Synthesis and photocytotoxic activity of new α-methylene-γ- butyrolactone-psoralen heterodimers

Article abstract of DOI:10.1016/j.bmc.2004.04.019

The synthesis of a new α-methylene-γ-butyrolactone-psoralen heterodimer 2 is reported. Its photoantiproliferative activity and skin phototoxicity were compared with that of 5-methoxypsoralen (5-MOP) and another heterodimer 1. Both derivatives show a significant phototoxicity toward malignant cell lines including melanoma cells A375 compared to their intrinsic cytotoxicity in the dark. Both compounds were found to be nonphototoxic on mice skin and therefore could be active potential drugs in photochemotherapy.

Full text of DOI:10.1016/j.bmc.2004.04.019

Bioorganic & Medicinal Chemistry 12 (2004) 3619–3625
Synthesis and photocytotoxic activity of new a-methylene-c-
butyrolactone-psoralen heterodimers
a
Sandrine Ropp, Julia Guy, Valerie Berl, Pierre Bischoff and Jean-Pierre Lepoittevin
a
a
b
a,
*
ꢀ
aLaboratoire de Dermatochimie, UMR 7123, Universitꢀe Louis Pasteur,
Clinique Dermatologique, CHU, F-67091 Strasbourg Cedex, France
bLaboratoire de Cancꢀerologie Expꢀerimentale et de Radiologie, EA 34301,
Institut de Recherche contre les Cancers de l’Appareil Digestif, CHU, F-67091 Strasbourg Cedex, France
Received 2 March 2004; accepted 15 April 2004
Available online 18 May 2004
Abstract—The synthesis of a new a-methylene-c-butyrolactone-psoralen heterodimer 2 is reported. Its photoantiproliferative
activity and skin phototoxicity were compared with that of 5-methoxypsoralen (5-MOP) and another heterodimer 1. Both deriv-
atives show a significant phototoxicity toward malignant cell lines including melanoma cells A375 compared to their intrinsic
cytotoxicity in the dark. Both compounds were found to be nonphototoxic on mice skin and therefore could be active potential
drugs in photochemotherapy.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
(Fig. 1) and shown that this molecule was not photo-
toxic for the skin. Psoralens such as 5-methoxypsoralen
(5-MOP) and 8-methoxypsoralen (8-MOP) are widely
used in PUVA therapy for the treatment of a variety of
epidermal proliferative diseases.2;3 Their biological
properties are generally related to their ability to bind
covalently to nucleic acids.10 On the other side, a-
methylene-c-butyrolactones are very good electrophiles
and are known to react with nucleophilic residues on
proteins to form covalent bonds.11;12
The use of drugs in association with ultra-violet light for
the treatment of skin diseases can be traced back to
ancient Egypt, India and Greece, where plant extracts,
containing psoralens, were applied on the skin in asso-
ciation with light to treat psoriasis and vitiligo.1 This
concept, known as photochemotherapy, represents a
common basis for different therapeutic procedures, such
as PUVA (psoralen+UVA)2;3 and photodynamic ther-
apy.4 The mechanism of cellular damage mediated by
photochemotherapy includes a variety of biochemical
and molecular reactions, leading to inhibition of the
hyperproliferation of skin keratinocytes or tissue
destruction. Unfortunately, most photosensitizing
chemicals are also phototoxic for the skin; contact with
these molecules in the presence of UV irradiation results
in sunburn, erythema and, eventually oedema.4;5
Moreover, we have shown that a-methylene-c-butyro-
lactones had an interesting photoreactivity potential and
O
O
CH
3
O
O
5
O
O
In recent years, several highly photoreactive molecules
have been synthesized with the aim of developing new
photochemotherapeutic drugs with less side effects.6–8
Thus, we have reported9 the synthesis of a new hetero-
dimer 1 based on a furocoumarin moiety bond to a a-
methylene-c-butyrolactone by a polymethylenic chain
O
O
O
O
1
3
O
O
O
H
N
O
N
2
3
O
3
H
2
O
O
O
Figure 1. Structure of heterodimeric compounds 1 and 2 and of the
reference compound 5-methoxypsoralen 3.
