Synthesis, biological evaluation, and in vivo imaging of the first camptothecin-fluorescein conjugate

Article abstract of DOI:10.1021/bc3005304

The first synthesis and photophysical properties of a fluorecently labeled camptothecin derivative, namely, camptothecin-FI (CPT-FI), an antitumoral agent that targets topoisomerase I, are reported. The preparation of this fluorescent conjugate is based o

Full text of DOI:10.1021/bc3005304

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Article
Synthesis, Biological Evaluation and in vivo Imaging
of the first Camptothecin-Fluorescein Conjugate
Arnaud Chevalier, Martine Dubois, Vadim Lejoncour, Sébastien Dautrey, Céline
Lecointre, Anthony Romieu, Pierre-Yves Renard, Hélène Castel, and Cyrille Sabot
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/bc3005304 • Publication Date (Web): 10 Jun 2013
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Synthesis, Biological Evaluation and in vivo Imaging of
the first CamptothecinꢀFluorescein Conjugate†
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Arnaud Chevalier,† Martine Dubois,$,Vadim Lejoncour,$ Sébastien Dautrey,† Céline Lecointre,$ Anthony
Romieu,† Pierre-Yves Renard,† Hélène Castel,*,$ and Cyrille Sabot*,†
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† Normandie Univ, COBRA, UMR 6014 et FR 3038;
Univ Rouen; INSA Rouen; CNRS, 1 rue Tesnière
76821 MontꢀSaintꢀAignan, Cedex, France.
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$ Inserm U982, Laboratory of Neuronal and Neuroendocrine Communication and Differentiation
(DC2N), Astrocyte and Vascular Niche, Institute of Research and Biomedical Innovation (IRIB), PRES
Normandy University, University of Rouen, 76821 MontꢀSaintꢀAignan Cedex, France; NorthꢀWest
Cancéropole (CNO), 59008 Lille Cedex, France.
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RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
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CORRESPONDING AUTHOR FOOTNOTE (Word Style “FA_Corresponding_Author_Footnote”). To
whom correspondence should be addressed. For H.C: eꢀmail: helene.castel@univꢀrouen.fr; phone: + 33
(0)2 35 14 66 30. For C.S.: email: cyrille.sabot@univꢀrouen.fr; phone: + 33 (0)2 35 52 24 39.
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Abstract (Word Style “BD_Abstract”). The first synthesis and photophysical properties of a
fluorecentlyꢀlabeled camptothecin derivative namely camptothecinꢀFI (CPTꢀFI), an antitumoral agent
that targets topoisomerase I, are reported. The preparation of this fluorescent conjugate is based on a
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highly convergent and flexible approach which enables the rapid chemical modification of the AB ring
system of this fragile pentacyclic alkaloid, aimed at introducing an anchoring point to graft the
fluorophore. The selection of a fluorescein analogue as the reporter group has enabled us to get the first
greenꢀemitting CPT conjugate exhibiting valuable spectral properties and retaining biological properties
of native CPT. Indeed, in biological models, i.e., glioma cell lines U87 and/or T98, the kinetic of cell
endocytosis as well as the efficacy of CPTꢀFI were compared to those of CPT. CPTꢀFI fluorescence was
measured in the cytosolic compartment of T98 glioma cells from 5 min treatment and remained
detectable until 48 h. As CPT, CPTꢀFI drastically inhibited glioma growth and cell cycle but exhibited a
reduced affinity as compared to the native CPT. In vivo and ex vivo imaging studies of CPTꢀFI
intratumoraly injected into a model of NIHꢀ3T3 murine tumor xenografts in nude mice, showed that
accumulation around the injected site area, which is very promising to target tumors and follow
biodistribution in vivo.
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Introduction
Since its first discovery in 1966,1 the pentacyclic alkaloid 20(S)ꢀcamptothecin has emerged as an
important lead compound in the field of anticancer drug development. CPT acts by stabilizing the DNAꢀ
topoisomerase I binary complex and enhancing apoptosis through blocking the advancement of
replication forks.2 Also, CPT and its numerous derivatives developed over the years, display various
