Synthesis and biological activities of fluorinated 10-hydroxycamptothecin and SN38

Article abstract of DOI:10.1016/j.jfluchem.2013.10.016

It is an important strategy for fluorine substitution in drug design because of its small size and high electronegativity. Fluorinated 10-hydroxycamptothecin and SN 38 were prepared and screened for antiproliferative activities. Among them, fluorinated co

Full text of DOI:10.1016/j.jfluchem.2013.10.016

Journal of Fluorine Chemistry 157 (2014) 48–51
Contents lists available at ScienceDirect
Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
Short Communication
Synthesis and biological activities of fluorinated 10-
hydroxycamptothecin and SN38
Yuelin Wu a,b, Lingjian Zhu b, Chunquan Sheng b, Jianzhong Yao b, Zhenyuan Miao b,
*
,
c
a,
Wannian Zhang b, , Yunyang Wei , Ruofu Shi
*
*
a School of Environment and Biological Engineering, Nanjing University of Science & Technology, 200 Xiaolingwei, Nanjing 210094, People’s Republic of China
b School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of China
c School of Chemical Engineering, Nanjing University of Science & Technology, 200 Xiaolingwei, Nanjing 210094, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Article history:
It is an important strategy for fluorine substitution in drug design because of its small size and high
electronegativity. Fluorinated 10-hydroxycamptothecin and SN 38 were prepared and screened for
antiproliferative activities. Among them, fluorinated compound MF-6 showed higher antiproliferative
activities against A549, HCT116 and MDA-MB-435 cancer cells than unfluorinated compound. The result
of Topoisomerase I activity also confirmed that the C-21 carbonyl group of camptothecin structure is
unnecessary to antitumor activity.
Received 13 August 2013
Received in revised form 16 October 2013
Accepted 22 October 2013
Available online 30 October 2013
Keywords:
ß 2013 Elsevier B.V. All rights reserved.
Camptothecin
Fluorinate
Biological activity
1. Introduction
It is well known that the E-ring of camptothecin with an
integrity
a-hydroxylactone is essential to antitumor activity of
Fluorinated molecules were the most important organo-
halogen drugs among the marked pharmaceuticals because of
their special properties. There are more than 20% of drugs
containing at least one fluorine atom in all the clinical drugs [1–
3]. Very important anticancer drugs, such as cytotoxic drugs
fluorouracil derivatives and protein kinase inhibitors sorefenib,
gefitinib and vandetanib, are fluorinated drugs [4,5]. In addition,
fluorine substitution has been a successful strategy in drug design
from natural bioactive products. Taxoid, as the famous anticancer
drug, had been investigated with strategic fluorine incorporation
for elevation of cytotoxicity and metabolic stability [6]. Another
well-known anticancer natural product camptothecin had also
been designed by fluorine substitution for increased activity.
Lavergne et al. achieved the fluorinated camptothecin analogue
diflomotecan with excellent antitumor activities both in vitro and
in vivo by introducing two fluorine atom to the A aromatic ring of
homocamptothecin [7]. Exatecan, another successful fluorinated
camptothecin derivative with fluorine at position 10 on ring A, is
currently in Phase III [8].
camptothecin. Although 20-fluorocamptothecin with lower lac-
tone stability is less active in cytotoxicity and Topoisomerase I
assays [9], 21-fluorocamptothecins with excellent antitumor
activities were fortunately obtained which were substituted with
fluorine on position 21 instead of carbonyl group of camptothecin
E-ring [10]. Our previous results indicated that compound MF-1
exhibited higher activity against all three cancer cell lines than its
diastereoisomer MF-2. Herein, we report the synthesis and
biological activities of two fluorinated camptothecin drugs 10-
hydoxycamptothecin and SN 38 which is the active metabolite of
Irinotecan (Fig. 1).