* Corresponding author. Tel.: +33-388-350-664; fax: +33-388-140-447;
e-mail: jplepoit@chimie.u-strasbg.fr
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.019

3620
S. Ropp et al. / Bioorg. Med. Chem. 12 (2004) 3619–3625
can form intramolecular photoadducts with psoralens9
and intra or intermolecular photoadducts with thy-
mine.13;14
acid15 was converted under basic conditions into deriv-
ative 7 by reaction with the 4-bromobutyric acid deriv-
ative 6. The deprotection was achieved by reduction
with zinc dust in acidic solution and the acid derivative 4
was finally obtained in 91% yield.16
The use of a psoralen to target this photoactivable/
electrophilic group toward DNA could lead to the for-
mation of diadducts of a new type, between two different
sites on DNA and/or between DNA and structural
proteins.
A peptidic coupling of N-Cbz-c-aminobutyric acid and
4,4-diethoxybutylamine using DCC/DMAP17;18 gave
rise to compound 8 in 90% yield. The amine 5 was ob-
tained in 99% yield from the benzyloxycarbonyl-pro-
tected precursor 8 using ammonium formate/10%
palladium on carbon procedure.19
Based on these considerations, we have synthesized a
new heterodimeric derivative 2 in which the psoralen is
bounded to a a-methylene-c-butyrolactone via a poly-
amide chain in order to increase its affinity toward DNA
(Fig. 1).
The two components of the target molecule were first
coupled using BOP/NMM as coupling reagent.20 This
method gave rise to compound 9 in good yield (78%) but
with a reaction time of three days. Using EDCI/DMAP
as coupling reagent,21 compound 9 was obtained after
only 6 h with quantitative yield.
In this paper we report the synthesis of 2 and the skin
phototoxicity of compounds 1 and 2 in comparison with
that of the 5-methoxypsoralen 3 used as reference
compound. We also report the cytotoxicity properties of
these molecules on three human cancer cell lines, with or
without irradiation.
Then the a-methylene-c-butyrolactone ring was gener-
ated in good yield directly from the protected aldehyde
using ‘Reformatsky type’ conditions as previously
described.13 For this purpose, we have used the com-
mercially available a-bromomethacrylic acid. The yield
of the reaction could be improved (77%) by use of the
methyl-a-bromomethacrylate, prepared in one step from
the acid.22
2. Results and discussion
2.1. Chemistry
The heterodimer 2 was synthesized according to a con-
vergent strategy (Scheme 1). The two parts of the mol-
ecule, 4 and 5 were prepared separately before coupling
and the a-methylene-c-butyrolactone moiety was gen-
erated in the last step.
2.2. Effect on the growth of human cancer cell lines
The antiproliferative activity of 1, 2, and 3 was assessed
under two different experimental conditions: by incu-
bation in the dark or after UVA irradiation. The growth
inhibitory activity of these compounds against three
5-Hydroxypsoralen (bergaptol), prepared by dealkyl-
ation of bergamottin in a mixture of acetic and sulfuric
O
2
O
OEt
OEt
O
2
a
+
OH
CCl
CbzHN
O
OH
Br
H N
Br
3
2
86 %
2
2
6
d, e
50%
b, c
50%
O
O
2
OEt
OEt
O
OR
2
RHN
N
H
2
+
7 : R = CH CCl
2
3
8 : R = Cbz
5 : R = H
4 : R = H
O
f
50%
O
O
O
O
O
OEt
OEt
O
H
H
N
N
O
N
O
N
H
2
2
3
O
3
O
3
3
H
g
60 %
O
O
O
O
O
O
2
9
Scheme 1. Synthesis of 2. Reagents and conditions: (a) 1.5 equiv CCl3CH2OH, 0.75 equiv p-TsOH, toluene, reflux, 58 h; (b) 0.9 equiv bergaptol,
1.3 equiv K2CO3, acetone, reflux, 18 h; (c) 19 equiv Zn, AcOH, CH2Cl2, rt, 19 h; (d) 1 equiv DCC, 0.9 equiv DMAP, CH2Cl2, rt, 18 h; (e) Pd/C,
3.5 equiv ammonium formate, MeOH, rt, 6 h; (f) 1.2 equiv EDCI, 1.1 equiv DMAP, CH2Cl2, rt, 6 h; (g) 1.6 equiv SnCl2ÆH2O, 1.6 equiv a-bromo-
methacrylic acid, THF, Hþ, reflux, 48 h.

