Inhibition of P-glycoprotein-mediated multidrug efflux by aminomethylene and ketomethylene analogs of reversins

Article abstract of DOI:10.1016/j.bmcl.2006.07.059

Several aminomethylene analogs and a ketomethylene analog of reversins were synthesized in order to evaluate their ability to inhibit P-glycoprotein-mediated drug efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein. These analogs retained

Full text of DOI:10.1016/j.bmcl.2006.07.059

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5700–5703
Inhibition of P-glycoprotein-mediated multidrug efflux by
aminomethylene and ketomethylene analogs of reversins
Ali Koubeissi,a Imad Raad,a Laurent Ettouati,a,* David Guilet,a
Charles Dumontetb and Joelle Parisa,*
aUniversite´ Claude Bernard Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques,
´
`
´
EA 3741 Ecosystemes et Molecules bioactives, 69373 Lyon cedex 08, France
bUniversite´ Claude Bernard Lyon 1, Faculte´ de Me´decine, Laboratoire de Cytologie analytique, INSERM U590,
69373 Lyon cedex 08, France
Received 28 June 2006; revised 17 July 2006; accepted 17 July 2006
Available online 6 September 2006
Abstract—Several aminomethylene analogs and a ketomethylene analog of reversins were synthesized in order to evaluate their
ability to inhibit P-glycoprotein-mediated drug efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein. These ana-
logs retained good activity compared to cyclosporin A and the original reversins.
Ó 2006 Elsevier Ltd. All rights reserved.
Multidrug resistance (MDR) to anticancer agents
remains a major cause of treatment failure in cancer che-
motherapy. MDR describes the cross-resistance of
tumor cell lines to several structurally unrelated chemo-
therapeutic agents after exposure to a single cytotoxic
drug. This phenomenon is often associated with overex-
pression of several proteins.1 Among them P-glycopro-
tein (Pgp) is the most important one that belongs to
the ABC superfamily of transporters which acts as a
drug efflux pump.2,3
groups.6 Among them reversin 121 1a showed the high-
est affinity and specificity for Pgp.
Numerous molecules have shown some activity on Pgp.4
Among them short linear hydrophobic peptides were
described as chemosensitizers.5a Sepro˜di et al. showed
that small hydrophobic peptide derivatives modulate
Pgp-ATPase activity and inhibited the drug extrusion
function of Pgp.5b,c These compounds are a family of
di- and tripeptide derivatives sharing some common
physico-chemical and structural features. Some of them
are dimerized aminoacids from diacid derivatives. The
enhanced affinity to Pgp of these chemosensitizers finally
coined reversins is ascribed to the hydrophobic nature of
the side chains protected with bulky aromatic or alkyl
At 1–2 lM this reversin was more effective than cyclo-
sporin A for blocking colchicine transport in isolated
membranes and reconstituted systems. In order to im-
prove proteolytic stability and bioavailability of rever-
sins, we planned to synthesize aminomethylene and
Keywords: Multidrug resistance; P-glycoprotein; Reversin; Amino-
methylene; Ketomethylene; Pseudopeptide; Chemosensitizer.
*
Corresponding authors. Tel.: +33 4 78 77 70 82; fax: +33 4 78 77 75
49; e-mail addresses: laurent.ettouati@univ-lyon1.fr; joelle.paris@
univ-lyon1.fr
Scheme 1. Reagents and condition: (a) NaBH3CN, MeOH, AcOH, rt,
1 h, 52% (n = 1) and 57% (n = 2).
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.059