2. Results and discussion
The synthesis of fluorinated camptothecin derivatives is
outlined in Scheme 1. Compounds MF-3 and MF-4 as 10-
hydroxycamptothecin derivatives was synthesized in three and
four steps respectively. In order to avoid the side fluorinated
impurities during fluorination by DAST, hydroxyl group on A ring
of natural 10-hydroxycamptothecin was protected with benzyl
group. Thus, compound MF-3 was obtained based on our previous
fluorination method [10]. Compound MF-3 reduced the benzyl
group to afford compound MF-4 in 90% yield. Compounds MF-5
and MF-6 was easily achieved by the same route from active
metabolite of Irinotecan with good yield.
*
Corresponding authors at: School of Environment and Biological Engineering,
Nanjing University of Science & Technology, 200 Xiaolingwei, Nanjing 210094,
People’s Republic of China. Tel.: +86 25 84315512; fax: +86 25 84315512.
E-mail addresses: miaozhenyuan@hotmail.com (Z. Miao),
zhangwnk@hotmail.com (W. Zhang), srf_173@163.com (R. Shi).
0022-1139/$ – see front matter ß 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jfluchem.2013.10.016

Y. Wu et al. / Journal of Fluorine Chemistry 157 (2014) 48–51
49
Fig. 1. The structures of camptothecin drugs and fluorinated camptothecin isomers.
Table 1
In vitro antiproliferative activities of fluorinated camptothecin derivatives (inhibition
rate at 100mg/mL).
Compounds
A549
MDA-MB-435
HCT116
MF-3
26.83 Æ 3.38
29.23 Æ 3.42
56.24 Æ 2.19
83.64 Æ 3.53
88.23 Æ 3.03
83.41 Æ 1.92
55.36 Æ 1.90
77.19 Æ 1.09
85.22 Æ 1.99
98.13 Æ 1.87
75.06 Æ 1.93
75.23 Æ 1.04
83.76 Æ 2.76
88.28 Æ 0.60
79.33 Æ 0.77
81.72 Æ 1.83
75.82 Æ 1.43
80.34 Æ 4.01
MF-4
MF-5
MF-6
10-HCPT
SN 38
Reagents and conditions of reactions: (a) K2CO3, dried DMF,
benzyl bromide, 50 8C; (b) KBH4, CH3OH, rt; (c) DAST, CH2Cl2,
À95 8C–0 8C; (d) Pt/C, H2, CH3OH, rt.
The in vitro antiproliferative activity of fluorinated camptothe-
cin derivatives were evaluated in three cancer cells A549, MDA-
MB-435 and HCT116 by MTT assay. The results are summarized in
Table 1 and all the target compounds indicated potent anti-
proliferative activities. As expected, compounds MF-4 and MF-6
showed higher activity against three cancer cells than that of their
corresponding compounds with benzyl group at the concentration
of 100
mg/mL. In addition, most of fluorinated compounds were
more selective to breast cancer cells MDA-MB-435 and colon
cancer cells HCT116 than non-small-cell lung cells A549 which
was not consistent to our previous work [10]. For example,
compound MF-4 possessed high activities against MDA-MB-435
and HCT116 cells with 77.19% and 88.28% proliferative inhibition
while the inhibition of A549 cells was 29.23%. More importantly,
compound MF-6 demonstrated exclusively the most potent
activity in all three cancer cell lines than both fluorinated
camptothecin derivatives and clinical drug 10-HCPT and SN 38.
As demonstrated in Fig. 2, the same trend was observed for the
Fig. 2. Antiproliferative activities of fluorinated camptothecin derivatives at the
concentration of 100–0.001
mg/mL.
comparison of MF-6 and10-HCPT and SN 38 for the antiprolifera-
tive activity at every concentration.
Encouraged by this result, Topo I-mediated DNA cleavage
assays of the most potent fluorinated camptothecin derivative MF-
6 and fluorinated camptothecin isomers MF-1 and MF-2 were
investigated. As expected, compound MF-6 was found to be active
R
R
N
R
HO
BnO
BnO
O
O
O
N
N
N
b
a
N
N
O
O
O
HO
HO
HO
2
O
O
OH
1
R=H, 1a
R=Et, 1b
R=H, 10-HCPT
R=Et, SN 38
R=H, 2a
R=Et, 2b
R
R
BnO
HO
O
O
N
c
N
d
N
N
O
O
HO
HO
F
F
R=H, MF-3
R=Et, MF-5
R=H, MF-4
R=Et, MF-6
Scheme 1. Synthesis of fluorinated 10-hydroxycamptothecin derivatives.