S. Ropp et al. / Bioorg. Med. Chem. 12 (2004) 3619–3625
Table 1. Cell growth inhibition for the compounds
3621
Compd
Cell lines IC50 (lM)
HL-60 (promyelocytic leukemia)a
K1 (thyroid carcinoma)b
A375 (malignant melanoma)b
Dark
UVA
Dark
2.84
UVA
Dark
4.82
27.6
>35.00
UVA
1
2
3
2.73
15.76
>40.00
0.31
2.50
0.29
1.13
6.96
0.70
3.50
>15.00
>39.00
29.50
23.70
a Concentration inducing a 50% growth inhibition assessed by the Uptiblue test.
b Concentration inducing a 50% growth inhibition assessed by the SRB test.
human cancer cell lines, HL-60 (promyelocytic leuke-
mia), K1 (thyroid carcinoma), A375 (malignant mela-
noma), is summarized in Table 1. The results are
expressed in concentrations able to inhibit 50% of pro-
liferation (IC50). Different behaviors were observed for
heterodimer derivatives 1, 2 and the reference 5-MOP 3
as well as between adherent or nonadherent cell lines.
On nonadherent cell lines, like HL-60, 5-MOP, 3 was
found to be the most cytotoxic derivative after irradia-
tion with UVA (Table 1). The photocytotoxicity of 1
was very similar to that of 3 (Table 1), but the hetero-
dimer 1 was also highly cytotoxic in the dark (Fig. 2).
The heterodimer 2 was found to be less photocytotoxic
Figure 3. Phototoxicity of compounds 2 and 3 (2.77 mM) and control
(vehicule) after irradiation with 6 J cmꢀ2 UVA, using the MEST.
than 1 and 3, but was also less cytotoxic in the dark
than 1.
On adherent cell lines, like K1 or A375, 5-MOP 3 was
the less photocytotoxic derivative while heterodimers 1
with an irradiation dose of 6 J cmꢀ2, whereas the refer-
ence compound 3 (5-MOP) induced a marked oedema.
Concentration of 2 could be increased up to 27.7 mM
and a UVA dose of 6 J cmꢀ2 without any phototoxicity.
and
2 showed a significant photoantiproliferative
activity. It should be noticed that 1 presented also a
significant cytotoxicity in the dark while 2 was less
cytotoxic.
Following the same procedure, compound 1 was found
to be nonphototoxic when irradiated at a concentration
2.3. Phototoxicity study
of 27.7 mM with a UVA dose of 6 J cmꢀ2
.
The skin phototoxicity of compounds 1 and 2, together
with that of 3, was tested on mice, using the mouse ear
swelling test (MEST)23 to quantify the inflammatory
reaction.
3. Conclusion
As seen in Figure 3, heterodimer 2 showed no photo-
toxicity when tested at a concentration of 2.77 mM and
We have synthesized a new heterodimeric derivative 2
based on a psoralen linked to an a-methylene-c-but-
yrolactone by a polyamide chain in order to increase its
affinity with DNA. The cytotoxicity and photocytotox-
icity properties of compounds 1 and 2 were evaluated on
three human cancer cell lines and compared with the one
of 5-methoxypsoralen 3. The new heterodimer 2 showed
an interesting cytotoxic activity on all tumor cell lines
tested with a higher cytotoxicity after irradiation and a
low toxicity in the dark. Heterodimer 1 and 5-MOP 3
are cytotoxic after irradiation, but they are also highly
toxic in the dark.
This toxicity could be due to the presence of the a-
methylene-c-butyrolactone group, which is a well-
known Michael acceptor and can react rapidly with
nucleophilic residues in proteins to form covalent
bonds.11 An inhibition of cellular enzymes activities and
metabolism could be observed24;25 leading to an anti-
proliferative effect.
Figure 2. Cytotoxicity of compounds 1 and 2 compared to 5-MOP 3
(control) in the dark determined on HL-60 cells using the Uptiblue
test.

3622
S. Ropp et al. / Bioorg. Med. Chem. 12 (2004) 3619–3625
0
0
The phototoxicity of these derivatives was evaluated on
mouse skin, as the ability of furocoumarins such as 5-
methoxypsoralen 3 to induce severe erythemas on the
skin is a well-known and serious side effect in PUVA
therapy. Heterodimeric derivatives appeared to be un-
able to cause skin phototoxicity, even when tested in
harsh experimental conditions (MEST experiment).
CDCl3): 170.9 (C1), 94.9 (C2 ), 74.0 (C1 ), 32.2 (C4), 28.0
(C2), 27.5 (C3). IR (film), m (cmꢀ1) 1754 (C@O ester), 720
(C–Cl). Anal. Calcd for C6H8BrCl3O2 (298.4): C, 24.15;
H, 2.70. Found: C, 24.34; H, 2.68.