A. Koubeissi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5700–5703
5701
ketomethylene analogs of typical reversin representa-
tives. We chose reversin 121 1a as our working model
for dipeptide-type reversins and reversin 213 2a as a
dimerized aminoacid containing succinyl unit. The re-
duced analogs 3a–b were synthesized using the classical
reductive amination strategy7 starting from chiral pool-
derived aminoaldehydes according to Scheme 1.8
Figure 1. Structures of by-products 9 and 10 obtained respectively in
the reductive amination of 7b and 7c with aldehyde 6.
tamic acid derivative 7c (Fig. 1).13 Undescribed deriva-
tives 1b and 1c along with compound 2b were also
synthesized by standard procedures for sake of
comparison.
We thought it would be worthwhile to test the intro-
duction of a spacer between the Asp and Lys residue
of reversin 121 1. Thus, we chose to synthesize the
protected Asp-w(CO–CH2)Gly-Lys ketomethylene ana-
log 16 as outlined in Scheme 3. The synthesis started
from L-homoserine which was protected on the side
chain as a tert-butyl dimethyl silyl ether followed by
protection of the free a-amine as tert-butyloxycarbon-
yl derivative and finally transformed in Weinreb amide
11 in 40% overall yield over three steps. The alkene 12
was then obtained from Weinreb amide 11 by alkyl-
ation with butenyl magnesium bromide as described14
in 55% yield. Transformation of the protected hydrox-
yl of 12 in benzyl ester 13 was carried out by oxida-
tion of the deprotected hydroxyl with PDC and then
esterification of the cesium salt in 53% overall yield
as described.15a,15b Oxidation of alkene 13 with Ru-
Cl3ÆxH2O/NaIO4 afforded free acid 14 in 96% yield.
The ketomethylene analog 16 was finally obtained by
coupling the succinimidyl derivative 15 and diprotect-
ed L-lysine hydrochloride in 94% yield.
As far as reduced analogs of reversin 213 2a were
concerned we started from 4-pentenoic acid which was
activated as succinimidyl ester and then coupled to L-
glutamic acid dibenzyl ester tosylate salt to obtain the
amide 4 (Scheme 2). The corresponding aldehyde 6
was obtained by a two-step reaction with osmium
tetroxide/4-methylmorpholine N-oxide9 followed by
oxidative cleavage of the diol 5 with sodium periodate.
It is noteworthy that aldehyde 6 exists in equilibrium
with the corresponding hemiaminals 60 as previously de-
scribed in similar reactions.10a,b Structure of the diaste-
reoisomeric hemiaminals 60 was assessed by ESIMS
and HSQC and HMBC experiments.10c The last step
consisted in obtaining the reduced analogs 8a–c of
reversin 213 2a. However in the standard reductive ami-
nation conditions, the reaction between diprotected
L-glutamic acid 7b and aldehyde 6 did not afford the cor-
responding secondary amine but instead the pyrrolidi-
none 9 in 38% yield (Fig. 1). This result is explained
by nucleophilic attack of the generated secondary amine
on the adjacent benzyl ester side chain as observed
for similar derivatives.11 So as to avoid cyclization,
several protected derivatives of L-glutamic acid such as
the 2,4-dimethyl-3-pentyl (Dmp)12a and cyclohexyl
(cHex)12b esters 7c and 7d12c known to prevent asparti-
mide formation were used to obtain, respectively, ana-
logs 8b and 8c. Moreover by using an excess of
aldehyde 6 a double addition product 10 was obtained
in 47% yield in the case of reductive amination of L-glu-
The efficiency of reversin analogs to inhibit Pgp-medi-
ated daunorubicin efflux was investigated by monitoring
the intracellular accumulation of this drug in K562/R7
human leukemic cells overexpressing Pgp in the presence
of daunorubicin.16a Cyclosporin A was used as a posi-
tive control (Table 1). These analogs showed strong
inhibitory activity comparable to that of reversin 121
1a except in the case of the tertiary amine 10 which
caused an important decrease in activity. It was note-
worthy that the replacement of the amide bond with
Scheme 2. Reagents and conditions: (a) N-hydroxysuccinimide, DMAP, DCC, THF, 0 °C, 10 min then rt, 48 h, 94%; (b) DIEA, DMF, rt, 24 h,
quant yield; (c) OsO4 2.5% in t-BuOH, NMO, THF, H2O, rt, 24 h, 86%; (d) NaIO4, THF, H2O, 3 h, quant yield; (e) NaBH3CN, MeOH, AcOH, rt,
1–2 h.

