Synthesis and bioevaluation of 22-hydroxyacuminatine analogs

Article abstract of DOI:10.1016/j.bmcl.2008.01.082

A series of 22-hydroxyacuminatine analogs was prepared by using different Friedlaender condensations. Several of the new compounds were tested for antiproliferative activity on cancer cell lines and for topoisomerase I inhibitory activity.

Full text of DOI:10.1016/j.bmcl.2008.01.082

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2143–2146
Synthesis and bioevaluation of 22-hydroxyacuminatine analogs
a
a
b
a
´
´
´
Franc¸ois Grillet, Barbora Baumlova, Gregoire Prevost, Jean-Franc¸ois Constant,
Sophie Chaumeron,b Dennis C. H. Bigg,b Andrew E. Greenea and Alice Kanazawaa,*
aDe´partement de Chimie Mole´culaire, UMR-5250, ICMG FR-2607, CNRS-Universite´ Joseph Fourier, BP-53, 38041 Grenoble, France
bIpsen Reseach Laboratories, Institut Henri Beaufour, 5 avenue du Canada, 91960 Les Ulis, France
Received 21 December 2007; revised 21 January 2008; accepted 22 January 2008
Available online 30 January 2008
Abstract—A series of 22-hydroxyacuminatine analogs was prepared by using different Friedla¨nder condensations. Several of the new
compounds were tested for antiproliferative activity on cancer cell lines and for topoisomerase I inhibitory activity.
Ó 2008 Elsevier Ltd. All rights reserved.
Camptothecin-family alkaloids and their derivatives
R2 R3
have attracted enormous attention from both academic
and pharmaceutical research groups due to their potent
antiproliferative activities.1 The archetype of this family,
camptothecin (1),2 acts as a selective poison of DNA
topoisomerase I (Fig. 1).3 Several AB-ring substituted
analogs of this molecule are among the most potent
drugs used in cancer chemotherapy: Topotecan (2)4
and Irinotecan (3)5 have been approved as antineoplas-
tic agents and a number of other analogs of 1 are cur-
rently in clinical trials.6
O
R1
O
N
N
N
N
HO
4
O
OH
1: R1=R2=R3=H
22-Hydroxyacuminatine
Camptothecin
2: R1=OH, R2=CH2NMe2, R3=H
3: R1=OCOPipPip, R2=H, R3=Et
Topotecan
Irinotecan
Figure 1. Structures of camptothecin, its analogs, and 22-
hydroxyacuminatine.
22-Hydroxyacuminatine (4)7 is structurally similar to
camptothecin, albeit lacking the lactone motif in the E
ring. This alkaloid was isolated in extremely low yield
(0.000006%) from Camptotheca acuminata and was
shown to have significant in vitro cytotoxic activity
against the murine leukemia P-388 and KB (ED50 1.32
and 0.61 lg/mL, respectively).
O
O
6 steps
OH
N
N
O
O
CO2Me
So far, only a few total syntheses of this alkaloid have
been described and most of them are not easily adapted
for producing analogs.8
5
6
tol
N
H
N
We have recently developed an efficient and flexible ap-
proach to 22-hydroxyacuminatine starting from hydro-
xy pyridone 5.8c As summarized in Scheme 1, the
synthesis of the natural product 4 was achieved by
reduction of ester 8, which could be produced by a
NH2
7
N
4
MeO2C
8
Scheme 1. Reagents and conditions: see Ref. 8c.
Keywords: Camptothecin; 22-Hydroxyacuminatine analogs; Synthesis;
Topoisomerase I; Cytotoxicity.
*
Friedla¨nder condensation of the key tricyclic compound
6 with an o-aminobenzaldehyde surrogate 7.