4.1.2. 5-(Trichloroethyloxybutyrate)psoralen (7). K2CO3
(1.70 g, 12.30 mmol) was added to a solution of berga-
ptol (2.00 g, 10 mmol) in dry acetone (110 mL). After
stirring under reflux for 1.5 h, a solution of ester 6
(3.26 g, 11.40 mmol) in acetone (20 mL) was added and
stirring continued under reflux for 21 h. The mixture was
cooled down to rt and acetone was removed under
vacuum. The residue was taken-up in EtOAc (100 mL)
and washed with water (3 · 50 mL) and 10% aqueous
HCl (3 · 50 mL). The organic layer was dried over
MgSO4 and concentrated under vacuum to afford 7,
which precipitated from hot EtOAc/Et2O as a white
powder (3.5 g, 80%). Mp 110–112 ꢁC. 1H NMR
(300 MHz, CDCl3): 8.16 (d, 1H, H4, J ¼ 9 Hz), 7.63 (d,
The precise cellular target affected by these compounds
is so far not known. Further work is currently under
investigation to fully elucidate the mechanism of action
of these compounds.
4. Experimental
4.1. Chemistry
1H and 13C NMR spectra were recorded on Bruker
AC200, Avance 300, or AM400-MHz spectrometers in
CDCl3 unless otherwise specified. Chemical shifts are
reported in ppm (d) with respect to TMS, and CHCl3
was used as internal standard (d ¼ 7:27 ppm). Multi-
plicities are indicated by s (singlet), d (doublet), t (trip-
let), m (multiplet). Infrared spectra were obtained on a
Perkin–Elmer FT-IR 1600 spectrometer; peaks are
reported in reciprocal centimeters. UV spectra were
obtained using a Hewlett Packard HP 8452 diode-array
spectrophotometer. Melting points were determined in
capillary tubes on a Buchi Tottoli 510 apparatus and are
uncorrected. Elemental analyses were performed on a
Perkin–Elmer 240B and were within ꢁ0.4% of calcu-
lated values in all cases. Dried solvents were freshly
distilled before use. Tetrahydrofuran and ethyl ether
were distilled from sodium benzophenone. Methylene
chloride was dried over P2O5 before distillation. All air-
or moisture-sensitive reactions were conducted in flame-
dried glassware under an atmosphere of dry argon.
Chromatographic purifications were conducted on silica
gel columns (Merck 60, 0.040–0.063 mm) or alumina gel
(Merck 90, activity II–III, 0.063–0.200 mm) according to
the flash chromatography technique. Analytical TLC
was performed on pre-coated silica gel plates (Merck 60
F254).
0
0
1H, H5 , J ¼ 3 Hz), 7.3 (s, 1H, H8), 6.99 (d, 1H, H4 ,
J ¼ 3 Hz), 6.33 (d, 1H, H3, J ¼ 9 Hz), 4.81 (s, 2H,
00
00
CH2CCl3), 4.57 (t, 2H, H4 , J ¼ 6 Hz), 2.83 (t, 2H, H2 ,
J ¼ 6 Hz), 2.28 (m, 2H, H3 ). 13C NMR (75 MHz,
00
00
CDCl3): 170.2 (C1 ), 161.1 (C2), 158.3 (C9), 152.7 (C7),
0
148.5 (C5), 145.1 (C5 ), 139.1 (C4), 113.5 (C10), 112.9
0
(C3), 106.9 (C6), 104.9 (C4 ), 94.3 (C8 and CCl3), 74.1
00
00
00
(CH2CCl3), 71.5 (C4 ), 30.4 (C2 ), 25.2 (C3 ). IR
(CHCl3), m (cmꢀ1) 1726 (C@O coumarin and ester), 1216
(C–O). UV (EtOH), k (nm): 222 (e ¼ 23; 576), 250
(e ¼ 18; 150), 260 (e ¼ 16; 266), 268 (e ¼ 16; 052), 310
(e ¼ 14; 419). Anal. Calcd for C17H13Cl3O6 (419.64): C,
48.86; H, 3.12. Found: C, 48.92; H, 3.30.
