4.63 (t, J=6.2 Hz, 1H), 3.95 (s, 3.52–3.28 (m, 12H), 3.00 (br t, 2H), 1.43 (s, 27H). 13C
3H), 3.47 (s, 6H), 3.61–3.36 (m, 6H). MS (ESI) m/z: 941 NMR (75 MHz, CDCl3) d 170.7, 170.4, 170.4, 155.3
(M+1)+. (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,
3.1.37. Compound 35. A suspension of LAM-501 (2) (50 125.5, 123.8, 123.1 (3C), 122.5, 122.0 (3C), 119.5 (3C),
mg, 0.0997 mmol), Boc-l-Phe-OH (106 mg, 0.3988 118.6 (3C), 116.0, 115.8, 114.7, 111.7, 111.3 (3C), 109.5
mmol), EDC.HCl (76 mg, 0.3988 mmol) and DMAP (7 (3C), 105.3, 80.0 (3C), 56.0 (2C), 55.6, 55.4, 54.4 (2C),
mg, 0.0598 mmol) in anhydrous CH2Cl2 (4.3 mL) was 42.3, 28.2 (12C), 27.7. MS (ESI) m/z: 1382 (M+23)+.
stirred under Argon atmosphere at 23  C for 6 h. The Rf: 0.13 (CH2Cl2:MeOH, 30:1).
resulting pale yellow solution was washed with H2O
(10 mL) and saturated aqueous solution of NaHCO3 3.1.40. Compound 38. TFA (1 mL) was added to a
(10 mL) and both aqueous phases were extracted with solution of 37 (25 mg, 0.018 mmol) in CH2Cl2 (3 mL) at
CH2Cl2 (10 mL). The combined organic layers were 0  C under Argon atmosphere. The reaction mixture
dried over anhydrous Na2SO4, filtered, and the solvent was stirred to 23  C for 1.5 h. The solvent was evapo-
removed under vacuum. The residue was purified by rated under reduced pressure and, in order to eliminate
chromatography on silica gel (CH2Cl2:MeOH, 100:1) to the remaining TFA, the mixture was treated with
give 35 as a white solid (87 mg, 68%). 1H NMR CH2Cl2 (315 mL) and evaporated to dryness to give
(300 MHz, CDCl3) d 7.34–7.26 (m, 15H), 7.16 (br s, 38 as a white solid (27 mg, quant.) 1H NMR (300 MHz,
2H), 7.10 (s, 1H), 7.05 (s, 1H), 6.91 (s, 1H), 6.78–6.68 CD3OD) d 7.68 (d, J=8.1 Hz, 1H), 7.62 (s, 1H), 7.60 (s,
(m, 2H), 4.99 (t, J=8.6 Hz, 2H), 4.88–4.72 (m, 6H), 3.81 1H), 7.44 (s, 1H), 7.42 (s, 3H), 7.34 (s, 1H), 7.30 (s, 1H),
(s, 3H), 3.41 (s, 3H), 3.34 (s, 3H), 3.30–3.12 (m, 8H), 7.29 (s, 1H), 7.21–7.16 (m, 5H), 7.12–7.09 (m, 3H), 6.98
1.44 (s, 9H), 1.43 (s, 18H). 13C NMR (75 MHz, CDCl3) (s, 1H), 6.85 (d, J=2.0 Hz, 1H), 6.77–6.76 (m, 2H),
d 170.1, 169.9 (2C), 155.0, 154.9, 152.0, 149.7, 147.5, 4.73–4.69 (m, 3H), 4.60 (br t, 2H), 3.87 (s, 3H), 3.77–
144.7, 139.6, 139.0, 138.4, 135.8 (2C), 134.8, 134.2, 129.4 3.34 (m, 6H), 3.43 (s, 3H), 3.34 (s, 3H). MS (ESI) m/z:
(9C), 129.2, 128.5 (6C), 127.1 (2C), 126.9, 125.9, 125.7, 1060 (M+1)+.
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, 3.1.41. Compound 39. A suspension of LAM-D (1) (300
38.0 (3C), 28.2 (9C), 27.9. MS (ESI) m/z: 1265 mg, 0.601 mmol), 6-tert-Butoxycarbonylamino-hex-
(M+23)+. Rf: 0.65 (CH2Cl2:MeOH, 30:1). anoic acid (834 mg, 3.604 mmol), EDC.HCl (691 mg,
0.3.604 mmol) and DMAP (44 mg, 0.360 mmol) in
3.1.38. Compound 36. TFA (1 mL) was added to a CH2Cl2 anh. (50 mL) was stirred under Argon atmos-
solution of 35 (15 mg, 0.012 mmol) in anhydrous phere at 23  C for 3 h. The resulting pale yellow solution
 C. Tardy et al. / Bioorg. Med. Chem. 12 (2004) 1697–1712 1711

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

sequencing standards generated by treatment of the References and notes

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grants (to C.B.) from the Ligue Nationale Française Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
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