50
Y. Wu et al. / Journal of Fluorine Chemistry 157 (2014) 48–51
4.1.1. 10-Benzyloxy-(20S, 21S)-diolcamptothecin(2a)
Light Yellow solid, Yield 89%, m.p.
25 = +856.6(c 0.030, DMF). 1H NMR (600 MHz, DMSO-d6)
d:
238–240 8C,
[a
]
D
0.90 (t, 3H, J = 7.5 Hz, CH3CH2), 1.71 (q, 2H, J = 7.2 Hz, CH3CH2),
4.48–4.62 (q, 2H, J = 17.1 Hz, OCH2), 4.90 (s, 1H, CH), 4.99 (d, 1H,
J = 5.4 Hz, OH), 5.21 (s, 2H, NCH2), 5.29 (s, 2H, CH2O), 6.71 (d, 1H,
J = 5.4 Hz, OH), 7.27 (s, 1H, C14-H), 7.36 (d, 1H, J = 2.7 Hz, C11-H),
7.42–7.44 (m, 2H), 7.54–7.62 (m, 4H), 8.07 (d, 1H, J = 9.0 Hz, C12-H),
8.50 (s, 1H, C7-H); MS (ESI): 457 (M+H). Anal. calcd for
Fig. 3. Effect of the selected compounds on Topo I-mediated DNA relaxation in
single concentration with 100
mM. Lane 1, supercoiled plasmid DNA; Lane 2,
DNA + Topo I; Lane 3, DNA + Topo I + CPT; Lane 4–6, DNA + Topo I + fluorinated
camptothecin derivatives (MF-1, MF-2 and MF-6, respectively).
C29H28ClN3O4: C, 71.04; H, 5.30; N, 6.14; found C, 71.13; H,
5.29; N,6.15.
against Topo I-mediated relaxation of supercoiled DNA at a high
4.1.2. 7-Ethyl-10-benzyloxy-(20S, 21S)-diolcamptothecin (2b)
concentration of 100
m
M which was similar to natural camp-
Light
Yellow
solid,
Yield
91%,
m.p.
273–275 8C,
tothecin (Fig. 3). As is evident, this result confirmed that the S-
isomer of fluorinated camptothecin derivative indicated higher
activity than that of R-isomer which was presented by our previous
study [10].
[a
]
25 = +1537.0(c 0.020, DMF). 1H NMR (600 MHz, DMSO-d6)
d:
D
0.90 (t, 3H, J = 7.5 Hz, CH3CH2), 1.24 (t, 3H, J = 7.8 Hz, CH3CH2), 1.71
(q, 2H, J = 7.2 Hz, CH3CH2), 3.14 (q, 2H, J = 7.2 Hz, CH3CH2), 4.48–
4.63 (q, 2H, J = 17.1 Hz, OCH2), 4.90 (s, 1H, CH), 4.99 (d, 1H,
J = 5.4 Hz, OH), 5.22 (s, 2H, NCH2), 5.36 (s, 2H, CH2O), 6.71 (d, 1H,
J = 5.4 Hz, OH), 7.28 (s, 1H, C14-H), 7.36 (d, 1H, J = 2.7 Hz, C11-H),
3. Conclusion
7.42–7.43 (m, 2H), 7.55–7.57 (m, 4H), 8.06 (d, 1H, J = 9.0 Hz, C12
-
H); MS (ESI): 485 (M+H). Anal. calcd for C29H28ClN3O4: C, 71.47; H,
5.57; N, 5.95; found C, 71.34; H, 5.56; N,5.96.
