Synthesis of new Targretin analogues that induce apoptosis in leukemia HL-60 cells

Article abstract of DOI:10.1016/S0960-894X(02)00803-X

Four new analogues of Targretin where the carboxylic acid function was replaced by an N,N-dimethyl-S-aryl carbamate or N,N-dimethyl-O-arylthiocarbamate function, were synthesized. Compounds 5, 6 and 7 have shown to be more potent than the parent compound

Full text of DOI:10.1016/S0960-894X(02)00803-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3529–3532
Synthesis of New Targretin1 Analogues that Induce Apoptosis
in Leukemia HL-60 Cells
Jean-Yves Winum,a,* Stephen Baghdiguian,b Therese Commes,c
Alain Leydeta and Jean-Louis Monteroa,*
a
´
´
Laboratoire de Chimie Biomoleculaire, UMR 5032, Universite Montpellier II - CNRS - Laboratoires Mayoly Spindler,
ENSCM, 8 Rue de l’Ecole Normale, 34296 Montpellier Cedex, France
b
´
Laboratoire de Dynamique Moleculaire des Interactions Membranaires, UMR 5539 Universite Montpellier II – CNRS,
´
Place E. Bataillon, 34095 Montpellier Cedex, France
´ ´
Institut de Genetique Humaine, UPR CNRS 1142, 34396 Montpellier Cedex, France
c
Received 11 March 2002; accepted 20 September 2002
Abstract—Four new analogues of Targretin where the carboxylic acid function was replaced by an N,N-dimethyl-S-aryl carbamate
or N,N-dimethyl-O-arylthiocarbamate function, were synthesized. Compounds 5, 6 and 7 have shown to be more potent than the
parent compound to induce apoptosis of HL-60 cells.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
Retinoids regulate many important biological processes
such as mediation of cell growth and differentiation in
both normal and neoplastic cells, and modulation of
programmed cell death also known as apoptosis.1 The
broad spectrum of physiological activities of retinoids is
mediated by two types of receptors, the retinoic acid
receptors (RAR a,b,g) and the retinoid X receptors
(RXR a,b,g). All-trans retinoic acid (RA) binds only
RAR’s, while 9-cis RA is an agonist for both RAR’s
and RXR’s.1,2 These two compounds have a significant
implication for the treatment of dermatological dis-
eases, and cancers. They induce remissions in high pro-
portion of patients with acute promyelocytic leukemia
(APL).3 But side effects due to their ability to activate
multiple retinoid receptors provide for identifying reti-
noids receptor selective having specific cancer chemo-
protective and chemotherapeutic activities without
concomitant unfavorable side effects. Recently, LGD-
1069 (Targretin1) (Fig. 1) was identified as a highly
selective RXR agonist with low affinity for RAR’s.
This compound, now in clinical trials, has proven its
Figure 1. Targretin and its synthesized analogues.
efficiency for the treatment of mammary adenocarci-
nome, and cutaneous T-cell lymphoma.4
The goal of this study was to investigate the influence of
new polar heads on the activity of Targretin1 against
human promyelocytic leukemia HL-60 cell line.5 It
appears interesting to synthesize analogues in which
carboxylic acid function was replaced respectively by
O-arylthiocarbamate and S-arylcarbamate moiety (Fig. 1).
The different compounds have been evaluated on their
capacity to induce apoptosis of leukemia HL-60 cells.
Chemistry
Synthetic routes to the different analogues are outlined
in Figure 2. Following literature protocols,6 starting
derivative 1 was synthesized in high yield. Synthesis of
*Corresponding authors. Tel./fax: +33-467144343; e-mail: winumj@
univ-montp2.fr (J.-Y. Winum); montero@univ-montp2.fr (J.-L.
Montero).
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00803-X

3530
J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3529–3532
bacterial and viability (higher than 95%) was deter-
mined by trypan blue exclusion. Treatment of cells with
0.02% DMSO as control (a concentration achieved in
cells treated with 2 mM retinoids) did not result in any
significant effect on inhibition of cell growth or on
induction of apoptosis.
Retinoid stock solutions in dimethylsulfoxide were
stored at À20 ꢀC. Cells treated with retinoids (2 mM)
were seeded at a density of 2Â105 cells/mLand were
collected 4 days or 7 days later.