Corresponding author. Tel.: +33 4 76 63 54 32; fax: + 33 4 76 63 57
54; e-mail: Alice.Kanazawa@ujf-grenoble.fr
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.082

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F. Grillet et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2143–2146
In this paper, we report the synthesis of a series of ana-
logs of 4, taking advantage of the flexibility inherent in
our previous work. For several derivatives, in vitro anti-
proliferative activities on human cancer cell lines and
topoisomerase I inhibitory activity have been measured.
by using Dibal-H in dichloromethane at low tempera-
ture, as for the preparation of natural product 4. How-
ever, due to their very poor solubility, the reduction of
these esters proved to be inefficient. Only compound
13b could be transformed into 14b in reasonable yield.12
The synthesis of the keto pyridone 6 was conducted by
using our previously described route, but with a modifi-
cation of the oxidation at the benzylic-like position. The
transformation of 9 into 6, initially achieved in two
steps, is now effected in a single step under aerobic con-
ditions in the presence of N-hydroxyphthalimide
(NHPI) and CuCl.9 This new procedure avoids the use
of toxic selenium dioxide and improves the reproducibil-
ity of the transformation (Scheme 2).
In order to synthesize also a series of analogs bearing
substituents on both the A and B rings, a number of
o-aminoketones 16a–g were prepared. The synthesis of
these substrates was effected by direct acylation of
substituted anilines,13 as outlined in Scheme 4. The
Friedla¨nder condensations were then performed as in
the previous series, but the corresponding pentacyclic es-
ters 17a–g were obtained in higher yields.14 Esters 17b,
17e, and 17g were next reduced by exposure to Dibal-
H to afford the corresponding alcohols.15
Next, a series of masked substituted o-aminobenzalde-
hydes 12a–d was prepared by condensation of p-tolui-
dine with o-nitrobenzaldehydes, followed by sodium
sulfide reduction.10 The resulting arylimines were then
treated with keto pyridone 6 under Friedla¨nder condi-
tions to afford the corresponding pentacyclic esters
13a–d11 in serviceable yields (Scheme 3).
Some of the pentacyclic compounds herein obtained
were selected for biological evaluation (see Table 1).
The antiproliferative activities against three human can-
cer cell lines (DU145, Mia PaCa, and HT29) were com-
pared with those of 22-hydroxyacuminatine (4) and SN-
38.
It had been hoped that these esters could be converted
into the corresponding hydroxymethylated derivatives
As can be seen, no effect on cellular proliferation could
be detected against the colorectal cancer cell line HT29.
Only compounds 18b and 18g showed slight activity
against prostate cancer cell line DU145. However, most
of the tested compounds were active against pancreatic
carcinoma Mia PaCa, with IC50 values ranging from
0.81 to 1.99 lM.16
O
O
a or b
N
N
O
CO2Me
CO2Me
In the ester series, the introduction of substituents on
the quinoline ring system does not seem to be beneficial
9
6
Scheme 2. Reagents and conditions: (a) SeO2, dioxane, reflux; Dess–
Martin periodinane, CH2Cl2 (75%, 2 steps); (b) O2, CuCl, NHPI,
CH3CN, 35 °C (75%).
Scheme 3. Reagents and conditions: (a) p-toluidine, EtOH, reflux, 2 h
(63–99%); (b) Na2SÆ9H2O, EtOH, reflux, 2 min (60–69%); (c) 6, PhMe,
p-TsOH, reflux Dean–Stark (30–53%); (d) Dibal-H, CH2Cl2, À78 °C
(56%).
Scheme 4. Reagents and conditions: (a) BCl3, R3CN, GaCl3, CH2Cl2,
reflux, 24 h (45–81%); (b) 6, p-TsOH, PhMe, reflux Dean–Stark, 5 h
(64–93%); (c) Dibal-H, CH2Cl2, À78 °C, 5 h (26–60%).

F. Grillet et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2143–2146
2145
Table 1. In vitro cytostatic activity against various cell lines
2. Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.;
McPhail, A. T.; Sim, G. A. J. Am. Chem. Soc. 1966, 88,
3888.