In this report, we have demonstrated that it is a successful
strategy for fluorine substitution on position 21 of camptothecin
clinical drugs. Fluorinated 10-hydroxycamptothecin and SN 38
4.2. General procedure for synthesis of fluorinated camptothecins
were prepared by
a same synthetic route. As the potent
topoisomerase I inhibitor, MF-6 showed higher antiproliferative
activities against A549, MDA-MB-435 and HCT116 cancer cells
than 10-HCPT and SN38. Moreover, this result also confirmed that
the C-21 carbonyl group of camptothecin structure is unnecessary
to antitumor activity.
A solution of 2a (1.4 mmol) in dichloromethane (30 mL) under
nitrogen at – 95 8C was added DAST (1.6 mmol) as soon as possible.
After 10 min, the reaction was stirred for another 2 h at 0 8C. Then,
the reaction was quenched with NaHCO3 saturated solution then
extracted with dichloromethane. After the removal of solvent, the
residue was purified by flash chromatography on silica gel (CH2Cl2/
CH3OH55 100:1) to afford targeted compound as a yellow solid
MF-3.
To a solution of MF-3 (1.1 mmol) in ethanol (15 mL), Pt/C
(5%, 10 mg) was added as catalyst and hydrogenation followed
at 3 bar for 12 h at room temperature. The resulting suspension
was filtered and concentrated under reduced pressure to afford
MF-4.
4. Experimental
Chemistry. General Methods. All starting materials were com-
mercially available and analytical pure. Melting points were
measured on an uncorrected X-5 digital melting point apparatus
(Gongyi City Yuhua Instrument Co., Ltd.; China). 1H NMR, 13C NMR
and 19F NMR spectra were recorded on a Bruker Avance 300
spectrometer, a Bruker Avance 500 spectrometer or a Bruker
Avance 600 spectrometer (Bruker Company, Germany), using TMS
as an internal standard and CDCl3 or DMSO-d6 as solvents.
4.2.1. 10-Benzyloxy-(20S, 21S)-fluorcamptothecin (MF-3)
Chemical shifts (
d
values) and coupling constants (J values) are
Yellow solid, Yield 35%, m.p. 185–187 8C, [
0.056, CH2Cl2). 1H NMR (500 MHz, DMSO-d6)
d: 0.90 (t, 3H,
a]
D
20 = +670.5(c
given in ppm and Hz, respectively. The mass spectra were recorded
on an Esquire 3000 LC–MS mass spectrometer. TLC analysis was
carried out on silica gel plates GF254 (Qindao Haiyang Chemical,
China). Flash column chromatography was carried out on silica gel
300–400 mesh using a Biotage instrument. Anhydrous solvent and
reagents were all analytical pure and dried through routine
protocols.
J = 7.5 Hz, CH3CH2), 1.70 (q, 2H, J = 7.2 Hz, CH3CH2), 4.70–4.78 (q,
2H, J = 17.1 Hz, OCH2), 4.90 (s, 1H, CH), 5.24 (d, 1H, J = 4.6 Hz,
NCH2), 5.29 (s, 2H, OCH2), 5.69 (d, 1H, J = 54.2 Hz, CH-F), 5.86 (s, 1H,
OH), 7.33 (s, 1H, C14-H), 7.37 (d, 1H, J = 2.7 Hz, C11-H), 7.42–7.43
(m, 2H), 7.54–7.63 (m, 4H), 8.08 (d, 1H, J = 9.2 Hz, C12-H), 8.52 (s,
1H, C7-H); 13C NMR (125 MHz, DMSO-d6)
d: 7.64, 32.21, 50.33,
60.84, 70.09, 70.23, 97.46, 107.02, 108.04, 108.82, 120.53, 123.52,
128.37, 128.47, 128.93, 129.61, 129.89, 130.38, 130.90, 136.94,
144.31, 144.46, 148.54, 151.01, 157.33; 19F NMR (470 MHz, DMSO-
4.1. General procedure for synthesis of diol-CPTs
To a suspension of 10-hydroxycamptothecin (10.0 mmol) and
K2CO3 (15.0 mmol) in dried DMF (50 mL), benzyl bromide
(15.0 mmol) was added dropwise at 0 8C. The reaction was stirred
for another 4 h at 50 8C. After being cooled, the reaction mixture
was diluted with ice-water and extracted with dichloromethane.