5702
A. Koubeissi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5700–5703
Scheme 3. Reagents and conditions: (a) TBDMSCl, DBU, CH3CN, 0 °C then rt, 24 h, 82%; (b) (Boc)2O, Et3N, acetone, H2O, rt, 24 h, 80%; (c) N,O-
dimethylhydroxylamine hydrochloride, TBTU, HOBt, DIEA, CH2Cl2, rt, 12 h, 61%; (d) butenyl magnesium bromide, THF, ꢀ78 °C then 3.5 h, rt,
30 min, 55%; (e) TBAF, THF, rt, 1 h, 87%; (f) PDC, DMF, rt, 3 h, 71%; (g) i—Cs2CO3, MeOH, H2O then reduced pressure; ii—benzyl bromide,
DMF, rt, 4.5 h, 86%; (h) RuCl3ÆxH2O NaIO4, H2O, CH3CN, rt 1 h, 96%; (i) N-hydroxysuccinimide, DMAP, DCC, THF, 0 °C, 10 min then rt, 48 h,
49%; (j) DIEA, DMF, rt, 24 h, 94%.
Table 1. Mean inhibitory activity (%) of reversins and synthesized
analogs on human leukemic cells K562/R716b
References and notes
Compounda
Mean inhibiting activityb
%
1. Gottesman, M. M.; Fojo, T.; Bates, S. E. Nat. Rev. Cancer
2002, 2, 48.
2. Gottesman, M. M.; Pastan, I.; Ambudkar, S. V. Curr.
Opin. Genet. Dev. 1996, 6, 610.
1a Reversin 121
72.73 ( 5.27)
52.70 ( 5.27)
85.43 ( 5.69)
1b
1c
´
3. Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-
Genthe, C.; Gottesman, M. M. Nat. Rev. Drug Disc. 2006,
5, 219.
4. Teodori, E.; Dei, S.; Scapecchi, S.; Gualtieri, F. Il Farmaco
2002, 57, 385.
5. (a) Sharom, F. J.; DiDiodato, G.; Yu, X.; Ashbourne, K.
J. D. J. Biol. Chem. 1995, 270, 10334; (b) Sepro¨di, J.;
2a Reversin 213
93.74 ( 2.17)
94.45 ( 1.36)
74.14 ( 4.06)
79.08 ( 5.13)
82.22 ( 4.57)
54.71 ( 3.63)
98.33 ( 1.35)
3.35 ( 3.44)
86.62 ( 4.86)
2b
3a
3b
8a
8b
8c
10
16
´
´
´
Mezo˜, I.; Vadasz, Zs.; Szabo, K.; Sarkadi, B.; Teplan, I.
In Peptides 1996; Proceedings of the 24th European
Symposium, 1996, Edimburgh; Ramage, R., Epton, R.,
Eds; Mayflower Scientific Ltd: Kingswinford, 1998; pp
a Compounds were tested at a 10 lM concentration.
´
´
´
801–802.; (c) Vadasz, Zs.; Szabo, K.; Sarkadi, B.; Teplan,
b Cyclosporin A was used as positive control (mean inhibitory activity
of 100%) at a final concentration of 2 lM. Standard deviation is
given in parentheses.
´
´
I.; Mak, M.; Miklos, I.; Gyo¨rffy, E.; Sepro¨di, J. In
Peptides 1998; Proceedings of the 25th European Sympo-
sium, 1998, Budapest; Bajusz, S., Hudecz, F., Eds;
Akademiai Kiado Ed: Budapest, 1999; pp 640–641.
6. Sharom, F. J.; Yu, X.; Lu, P.; Liu, R.; Chu, J. W. K.;
´
´
Szabo, K.; Muller, M.; Hose, C. D.; Monks, A.; Varadi,
¨
an amino methylene isostere did not impair the capacity
to inhibit Pgp-mediated drug efflux (1a vs 3a and 2b vs
8c). Analogs with cyclohexyl ester presented an in-
creased inhibitory activity when compared to 2,4-di-
methyl-3-pentyl ester analogs (1b vs 1c and 8b vs 8c).
Moreover, exchanging benzyl ester by cyclohexyl ester
was tolerated as shown in Table 1 by comparing activity
for compounds 2a and 2b. Finally, the inhibitory activ-
ity of ketomethylene analog 16 was conserved indicating
that inserting a succinyl moiety into reversin 121 1a was
not detrimental to the Pgp-mediated drug efflux inhibi-
tory activity.
A. E.; Sepro¨di, J.; Sarkadi, B. Biochem. Pharmacol. 1999,
58, 571.
7. Martinez, J.; Bali, J.-P.; Rodriguez, M.; Castro, B.;
Magous, R.; Laur, J.; Lignon, M.-F. J. Med. Chem.
1985, 28, 1874.
8. The aminoaldehydes were synthesized by reduction of
aminoacids-derived mixed anhydrides with NaBH4 and
then oxidized by Swern oxidation.
9. VanRheenen, V.; Kelly, R. C.; Cha, D. Y. Tetrahedron
Lett. 1976, 17, 1973.
10. (a) Baldwin, J. E.; Hulme, C.; Edwards, A. J.; Schofield,
C. J.; Parkes, K. E. B. Tetrahedron Lett. 1993, 34, 1665;
(b) Delcros, J.-G.; Tomasi, S.; Carrington, S.; Martin, B.;
Renault, J.; Blagbrough, I. S.; Uriac, P. J. Med. Chem.
2002, 45, 5098; (c) Spectral data of diastereoisomeric
hemiaminals 60: IR (film, m, cmꢀ1): 3412 (OH and NH),
3066 (arom CH), 2954 (al CH), 1738 (C@O ester), 1674
(CO amide). ESIMS (negative mode, NaCl): 856,9
We have designed aminomethylene and ketomethylene
analogs with inhibitory activity of Pgp-mediated drug
efflux comparable to that of reversins 121 1a and 213
2a. We envisage now the synthesis of ketomethylene
analogs of reversin 121 and modified side chain analogs
to confirm and refine our first results.
1
(2M+Clꢀ), 446,0 (M+Clꢀ). H NMR (300 MHz, CDCl3,
d ppm): 7.36 (20H, m, 4-Phe), 5.31 (2H, m, 2 CHOH), 5.18