4.1.3. 5-(Oxybutyric-acid)psoralen (4). A solution of
AcOH/H2O (9/1, 4.00 mL) was added to ester 7 (1.00 g,
2.38 mmol), in CH2Cl2 (5 mL). The resulting mixture
was cooled down to 0 ꢁC and powder zinc (2.94 g,
45.00 mmol, 19 equiv) was added. After 40 min at 0 ꢁC
the reaction was allowed to warmup to rt and stirred for
an additional 26 h. The zinc was filtered and the organic
layer was washed with water (3 · 50 mL) and extracted
with saturated aqueous Na2CO3 (3 · 30 mL). The aque-
ous layer was acidified with 1 M HCl and then extracted
with EtOAc (5 · 30 mL). The combined organic layers
were dried over MgSO4 and concentrated under vacuum
to afford acid 4 as a white solid (623 mg, 91%). Mp 164–
4.1.1. 20,20,20-Trichloroethyl-4-bromobutyrate (6). In a
two-necked round bottom flask equipped with a Dean–
Stark apparatus 2,2,2-trichloroethanol (4.30 mL,
45.00 mmol, 1.50 equiv) and p-TsOH (4.00 g, 23.00
mmol, 0.75 equiv) were added to a solution of 4-bro-
mobutyric acid (5.00 g, 30.00 mmol) in toluene (150 mL).
After stirring under reflux for 48 h, toluene was removed
under vacuum. The residue dissolved in Et2O (150 mL)
was washed with saturated aqueous NaHCO3 (30 mL),
water (2 · 30 mL), brine (2 · 30 mL), dried over MgSO4
and concentrated. Purification by column chromato-
graphy on silica (hexane/EtOAc, 95/5) afforded a color-
1
166 ꢁC. H NMR (200 MHz, acetone-d6): 8.11 (d, 1H,
0
H4, J ¼ 9:8 Hz), 7.55 (d, 1H, H5 , J ¼ 2:4 Hz), 7.20 (s,
0
1H, H8), 6.90 (d, 1H, H4 , J ¼ 2:4 Hz), 6.26 (d, 1H, H3,
00
J ¼ 9:8 Hz), 4.47 (t, 2H, H4 , J ¼ 6:1 Hz), 2.62 (t, 2H,
H2 , J ¼ 7:1 Hz), 2.19 (m, 2H, H3 ). 13C NMR (50 MHz,
00
00
00
acetone-d6): 174.1 (C1 ), 160.0 (C2), 157.5 (C9), 152.0
0
(C7), 148.6 (C5), 145.9 (C5 ), 139.5 (C4), 112.8 (C10),
0
00
112.3 (C3), 106.0 (C6), 105.5 (C4 ), 93.2 (C8), 66.8 (C4 ),
00
30.1 (C2 ), 24.9 (C3 ). IR (CHCl3), m (cmꢀ1) 1736 (C@O
coumarin), 1704 (C@O acid), 1214 (C–O). UV (EtOH),
00
k
(nm): 222 (e ¼ 24; 851), 250 (e ¼ 18; 326), 260
1
less oil (7.7 g, 86%). H NMR (200 MHz, CDCl3): 4.75
0
(e ¼ 17; 008), 268 (e ¼ 17; 338), 310 (e ¼ 15; 034). Anal.
Calcd for C15H12O6 (228.25): C, 62.50; H, 4.20. Found:
C, 62.38; H, 4.28.
(s, 2H, H1 ), 3.49 (t, 2H, H4, J ¼ 6:4 Hz), 2.67 (t, 2H, H2,
J ¼ 7:1 Hz), 2.23 (m, 2H, H3). 13C NMR (50 MHz,

S. Ropp et al. / Bioorg. Med. Chem. 12 (2004) 3619–3625
3623
4.1.4. 4-Carbobenzyloxyamino-N-(40,40-diethoxybutyl)but-
anamide (8) . DMAP (2.10 g, 17.22 mmol) was added to
00
4.48 (m, 3H, H1 and H12 ), 3.62 (m, 2H, H13 ), 3.46 (m,
00
00
00
2H, H13 ), 3.26 (m, 4H, H5 and H9 ), 2.45 (t, 2H, H3 ,
00
00
00
00
00
a
solution of N-Cbz-c-aminobutyric acid (5.00 g,
J ¼ 7:3 Hz), 2.22 (m, 4H, H2 and H7 ), 1.81 (m, 2H,
0
0
0
0
0
0
0
0
21.00 mmol) in dry CH2Cl2 (100 mL). After cooling the
mixture to ꢀ10 ꢁC, DCC (3.85 g, 18.70 mmol) in dry