Compound
DU145
a
Mia PaCa
a
HT29
a
IC50 (lM)
IC50 (lM)
IC50 (lM)
3. (a) Hsiang, Y. H.; Hertzberg, R.; Hetch, S. M.; Liu, L. F.
J. Biol. Chem. 1985, 260, 14873; (b) Kohn, K. W.;
Pommier, Y. Ann. N.Y. Acad. Sci. 2000, 922, 11; (c)
Staker, B. L.; Hjerrild, K.; Fesse, M. D.; Behnke, C. A.;
Burgin, A. B., Jr.; Stewart, L. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 15387.
4. Kingsbury, W. D.; Boehm, J. C.; Jakas, D. R.; Holden, K.
G.; Hetch, S. M.; Gallagher, G.; Caranfa, M. J.; McCabe,
F. L.; Faucette, L. F.; Johnson, R. K.; Hertzbeg, R. P. J.
Med. Chem. 1991, 34, 98.
5. (a) Negoro, S.; Fukuoka, M.; Masuda, N.; Takada, M.;
Kusunoki, Y.; Matsui, K.; Takifuji, N.; Kudoh, S.;
Niitani, H.; Tagushi, T. J. Natl. Cancer Inst. 1991, 83,
1164; (b) Kawato, Y.; Aonuma, M.; Hirota, Y.; Kuga, H.;
Sato, K. Cancer Res. 1991, 51, 4187.
6. For compilations of derivatives that are in preclinical or
clinical trials, see (a) Butler, M. S. Nat. Prod. Rep. 2005,
22, 162; (b) Cragg, G. M.; Newman, D. J. J. Nat. Prod.
2004, 67, 232.
8
nab
na
1.01
1.70
1.99
na
na
na
na
na
na
na
na
na
na
na
na
na
0.012
14b
17a
17b
17c
17e
17f
17g
4
na
na
na
na
1.40
1.81
1.35
1.50
1.01
0.81
1.14
0.81
0.006
na
na
na
18b
18e
18g
SN-38
1.81
na
1.69
0.004
a Concentration necessary for 50% of cell growth inhibition after 96 h
of incubation.
b Not active at concentrations up to 2 lM.
7. Lin, L. Z.; Cordell, G. A. Phytochemistry 1989, 28, 1295.
8. (a) Ma, Z.; Lee, D. Y. W. Tetrahedron Lett. 2004, 45,
6721; (b) Xiao, X.; Antony, S.; Pommier, Y.; Cushman,
M. J. Med. Chem. 2006, 49, 1408; (c) Babjak, M.;
Kanazawa, A.; Anderson, R. J.; Greene, A. E. Org.
Biomol. Chem. 2006, 4, 407; (d) Zhou, H.-B.; Liu, G.-S.;
Yao, Z.-J. J. Org. Chem. 2007, 72, 6270.
9. (a) A solution of 0.62 mmol of compound 9, 0.62 mmol of
N-hydroxyphthalimide and 0.031 mmol of CuCl in 11 mL
of acetonitrile under O2 was stirred at 35 °C for 24 h. After
evaporation of the solvent under reduced pressure, the
residue was purified by silica gel column chromatography
to afford 6; For a review of NHPI-catalyzed aerobic
oxidation, see (b) Ishii, Y.; Sakaguchi, S.; Iwahama, T.
Adv. Synth. Catal. 2001, 343, 393.
for biological activity, while in the hydroxymethyl series,
the presence of these substituents showed negligible
influence.
In addition, topoisomerase I inhibitory activity assays
with the same derivatives17 were carried out using the
supercoiled DNA unwinding method.18 Only com-
pounds 8, 17g, and 18g revealed some inhibitory activ-
ity, but much lower than that of camptothecin (used
as the positive reference).
It should be pointed out that all of the tested com-
pounds have low solubility in DMSO. As the maximum
tested concentrations used in both biological studies
were limited by the final DMSO concentrations in the
assay mixtures (4% or 5%), in some cases the activity
could not be quantified.