A. Koubeissi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5700–5703
5703
(4H, m, 2 CH2O Bn), 5.12 (4H, m, 2 CH2O Bn), 4.77 (1H,
dd, J = 4.5 and 9.8 Hz, CHa), 4.64 (1H, t, J = 7.1 Hz,
CHa), 4.32 (1H, d, J = 4.5 Hz, exch OH), 3.41 (1H, d,
J = 7.8 Hz, exch OH), 1.8–2.8 (16H, m, 2 –CH2CH2– and
2 –CH2CH2– Glu). 13C NMR (75 MHz, CDCl3, d ppm):
176.3 (C@O), 175.8 (C@O), 173.7 (C@O), 173.1 (C@O),
172.8 (C@O), 171.0 (C@O), 136.0 (Cq Phe), 135.9 (Cq
Phe), 135.5 (Cq Phe), 135.2 (Cq Phe), 129.1 (CH–Phe),
129.07 (CH–Phe), 129.02 (CH–Phe), 128.9 (CH–Phe),
128.8 (CH–Phe), 128.7 (CH–Phe), 128.6 (2 CH–Phe), 84.4
(CHOH), 82 (CHOH), 68.3 (CH2O), 67.9 (CH2O), 67.1
(CH2O), 67.0 (CH2O), 54.7 (CHa), 54.3 (CHa), 31.3
(CH2), 30.0 (CH2), 29.5 (CH2), 29.3 (CH2), 29.0 (CH2),
28.8 (CH2), 25.7 (CH2), 24.6 (CH2).
benzyl bromide and cesium carbonate in MeOH/H2O in
70% yield and then Boc deprotected in the same condi-
tions as previously in 96% yield.
13. See Salvi, J. P.; Walchshofer, N.; Paris, J. Tetrahedron
Lett. 1994, 35, 1181, for similar formation of double
condensation product in reductive amination reactions.
14. Kaiser, M.; Siciliano, C.; Assfalg-Machleidt, I.; Groll, M.;
Milbradt, A. G.; Moroder, L. Org. Lett. 2003, 5, 3435.
˚
15. (a) Vabenø, J.; Nielsen, U.; Ingebrigtsen, T.; Lejon, T.;
Steffansen, B.; Luthman, K. J. Med. Chem. 2004, 47, 4755;
(b) As racemization of a similar benzylated ester was
noted in the cited reference 15a the enantiomeric purity of
13 was checked and found to be enantiomerically pure by
C18 RP-HPLC analysis after deprotection (TFA/CH2Cl2)
and derivatization with GITC.
11. Gareau, Y.; Zamboni, R.; Wong, A. W. J. Org. Chem
1993, 58, 1582.
16. (a) Comte, G.; Daskiewicz, J.-P.; Bayet, C.; Conseil, G.;
Viornery-Vanier, A.; Dumontet, C.; Di Pietro, A.; Barron,
D. J. Med. Chem. 2001, 44, 763; (b) One million K562/R7
human leukemic cells expressing high levels of P-glyco-
protein were incubated for 1 h at 37 °C in 1 mL of RPMI
1640 medium containing a final concentration of 10 lM
daunorubicin, in the presence or absence of inhibitor. The
cells were then washed twice with ice-cold phosphate-
buffered saline (PBSt) and kept on ice until analysis by
flow cytometry on a FACS-II. Assays were performed in
duplicate, with at least three separate experiments.
´
12. (a) Karlstro¨m, A. H.; Unden, A. E. Tetrahedron Lett.
1995, 36, 3909; (b) Tam, J. P.; Wong, T.-W.; Riemen, M.
W.; Tjoeng, F.-S.; Merrifield, R. B. Tetrahedron Lett.
1979, 42, 4033; (c) H-Glu(ODmp)-OBn 7c was synthesized
from commercial NaBoc-Glu-OBn by side-chain esterifi-
cation with DCC/DMAP and 2,4-dimethyl-3-pentanol in
dichloromethane in 70% yield and then Boc-deprotected
with conc H2SO4 in AcOEt in 95% yield. H-Glu(OcHex)-
OBn 7d was obtained from commercial NaBoc-Glu(O-
cHex)-OH by esterification of the a-carboxylic acid with