CH2Cl2 (30 mL) and aminobutanal diethylacetal
(3.00 mL, 17.58 mmol) were added. The reaction mixture
was stirred at ꢀ10 ꢁC for 20 min and then allowed to
warmup to rt for further 18 h. Formed urea was filtered
off and the reaction washed with brine (2 · 100 mL). The
organic layer was dried over MgSO4 and concentrated
under vacuum. Purification by column chromatography
on silica (hexane/EtOAc, 10/90) afforded 8 as a white
H11 ), 1.61 (m, 4H, H10 and H6 ), 1.18 (t, 6H, H14 ,
00
J ¼ 7:1 Hz). 13C NMR (75 MHz, CDCl3): 172.8 (C4 or
00
C8 ), 172.2 (C4 or C8 ), 161.3 (C2), 158.2 (C9), 152.5
00
00
0
(C7), 148.7 (C5), 144.9 (C5 ), 139.4 (C4), 1123.1 (C10),
0
112.4 (C3), 106.5 (C6), 105.1 (C4 ), 102.6 (C12 ), 93.8 (C8),
00
00
00
71.9 (C1 ), 61.5 (C13 ), 39.4, 34.1, 32.6, 31.1, 25.9, 25.1,
00
24.5, 15.3 (C14 ). IR (CHCl3), m (cmꢀ1) 1732 (C@O
coumarin), 1672 (C@O amide), 1209 (C–O). UV
(EtOH), k (nm): 208 (e ¼ 16; 496), 222 (e ¼ 17; 121), 250
(e ¼ 11;796), 260 (e ¼ 10;704), 268 (e ¼ 10;785), 310
(e ¼ 8884). Anal. Calcd for C27H36N2O8 (516.25): C,
62.78; H, 7.02; N, 5.42. Found: C, 62.76; H, 7.28; N,
5.35.
1
solid (6.0 g, 90%). Mp 60–62 ꢁC. H NMR (200 MHz,
CDCl3): 7.31–7.24 (m, 5H, –Ph), 7.10 (br s, 1H, NH),
6.20 (br s, 1H, NH), 5.03 (s, 2H, CH2Ph), 4.42 (t, 1H,
00
0
0
H4 , J ¼ 5:0 Hz), 3.73 (m, 2H, H5 ), 3.42 (m, 2H, H5 ),
0
3.17 (m, 4H, H4 and H1 ), 2.14 (t, 2H, H2, J ¼ 6:9 Hz),
0
1.83 (m, 2H), 1.50 (m, 4H), 1.18 (t, 6H, H6 , J ¼ 4:8 Hz).
4.1.7. N-{3-[3-(3-Methylene-5-oxo-tetrahydrofuran-2-yl)-
propylcarbamoyl]-propyl}-4-(7-oxo-7H-furo[3,2-g]chromen-
4-yloxy)-butyramide (2). To a solution of compound 9
(205 mg, 0.40 mmol) in THF (4 mL) was added 8 mL of
an acidic solution (37.5 mL CH3O(CH2)2OH, 20 mL
H2O, 10 mL CH3COOH, 0.5 mL HClconcd), a-bromo-
methacrylic acid (118 mg, 0.2 mmol, 1.8 equiv) and
SnCl2Æ2H2O (129 mg, 0.68 mmol, 1.7 equiv). The mixture
was heated under reflux for 21 h, and the solvents
removed under reduced pressure. The residue was taken
up in EtOAc (50 mL) and water (20 mL). pH was
adjusted with saturated aqueous NaHCO3 until 8 and
the aqueous layer was extracted with EtOAc (5 · 20 mL).
Combined organic layers were dried on MgSO4, filtered
and concentrated under vacuum to afford the compound
2, which crystallized from EtOAc/Et2O as a white solid
(124 mg, 61%). Mp 84-86 ꢁC. 1H NMR (400 MHz,
DMSO-d6): 8.21 (d, 1H, H4, J ¼ 9:8 Hz), 8.02 (d, 1H,
13C NMR (50 MHz, CDCl3): 172.0 (COCbz), 160.0 (C1),
0
136.6 (Carom), 128.4 (Carom), 128.0 (Carom), 102.6 (C4 ),
0
66.6 (CH2Ph), 61.5 (C5 ), 40.4, 39.3, 33.9, 31.1, 26.1,
0
24.6, 15.4 (C6 ). IR (CHCl3), m (cmꢀ1) 1711 (C@O
amide), 1668 (C@O carbamate), 1220 (C–O). Anal.
Calcd for C20H32N2O5 (380.23): C, 63.13; H, 8.48; N,
7.36. Found: C, 63.25; H, 8.51; N, 7.28.
4.1.5. 4-Amino-N-(40,40-diethoxybutyl)butanamide (5).