After the removal of solvent, the residue was purified by flash
chromatography on silica gel (CH2Cl2/CH3OH55100:2) to afford 1a
as a yellow solid.
To a suspension of 1a (0.5 mmol) in methanol (30 mL) was
slowly added KBH4 (10.0 mmol). The reaction was stirred for
another 4 h at room temperature. After the removal of solvent,
water (50 mL) was added to the residue. The resulting solution was
acidulated with acetic acid. The precipitate was collected and
washed with water to give white solid 2a.
d6)
d
: À140.87; MS (ESI): 459 (M+H). Anal. calcd for C29H28ClN3O4:
C, 70.73; H, 5.06; N, 6.11; found C, 70.85; H, 5.05; N,6.10.
4.2.2. 10-Hydroxy-(20S, 21S)-fluorcamptothecin (MF-4)
Brown solid, Yield 90%, m.p. >260 8C, [
DMF). 1H NMR (300 MHz, DMSO-d6)
: 0.91 (t, 3H, J = 7.4 Hz,
CH3CH2), 1.71 (q, 2H, J = 7.1 Hz, CH3CH2), 4.72 (s, 2H, OCH2), 5.20 (s,
2H, NCH2), 5.62 (d, 1H, J = 54.0 Hz, CH-F), 5.85 (s, 1H, OH), 7.26 (d,
1H, J = 2.7 Hz, C9-H), 7.29 (s, 1H, C14-H), 7.38–7.42 (dd, 1H,
J = 2.7 Hz, J = 6.6 Hz, C11-H), 8.01 (d, 1H, J = 9.0 Hz, C12-H), 8.42 (s,
1H, C7-H), 10.29 (s, 1H, OH); 13C NMR (125 MHz, DMSO-d6)
d: 7.61,
32.20, 50.22, 60.83, 70.29, 97.21, 108.73, 109.26, 120.25, 123.31,
129.61, 129.98, 130.93, 143.65, 144.48, 148.56, 150.15, 156.94,
157.36, 162.71; 19F NMR (470 MHz, DMSO-d6)
a]
D
20 = +224(c 0.09,
d
d
: À140.85; MS

Y. Wu et al. / Journal of Fluorine Chemistry 157 (2014) 48–51
51
(ESI): 367 (MÀH). Anal. calcd for C29H28ClN3O4: C, 65.21; H, 4.65;
100 mL of DMSO. The absorbance (OD) was read on a Wellscan
N, 7.60; found C, 65.13; H, 4.66; N,7.61.
MK-2 microplate reader (Lab systems) at 570 nm. The con-
centration causing 50% inhibition of cell growth (IC50) was
determined by the Logit method. All the experiments were
performed three times.
Topo I inhibitory activity assay. DNA relaxation assays were
employed according to the procedure described in previous studies
[10]. The reaction mixture contained 35 mM Tris–HCl (pH 8.0),
72 mM KCl, 5 mM MgCl2, 5 mM dithiothreitol, 5 mM spermidine,
4.2.3. 7-Ethyl-10-benzyloxy-(20S, 21S)-fluorcamptothecin (MF-5)
Yellow solid, Yield 32%, m.p. 211–213 8C, [
CH2Cl2). 1H NMR (500 MHz, DMSO-d6)
: 0.89 (t, 3H, J = 7.5 Hz,
a]
20 = +159.5(c 0.15,
D
d
CH3CH2), 1.01 (t, 3H, J = 7.7 Hz, CH3CH2), 1.24 (t, 3H, J = 7.7 Hz,
CH3CH2), 1.70 (q, 2H, CH3CH2), 1.72–2.35 (dm, 2H, CH3CH2), 3.16
(q, 2H, J = 7.2 Hz, CH3CH2), 4.72–4.78 (m, 4H, OCH2), 5.26–5.28 (q,
4H, NCH2), 5.37 (s, 4H, CH2O), 5.61–5.73 (dd, 2H, CH), 5.85 (s, 1H,
OH), 5.87 (s, 1H, OH), 7.25 (s, 1H, C14-H), 7.30 (s, 1H, C14-H), 7.35–
7.37 (m, 3H), 7.41–7.44 (m, 4H), 7.55–7.59 (m, 8H), 8.07–8.09 (d s,
1H, C12-H); 13C NMR (125 MHz, DMSO-d6)
d: 7.64, 8.01, 13.81,
22.63, 28.38, 32.21, 49.56, 60.67, 67.08, 70.22, 97.40, 104.28,
107.04, 108.26, 108.84, 120.39, 121.82, 122.89, 128.07, 128.27,
128.40, 128.95, 131.74, 137.11, 144.19, 144.85, 145.25, 147.08,
0.1% bovine serum albumin (BSA), pBR322 plasmid DNA (0.25
the indicated drug concentrations (1% DMSO), and 1 unit of Topo I
(TaKaRa Biotechnology Co., Ltd., Dalian) in a final volume of 20 L.