For hipodiploid measurement, cells (2.106 cells/assay)
were washed twice in PBS and permeabilized with
75% ethanol for 2 min at 4 ꢀC. The cells were incu-
bated overnight at 4 ꢀC with propidium iodide (40
mg/mL) and RNAse A (100 mg/mL) in PBS. Flow
cytometric analysis of retinoid-treated cells was done
on a FACSCAN flow cytometer. Fluorescence data
are expressed by percent of cells. Apoptotic cells were
the cells with a fluorescence value down to the Go/G1
pic. Research products: culture media were from Life
Technologies.
Figure 2. Synthesis of the different analogues starting from compound
1. Reagents and conditions: (a) p-methoxybenzoyl chloride, AlCl3
70%; (b) BBr3, CH2Cl2, À60 ꢀC, 98%; (c) Tebbe reagent 80%; (d) N,N-
dimethylthiocarbamoyl chloride, DABCO, DMF 70–75%; (e) 200 ꢀC,
8 h, 70–95%.
Targretin analogues was achieved from 1 by straight-
forward high yielding methods in four or five steps.
The Friedel and Craft acylation of 1 with p-methoxy-
benzoyl chloride provided compound 2, which after
treatment with boron tribromide at À60 ꢀC gave phenol
3 in good yield. Olefination of 3 with Tebbe reagent7
afforded the derivative 4. The S-arylcarbamate ana-
logues 6 and 8 were synthesized starting from the com-
Detection of Apoptosis by Confocal Microscopy
Four days after addition of retinoid analogues, cells
were collected using Cytospin cell preparation system
(Shandon, Pittsburgh, PA) and TUNELstained
according to the manufacturer recommendations
(Boehringer Mannheim). Positive controls were per-
formed by incubation of HL-60 cells with 0.5 mg/mL of
DNAse1 (Sigma) at room temperature for 10 min prior
TUNELstaining. DNAse1-treated cells incubated with
fluorescein-labeled nucleotide mixture, without addition
of terminal deoxynucleotidyl transferase (TdT), were used
as negative controls. The TUNEL-labelled cells were also
stained with DAPI (in order to visualize all nuclei)
and TRITC-phalloidin (for F-actin detection). The
slides were observed in a Leica TCS 4D Laser Con-
focal Microscope. Fluorescence confocal images
(TUNELand F-actin) were acquired simultaneously
using a 25X NA 0.75 Phaco 2 oil-immersion lens. In
some cases DAPI fluorescence images for the same
microscope field were also recorded using a Hitashi
KP-161 CCD camera.
pounds
3
and
4
using the Newman–Kwart
rearrangement.8 Treatment of 3 and 4 with N,N-di-
methylthiocarbamoyl chloride and DABCO led to the
O-arylthiocarbamates 5 and 7. Thermic rearrangement
at 200 ꢀC afforded the S-aryl carbamate 6 and 8. All
new compounds were fully characterized by usual spec-
troscopy methods.9 The O-arylthiocabamate and
S-arylcarbamate can be easily distinguished by 1H
NMR spectroscopy. Indeed, the 6 protons of the
N,N-dimethyl moiety appeared as a singlet in the case of
S-arylcarbamate and as a doublet for the O-arylthio-
carbamate derivatives when spectra are recorded at
room temperature.
Biological Assays
Compounds 5, 6, 7 and 8 were examined for their ability
to induce apoptosis on HL-60 cell line.
Quantitative Analysis
Materials and Methods
Three fields for each experimental condition were cho-
sen by random sampling and examined by fluorescence
microscopy (F-actin, red fluorescence and TUNEL
labelling, green fluorescence). The total number of cells
(F-actin labelling) and the number of TUNELpositive
nuclei were counted on digitized images using Image
Tool software. The results were expressed as percent of
the estimated total number of cells present in the field.