Pd/C (10%, 0.44 g) and ammonium formate (0.46 g,
7.22 mmol) were added to a solution of 8 (0.80 g,
2.11 mmol) in methanol (20 mL). After stirring at rt until
the starting material has disappeared, the catalyst was
removed by filtration on Celite and washed with MeOH
(5 · 10mL). The residue was concentrated under vacuum
to afford a colorless oil (518 mg, 99%), which we used
without further purification. 1H NMR (300 MHz,
0
H5 , J ¼ 2:4 Hz), 7.88 (br t, 1H, –NH–, J ¼ 5:4 Hz), 7.79
0
CDCl3): 6.09 (br s, 1H, NH), 4.47 (t, 1H, H4 ,
(br t, 1H, –NH–, J ¼ 5:5 Hz), 7.34 (s, 1H, H8), 7.28 (d,
0
0
0
J ¼ 5:2 Hz), 3.63 (m, 2H, H5 ), 3.47 (m, 2H, H5 ), 3.25
1H, H4 , J ¼ 2:4 Hz), 6.29 (d, 1H, H3, J ¼ 9:8 Hz), 5.98
0
00
00
00
(m, 2H, H1 ), 2.75 (t, 2H, H4, J ¼ 6:7 Hz), 2.24 (t, 2H,
(dd, 1H, H15 , J1 ¼ J2 ¼ 2:7 Hz), 5.65 (dd, 1H, H15
,
0
00
H2, J ¼ 7; 0 Hz), 1.81 (br s, 2H, NH2), 1.78 (m, 2H, H3 ),
13
J1 ¼ J2 ¼ 2:3 Hz), 4.55 (m, 1H, H12 ), 4.49 (t, 3H, H1 ,
0
0
00
00
00
1.60 (m, 4H, H3 and H2 ), 1.20 (t, 6H, H6 , J ¼ 7:0 Hz).
J ¼ 6:2 Hz), 3.05 (m, 5H, H5 , H9 , and H13 ), 2.56 (m,
0
C NMR (75 MHz, CDCl3): 172.7 (C1), 102.7 (C4 ),
0
0
0
0
0
0
1H, H13 ), 2.33 (t, 2H, H3 , J ¼ 7:3 Hz), 2.06 (m, 4H, H2
13
0
0
00 00 00
and H7 ), 1.61 (m, 2H, H6 ), 1.58 (m, 2H, H11 ), 1.46 (m,
61.4 (C5 ), 40.9, 39.2, 33.9, 31.1, 28.2, 24.6, 15.3 (C6 ). IR
(CHCl3), m (cmꢀ1) 3320 (–NH2), 1658 (C@O amide),
1560 (NH2).
00
2H, H10 ). C NMR (100 MHz, DMSO-d6): 171.5 (C4 ),
00
00
171.3 (C8 ), 169.9 (C16 ), 160.1 (C2), 157.6 (C9), 152.1
00
0
(C7), 148.6 (C5), 145.9 (C5 ), 139.5 (C4), 135.2 (C14 ),
00
00
121.3 (C15 ), 110.8 (C10), 112.3 (C3), 106.0 (C6), 105.5
0 00 00 00 00
(C4 ), 93.2 (C8), 76.9 (C12 ), 72.1 (C1 ), 38.6 (C5 or C9 ),
00 00 00 00 00 00
38.0 (C5 or C9 ), 33.3 (C7 or C11 ), 32.8 (C7 or C11 ),
4.1.6. N-[3-(4,4-Diethoxybutylcarbamoyl)-propyl]-4-(7-
oxo-7H-furo[3,2-g]chromen-4-yloxy)-butyramide
(9).
00 00 00 00 00
32.7 (C13 ), 31.7 (C3 ), 25.5 (C2 or C6 ), 25.4 (C2 or
DMAP (8 mg, 0.06 mmol) and EDCI (164 mg,
1.03 mmol) were added to a solution of acid 4 (268 mg,
0.93 mmol) in dry CH2Cl2 (15 mL) at ꢀ10 ꢁC. After
stirring for 20 min at ꢀ10 ꢁC, amine 5 (270 mg,
1.10 mmol) in dry CH2Cl2 (10 mL) was added. The
reaction was stirred at 10 ꢁC for 1 h 30 and 20 h at room.
The mixture was concentrated under vacuum and puri-
fied by column chromatography on alumina (CH2Cl2/
MeOH, 98/2) to afford 9 as a white solid (480 mg, 99%).