Reaction mixtures were incubated for 15 min at 37 8C and stopped
by addition of 2
mg),
m
m
L of 10Â loading buffer (0.9% sodium dodecyl
sulfate (SDS), 0.05% bromophenol blue, and 50% glycerol).
Electrophoresis was carried out in a 0.8% agarose gel in TAE
(Tris-acetate-EDTA) at 8 V/cm for 1 h. Gels were stained with
148.55, 150.30; 19F NMR (470 MHz, DMSO-d6)
d
:
À142.49,
À140.80; HRMS (EI) m/z: 486.1952. Anal.calcd for C29H28ClN3O4:
ethidium bromide (0.5 mg/mL) for 60 min. The DNA band was
visualized over UV light and photographed with Gel Doc Ez imager
(Bio-Rad Laboratories Ltd.)
C, 71.17; H, 5.33; N, 5.93; found C, 71.25; H, 5.32; N,5.92.
4.2.4. 7-Ethyl-10-hydroxy-(20S, 21S)-fluorcamptothecin (MF-6)
Yellow solid, Yield 95%, m.p. >260 8C, [
DMF). 1H NMR (300 MHz, DMSO-d6)
: 0.90 (t, 3H, J = 7.4 Hz,
a]
20 = +422.4(c 0.083,
D
Acknowledgments
d
CH3CH2), 1.01 (t, 3H, J = 7.5 Hz, CH3CH2), 1.71 (q, 2H, J = 7.3 Hz,
CH3CH2), 3.09 (q, 2H, J = 7.6 Hz, CH3CH2), 4.72–4.78 (q, 2H,
J = 8.3 Hz, OCH2), 5.22–5.29 (q, 2H, J = 18.2 Hz, NCH2), 5.62 (d,
1H, J = 54.0 Hz, CH-F), 5.87 (s, 1H, OH), 7.26 (d, 1H, J = 2.7 Hz, C9-H),
7.29 (s, 1H, C14-H), 7.38–7.42 (dd, 1H, J = 2.7 Hz, J = 6.6 Hz, C11-H),
8.02 (d, 1H, J = 8.9 Hz, C12-H), 10.28 (s, 1H, OH); 13C NMR
This work was supported in part by the National Natural
Science Foundation of China (Grant 30930107). We gratefully
acknowledge technical support from Prof. Xiuhua Chen (Shanghai
institute of pharmaceutical industry, P.R. China) for part of
biological activity assays.
(125 MHz, DMSO-d6) d: 7.63, 13.76, 22.70, 28.38, 32.21, 49.53,
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60.85, 70.30, 97.16, 105.25, 107.06, 108.29, 108.86, 110.07, 120.13,
121.58, 122.70, 128.04, 128.53, 131.84, 143.13, 144.06, 145.03,
145.43, 147.11, 148.57, 149.53, 149.61; 19F NMR (470 MHz, DMSO-
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mL of
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) solution (5 mg/mL) was added to each well and the plate
was incubated for an additional 4 h. The formazan was dissolved in