The main advantage of this method is in the significance
of the results that can be achieved. Our previous work
has proven that this methodology is reliable even with
very low level of apoptosis (<1%).10
Cell line and assessment of apoptosis by flow cytometry
HL-60 cell line (American Type Culture Collection,
CCL240 Rockville, USA) is a human myelomonocytic
cell line arrested at a myeloblastic stage. The cell line
was routinely cultured at 37 ꢀC, 5% CO2 in complete
medium (RPMI 1640–10% FCS (Life Technologies,
Cergy-Pontoise, France). Cell cultures were checked for
the absence of mycoplasma contaminations (Detection
kit, Boehringer Mannheim, Meylan, France), and per-
formed without antibiotics to avoid masking silent

J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3529–3532
3531
Figure 3. (a) Changes in the morphology of nucleus and actin fibers during apoptosis induced by TA (compound 6). 3, Phalloidin staining; 4,
TUNELstaining; 5, DAPI staining. 1 and 2 represent TUNELpositive and negative control, respectively. Scale bars: 100 mm (1, 2); 10 mm (3–5); (b)
induction of genomic DNA-nicking in apoptotic HL-60 cells treated with 2 mM Targretin (T) or Targretin analogues (TA) for 4 days. Cytospins
were prepared from control (1), T (2) or TA-treated cells (3: compound 8, 4: compound 7, 5: compound 6, 6: compound 5), and the TUNELassay
counterstained with rhodamine-phalloidin. Labeling of the apoptosis-specific DNA strand breaks appears in green, F-actin in red (scale bar, 70 mm);
(c) estimation of apoptosis using quantitative confocal microscopy (1: control, 2: Targretin, 3: compound 8, 4: compound 7, 5: compound 6, 6:
compound 5).
Results and Discussion
while the compound 8 is less effective (Fig. 3c). These
quantitative results were confirmed by flow cytometry
(Fig. 4) and were consistent with the levels of apoptosis
previously found in HL-60 cells treated with other reti-
noid analogues.6
The therapeutic actions of retinoids are due to their
ability to regulate cellular process such as cellular dif-
ferentiation, proliferation and modulation of apop-
tosis.1 To test if new synthesized Targretin analogues
induce apoptosis in HL-60 leukemia cells, DNA frag-
mentation analysis was performed. HL-60 cells incu-
bated in defined media and treated with 2 mM of
Targretin (T) or Targretin analogues (TA) demon-
strated a clear induction of apoptosis when analyzed by
TUNELlabeling and propidium iodide incorporation.
These results have been obtained through two indepen-
dent methods: quantitative confocal microscopy (Fig.
3b and c) and flow cytometry (Fig. 4), were conducted
under a double blind protocol. Fluorescence microscopy
observation of T and TA-treated HL-60 cells showed
typical characteristics of apoptosis including morpho-
logical change of actin microfilament11 (Fig. 3, a3
arrow), genomic DNA cleavage (Fig. 3, a4 arrow) and
nuclear fragmentation (Fig. 3, a5 arrow). Control cells
growing exponentially showed very limited TUNEL
labeling (Fig. 3, b1), whereas T and TA-treated cells
showed numerous TUNELpositive cells ( Fig. 3b 2–6).
The most potent inducer’s of apoptosis is the compound
6. In addition, the compounds 7, 6 and 5 induce levels of
apoptosis significantly higher (P<0.05) than the targretin
Figure 4. Estimation of apoptosis using flow cytometry (1) control,
(2) Targretin, (3) compound 8, (4) compound 7, (5) compound 6,
(6) compound 5.

3532
J.-Y. Winum et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3529–3532
Rizvi, N. A.; Marshall, J. L.; Dahut, W.; Ness, E.; Truglia,
Conclusion
J. A.; Loewen, G.; Gill, G. M.; Ulm, E. H.; Geiser, R.; Jau-
nakais, D.; Hawkins, M. J. Clin. Cancer Res. 1999, 5, 1658. (c)
Bedikian, A. Y.; Plager, C.; Papadopoulos, N.; Ellerhorst, J.;
Smith, T.; Benjamin, R. S. Oncol. Rep 2000, 7, 883 and refer-
ences therein.
5. Kizaki, M.; Dawson, M. I.; Heyman, R.; Elstner, E.; Mor-
oseti, R.; Pakkala, S.; Chen, D. L.; Ueno, H.; Chao, W.;
Morikawa, M.; Ikeda, Y.; Heber, D.; Pfahl, M.; Koeffler, H. P.
Blood 1996, 87, 1977.
6. Boehm, M. F.; Zhang, L.; Zhi, L.; McClurg, M. R.; Berger,
E.; Wagoner, H.; Mais, D. E.; Suto, C. M.; Davies, P. J. A.;
Heyman, R. A.; Nadzan, A. M. J. Med. Chem. 1995, 38, 3146
and references therein.
7. Tebbe, F. N.; Parshall, G. W.; Reddy, G. S. J. Am. Chem.
Soc. 1978, 100, 3611.