C6 ), 24.6 (C10 ). IR (CHCl3), m (cmꢀ1) 3480 (NH), 1756
(C@O lactone), 1731 (C@O coumarin), 1662 (C@O
amide). UV (EtOH), k (nm): 210 (e ¼ 28; 893), 250
(e ¼ 15; 399), 268 (e ¼ 14; 064), 310 (e ¼ 12; 100). Anal.
Calcd for C27H30N2O8 (510.20): C, 63.52; H, 5.92; N,
5.49. Found: C, 63.96; H, 6.28; N, 5.21.
00
00
1
Mp 116–118 ꢁC. H NMR (300 MHz, CDCl3): 8.13 (d,
4.2. Biology
0
1H, H4, J ¼ 9:5), 7.56 (d, 1H, H5 , J ¼ 2:3 Hz), 7.06 (s,
0
1H, H8), 6.96 (d, 1H, H4 , J ¼ 2:3 Hz), 6.76 (br s, 1H,
4.2.1. Cell cultures. HL-60 cells were grown in RPMI
1640 (Sigma Chemical Co.) supplemented with 15%
NH), 6.27 (br s, 1H, NH), 6.23 (d, 1H, H3, J ¼ 9:5 Hz),

3624
S. Ropp et al. / Bioorg. Med. Chem. 12 (2004) 3619–3625
heat-inactivated fetal calf serum (Seromed), 1% sodium
pyruvate. K1 cells were grown in complete DMEM
medium with phenol red supplemented with 8.6% heat-
inactivated fetal calf serum (Seromed), and Nutrient
Mixture F-12 (HAM). A375 cells were grown in DMEM
with phenol red supplemented with 15% heat-inacti-
vated fetal calf serum (Seromed). Penicillin–streptomy-
cin (1%) was added to the three media. Cells were
cultured at 37 ꢁC in a moist atmosphere with 5% carbon
dioxide.
Cytotoxicity data were expressed in IC50 values,
corresponding to the concentrations of the test agent
inducing 50% reduction of the measured parameter
(quantity of proteins. . .) compared with control cul-
tures.
4.2.4. Skin phototoxicity. Skin phototoxicity was tested
on BALB/c female mice according to the mouse ear
swelling test (MEST). An acetone/olive oil solution of
each compound (2.77 and 27.7 mM) was applied topi-
cally to the right ear of each mouse, the left ear was not
treated and used as an internal control. The animals
were kept in the dark for 1 h and then irradiated with a
UVA dose of 6 J cmꢀ2. After irradiation the ear’s
swelling was recorded every 24 h for six days.
4.2.2. Irradiation procedure. Irradiations were performed
with a Tecima-Dixwellꢂ block equipped with a mercury
vapor lamp Herausꢂ 400 W emitting between 320 and
400 nm. Irradiation intensity was checked on Dixwellꢂ
radiometers UVA-365 and UVB-312 before each
experiment.
Acknowledgements
4.2.3. Proliferation assays. Cells (4.5 · 103) were seeded
into each well of a 96-well cell culture plate. After 24 h of
incubation, various concentrations of the test agents in
DMSO solution were administered so that the final
DMSO concentration never exceeded 1%. Controls were
treated with the same concentration of DMSO in med-
ium solution.
Authors thank la Ligue Nationale contre le Cancer for a
fellowship to S.R. and the French MENRT for a fel-
lowship to J.G.
References and notes
After incubation for 5 h in the dark, the medium con-
taining phenol red was replaced with an equal volume of
PBS in the case of K1 and A375 cells and cells were
irradiated with a UVA dose of 0.8 J cmꢀ2. After irradi-
ation PBS was removed and cells were incubated in
DMEM medium for 60 h. A sulforhodamine B (SRB)
test was performed 64 h after irradiation to assess cell
viability of K1 and A375 cells. Cells in 96-well plates
were washed with PBS, fixed with 10% ice-cold trichlo-
roacetic acid at 4 ꢁC for 1 h, then washed with water five
times and dried at room temperature. The cellular pro-
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in 1% acetic acid at room temperature for 20 min and
then washed with 1% acetic acid four times and dried at
37 ꢁC for another 30 min. To dissolve the SRB bound to
cellular protein, 200 lL of 10 mM Tris were added to
each well and incubated at room temperature with
mechanical agitation until the color became homoge-
neous. SRB bound to protein was measured by absor-
bance at a 550 nm wavelength using MRX microplate
reader (Dynech, France).
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