10. Porter, H. K. Org. React. 1973, 20, 455.
11. (a) Compound 13a: mp 264.5–266.5 °C, lH NMR (CDC13)
d 3.14 (s, 6H), 4.06 (s, 3H), 5.29 (s, 2H), 7.13 (m, 2H), 7.54
(t, 1H, J = 7.7 Hz), 7.63 (d, 1H, J = 8.9 Hz), 8.10 (s, 1H),
8.27 (d, 1H, J = 7.4 Hz), 8.56 (s, 1H), 8.73 (d, 1H, J = 8.2
Hz), HRMS m/z calcd for C23H20N3O3 (MH+) 386.14992,
In summary we have used the flexibility of our synthetic
approach to prepare a variety of 22-hydroxyacumina-
tine analogs. We have also performed the first struc-
ture–activity relationship study of this cytotoxic
alkaloid. None of the tested compounds proved signifi-
cantly more active than the natural product 4.
1
found 386.15012; (b) Compound 13b: mp 277–279 °C, H
NMR (CDCl3) d 4.07 (s, 3H), 5.35 (s, 2H), 7.58 (t, 1H,
J = 7.5 Hz), 7.69 (d, 1H, J = 8.8 Hz), 7.83 (s, 1H), 8.13 (d,
1H, J = 8.8 Hz), 8.20 (s, 1H), 8.39 (d, 1H, J = 7.2 Hz), 8.69
(s, 1H), 8.73 (d, 1H, J = 7.8 Hz), HRMS m/z calcd for
C21H14N2O3CI (MH+) 377.06875, found 377.06892; (c)
Compound 13c: mp 267–268 °C, lH NMR (CDC13) d 4.06
(s, 3H), 5.30 (s, 2H), 6.17 (s, 2H), 7.12 (s, 1H), 7.51 (s, 1H),
7.56 (t, 1H, J = 7.9 Hz), 8.12 (s, 1H), 8.38 (d, 1H, J = 7.5
Hz), 8.63 (s, 1H), 8.76 (d, 1H, J = 7.3 Hz); (d) Compound
Acknowledgments
l
13d: mp 295–297 °C, H NMR (CDC13) d 4.03 (s, 3H),
We thank Professor P. Dumy for his interest in our
´
4.06 (s, 3H), 4.10 (s, 3H), 5.31 (s, 2H), 7.06 (s, 1H), 7.55
(m, 2H), 8.14 (s, 1H), 8.36 (m, 1H), 8.58 (s, 1H), 8.74 (d,
1H, J = 7.8 Hz), HRMS m/z calcd for C23H18N2O5Na
(MNa+) 425.11079, found 425.11154.
work. Financial support from the Universite Joseph
Fourier, the CNRS (UMR 5250, FR 2607), and the
French Ministry of Research (for F.G.) are gratefully
acknowledged.
12. Compound 14b: mp 297.5–299 °C, 1H NMR (CDCl3/
MeOD 4/1) d 5.13 (s, 2H), 5.38 (s, 2H), 7.58 (t, 1H, J = 7.6
Hz), 7.76 (m, 1H), 7.87 (d, 1H, J = 6.9 Hz), 7.92 (m, 1H),
7.97 (s, 1H), 8.13 (d, 1H, J = 9.1 Hz), 8.34 (s, 1H), 8.41 (d,
1H, J = 8.1 Hz), HRMS m/z calcd for C20H14N2O2Cl
(MH+) 349.07383, found 349.07390.
References and notes
1. For recent reviews on camptothecin and its derivatives, see
(a) Thomas, C. J.; Rahier, N. J.; Hetch, S. M. Bioorg.
Med. Chem. 2004, 12, 1585; (b) Pizzolato, J. F.; Saltz, L.
B. Lancet 2003, 361, 2235; (c) Du, W. Tetrahedron 2003,
59, 8649; (d) Lansiaux, A.; Bailly, C. Bull. Cancer 2003, 90,
239.