In summary, we report the synthesis of new analogues
of Targretin. The replacement of the carboxylic func-
tion by N,N-dimethylcarbamate or N,N-dimethylthio-
carbamate functions led to compounds which induce
apoptosis of HL-60 cells in higher level than the parent
compound. We show that the polar carboxylic function
which was thought to be essential for activity1,2 could be
replaced by non polar functions leading to more potent
compounds. These preliminary results are quite
encouraging and make compounds 5, 6, and 7 good
candidates for further biological investigations. This
work is currently underway to determine by which
nuclear receptors are mediated the effects of these com-
pounds and will be reported in due courses.
8. (a) Newman, M. S.; Karnes, H. A. J. Org. Chem. 1966, 31,
3980. (b) Kwart, H.; Evans, R. E. J. Org. Chem. 1966, 31, 410.
9. All new compounds were characterized by 1H NMR and
FAB mass spectrometry. Elemental values (C, H, O) were
1
Acknowledgements
within 0.4% of the theorical values. H NMR data of selected
1
compounds: Compound 5: H NMR (300 MHz, CDCl3) d 7.9
(d, J=12 Hz, 2H), 7.25 (s, 1H), 7.20 (s, 1H), 7.15 (d, J=12
Ministere de l’Enseignement Superieur et de la
Recherche (doctoral fellowship J-Y.W.), Association de
Recherche sur le Cancer (ARC Grants Nos. 9519 and
5761), and the Laboratoires Mayoly Spindler are grate-
fully acknowledged for support of this work.
Hz, 2H), 3.4–3.5 (2s, 6H), 2.35 (s, 3H), 1.7 (s, 4H), 1.3 (s, 6H),
1
1.2 (s, 6H). Compound 7: H NMR (300 MHz, CDCl3) d 7.35
(d, J=8 Hz, 2H), 7.15 (s, 1H), 7.05 (s, 1H), 6.95 (d, J=8 Hz,
2H), 5.75 (d, J=2 Hz, 1H), 5.2 (d, J=2 Hz, 1H), 3.35–3.5 (2s,
6H), 2 (s, 3H), 1.7 (s, 4H), 1.3 (s, 6H), 1.25 (s, 6H). Compound
6: 1H NMR (300 MHz, CDCl3) d 7.85 (d, J=8 Hz, 2H), 7.6 (d,
J=8 Hz, 2H), 7.25 (s, 1H), 7.2 (s, 1H), 3.1 (s, 6H), 2.3 (s, 3H),
1
References and Notes
1.75 (s, 4H), 1.3 (s, 6H), 1.2 (s, 6H). Compound 8: H NMR
(300 MHz, CDCl3) d 7.45 (d, J=8 Hz, 2H), 7.3 (d, J=8 Hz,
2H), 7.15 (s, 1H), 7.1 (s, 1H), 5.8 (d, J=2 Hz, 1H), 5.25 (d,
J=2 Hz, 1H), 3.1 (s, 6H), 2 (s, 3H), 1.7 (s, 6H), 1.25 (s, 6H).
10. Baghdiguian, S.; Martin, M.; Richard, I.; Pons, F.; Astier,
C.; Bourg, N.; Hay, R. T.; Chemaly, R.; Halaby, G.; Loiselet,
J.; Anderson, L. V.; Lopez de Munain, A.; Fardeau, M.;
Mangeat, P.; Beckmann, J. S.; Lefranc, G. Nature Med. 1999,
5, 503.
1. Sporn, M. B.; Robert, A. B.; Goodman, O. S. The Reti-
noids: Biology, Chemistry and Medicine, 2nd ed.; Raven Press:
New-York, 1994.
2. (a) Gale, J. Progress in Medicinal Chemistry 1993, 30, 1. (b)
Curley, R. W., Jr.; Robarge, M. J. Current Medicinal Chem-
istry 1996, 3, 325.
3. Slack, J. L.; Rusiniak, M. E. Ann. Hematol. 2000, 79, 227.
4. (a) Bischoff, E. D.; Gottardis, M. M.; Moon, T. E.; Hey-
man, R. A.; Lamph, W. W. Cancer Res. 1998, 58, 479. (b)
11. Iguchi, K.; Usami, Y.; Hirano, K.; Hamatake, M.; Shi-
bata, M.; Ishida, R. Biochem. Pharmacol. 1999, 57, 1105.