13. Sugasawa, T.; Adachi, M.; Sasakura, K.; Kitagawa, A. J.
Org. Chem. 1979, 44, 578.
14. (a) Compound 17a: mp 276–278.5 °C, 1H NMR (CDC13) d
2.73 (s, 3H), 3.98 (s, 3H), 4.05 (s, 3H), 5.31 (s, 2H), 7.45
(m, 2H), 7.56 (t, 1H, J = 7.7 Hz), 8.14 (d, 1H, J = 9.2 Hz),

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F. Grillet et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2143–2146
8.37 (d, 1H, J = 7.7 Hz), 8.64 (s, 1H), 8.77 (d, 1H, J = 7.4
Hz), HRMS m/z calcd for C23H19N2O4 (MH+) 387.13393,
found 387.13398; (b) Compound17b: mp 236–237 °C, H
for C23H21N2O3 (MH+) 373.15467, found 373.15464; (b)
Compound 18e: mp 312–314 °C, NMR 1H (CDCl3/
MeOD, 4/1) d 1.36 (t, 3H, J = 7.7 Hz), 2.47 (s, 3H), 3.15
(q, 2H, J = 7.7 Hz), 5.04 (s, 2H), 5.27 (s, 2H), 7.50
(t, 1H, J = 7.7 Hz), 7.58 (d, 1H, J = 8.7 Hz), 7.82 (m,
3H), 8.00 (d, 1H, J = 8.6 Hz), 8.35 (d, 1H, J = 7.9 Hz),
HRMS m/z calcd for C23H21N2O2 (MH+) 357.16049,
found 357.15975; (c) Compound 18g: mp 301–302 °C,
1
NMR (CDC13) d 1.40 (t, 3H, J = 7.5 Hz), 3.11 (q, 2H,
J = 7.5 Hz), 3.96 (s, 3H), 4.06 (s, 3H), 5.29 (s, 2H), 7.24 (s,
1H), 7.41 (m, 1H), 7.54 (t, 1H, J = 7.8 Hz), 8.11 (d, 1H,
J = 9.2 Hz), 8.35 (d, 1H, J = 7.5 Hz), 8.59 (s, 1H), 8.73 (d,
1H, J = 7.8 Hz), HRMS m/z calcd for C24H21N2O4 (MH+)
401.14958, found 401.14958; (c) Compound 17c: mp 307–
308 °C, 1H NMR (CDC13) d 1.39 (t, 3H, J = 7.7 Hz), 3.09
(q, 2H, J = 7.7 Hz), 4.06 (s, 3H), 4.43 (s, 4H), 5.29 (s, 2H),
7.48 (s, 1H), 7.56 (t, 1H, J = 7.7 Hz), 7.68 (s, 1H), 8.38 (m,
1H), 8.64 (s, 1H), 8.77 (d, 1H, J = 8.1 Hz), HRMS m/z
calcd for C25H21N2O5 (MH+) 429.14450, found
l
NMR H (DMSO-d6) d 1.33 (t, 3H, J = 7.5 Hz), 3.21
(q, 2H, J = 7.5 Hz), 4.94 (m, 2H), 5.38 (s, 2H), 5.47 (t,
1H, J = 5.4 Hz), 7.57 (t, 1H, J = 7.7 Hz), 7.69 (s, 1H),
7.84 (m, 2H), 8.19 (d, 1H, J = 9.0 Hz), 8.29 (m, 2H),
HRMS m/z calcd for C22H18N2O2Cl (MH+) 377.10574,
found 377.10513.
l
429.14351; (d) Compound 17d: mp 324–326 °C, H NMR
16. Since no significant improvement of activity relative to 4
on the three tumor cell lines was observed, tests on normal
cell lines were not performed.
17. Although 22-hydroxyacuminatine does not inhibit topoi-
somerase I,8b several non-lactonic derivatives of campto-
thecin do show inhibitory activity (Hautefaye, P.;
(DMSO-d6, 70°C) d 4.01 (s, 3H), 4.48 (s, 4H), 5.34 (s, 2H),
5.42 (s, 2H), 7.68 (m, 2H), 7.76 (s, 1H), 8.38 (m, 2H), 8.65
(m, 1H); (e) Compound 17e: mp 260–262 °C, 1H NMR
(CDCl3) d 1.42 (t, 3H, J = 7.7 Hz), 2.61 (s, 3H), 3.20 (q,
2H,J = 7.7 Hz), 4.07 (s, 3H), 5.33 (s, 2H), 7.59 (m, 2H),
7.85 (s, 1H), 8.14 (d, 1H, J = 8.6 Hz), 8.38 (d, 1H, J = 7.2
Hz), 8.68 (s, 1H), 8.78 (d, 1H, J = 8.6 Hz), HRMS m/z
calcd for C24H21N2O3 (MH+) 385.15467, found
385.15474; (f) Compound 17f: mp 297–299.5 °C, 1H
NMR (CDC13) d 2.64 (s, 3H), 4.07 (s, 3H), 5.05 (s, 2H),
5.45 (s, 2H), 7.62 (m, 2H), 7.91 (s, 1H), 8.18 (d, 1H, J = 8.6
Hz), 8.40 (d, 1H, J = 7.4 Hz), 8.72 (s, 1H), 8.78 (d, 1H,
J = 8.3 Hz), HRMS m/z calcd for C23H18N2O3Cl (MH+)
405.10005, found 405.10017; (g) Compound 17g: mp 296–
298 °C, 1H NMR (CDC13) d 1.42 (t, 3H, J = 7.7 Hz), 3.15
(q, 2H, J = 7.7 Hz), 4.06 (s, 3H), 5.33 (s, 2H), 7.59 (t, 1H,
J= 7.8 Hz), 7.70 (m, 1H), 8.04 (m, 1H), 8.17 (d, 1H, J = 9.0
Hz), 8.40 (m, 1H), 8.71 (s, 1H), 8.77 (d, 1H, J = 7.9 Hz),
HRMS m/z calcd for C23H18N2O3Cl (MH+) 405.10005,
`
´
´
Cimetiere, B.; Pierre, A.; Leonce, S.; Hickman, J.; Laine,
W.; Bailly, C.; Lavielle, G. Bioorg. Med. Chem. Lett. 2000,
13, 2731). Since the new compounds reported in this paper
can also be viewed as non-lactonic derivatives of campto-
thecin, they were screened as well for inhibition of
topoisomerase I.
18. Supercoiled pBR322 plasmid DNA (130 ng) was incu-
bated with 4 U of human topoisomerase I (TopoGen) at
37 °C for 45 min in 20 lL of relaxation buffer (50 mM
tris(hydroxymethyl)aminomethane (pH 7.8), 50 mM KCl,
10 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA and
1 mM ATP) in the presence of the tested compounds and
DMSO (5%). The reactions were terminated by addition
of SDS to 0.25% and proteinase K to 250 lg/mL and
incubation at 50 °C for a further 30 min. Three microliters
of the electrophoresis dye mixture was added to the DNA
samples, which were then separated by electrophoresis in a
1% agarose gel containing ethidium bromide (1 lg/mL).
Gels were run at room temperature for 2 h at 120 V and
scanned with a Typhoon 9410 imager.
found 405.10010.
l
15. (a) Compound 18b: mp 280–282 °C, NMR H (DMSO-
d6) d 1.35 (t, 3H, J = 7.2 Hz), 3.23 (m, 2H), 3.99 (s, 3H),
4.94 (m, 2H), 5.37 (s, 2H), 5.46 (m, 1H), 7.53 (m,
3H), 7.63 (s, 1H), 7.80 (d, 1H, J = 6.2 Hz), 8.11 (d, 1H,
J = 9.4 Hz), 8.30 (d, 1H, J = 8.3 Hz), HRMS m/z calcd