antitumor activities in a wide range of cancers, such as ovarian and lung (topotecan), colon (irinotecan)
or brain (gimatecan and belotecan).3,4,5 Despite significant progress, residual secondary effects are still
observed, such as for example hematological toxicity, headache, fever and fatigue, associated with the
CPTꢀbased chemotherapy. Fluorescent labeling of biomolecules or bioactive compounds is a common
way to gain insights into either unwanted organismꢀdrug interactions or pharmacological
mechanisms.6,7,8 However, despite the number of questions concerning the pharmacokinetic, real access
and penetration in the tumor and the number of side effects of usual chemotherapy (as well as new
targeted therapies), strategies for fluorescently labeling of cancer therapeutic drugs have scarcely been
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developed until now. Thus, we propose here the first strategy to develop original imaging tools, adapted
to in vivo imaging of the small animal, but also to study mechanisms of action of molecules at the
cellular level within tumors (role in apoptosis, proliferation, and vascularization) and in healthy tissues.
Despite the overwhelming number of CPT analogues reported in the literature over the past decade,
none of them has already been directly modified9 with a fluorescent reporter group.10,11,12 This can be
mainly explained to the absence of a suitable bioconjugatable reactive handle within this pentacyclic
alkaloid structure whose derivatization would not drastically affect its biological activity.13 In addition,
the facile hydrolysis of its lactone moiety represents a challenging bioconjugation issue especially for
considering further functionalization of this fragile molecular scaffold. Although most of CPT
derivatives exhibit a natural and strong blue fluorescence, their absorption and emission spectra (e.g.,
shape, intensity and wavelength) are highly dependent on the analogue considered and the stability of
their crucial Eꢀring lactone. Indeed, under physiological conditions, the lactone ring of CPT is rapidly
hydrolyzed into the usually less fluorescent and physiologically active carboxylate form.14,15,16 Even if
the intrinsic fluorescence properties of CPT could be compatible with in vitro bioassays,17 more
challenging bioꢀanalytical applications focusing on the tracking of biological events (following
administration of this bioactive compound) in cellulo or in vivo could not be easily implemented due to
the blue light absorption and scattering by the biological matrix and the background signal from cellular
autofluorescence.18 Thus, it is essential to dispose of a reliable fluorescent chemical reporter
conveniently link to the bioactive alkaloid, to efficiently track the biological processes, whatever the
analogues considered and its lactone form proportion in biological media. Consequently, in this present
work, we wish to provide a general framework to access to a fluorescentlyꢀlabeled CPT in order to
validate the possibility of fluorescently modified chemotherapy (or targeted therapy) without losing
valuable and characteristic biological antiꢀtumoral properties, determined in vitro and in vivo in animal
models.
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Experimental Procedures
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General
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All solvents were dried following standard procedures (dichloromethane (CH2Cl2): distillation over
P2O5, acetonitrile (CH3CN): distillation over CaH2, THF: distillation over Na°/benzophenone). N,Nꢀ
dimethylformamide (DMF) was purchased from Fisher Scientific and stored over 3Å molecular sieve.
1,2ꢀDimethoxyethane (DME) was dried by storage over 3Å molecular sieve. N,Nꢀ
Diisopropylethylamine (DIEA) was distilled over CaH2. Column chromatography purifications were
performed on Geduran® Si 60 silica gel (40ꢀ63 ꢁm) from Merck. TLC were carried out on Merck DC
Kieselgel 60 Fꢀ254 aluminum sheets. Compounds were visualized by one or more of the following
methods: (1) illumination with a short wavelength UV lamp (i.e., λ = 254 nm), (2) spray with a 0.2%
(w/v) ninhydrin solution in absolute ethanol, (3) spray with a 3.5% (w/v) phosphomolybdic acid
solution in absolute ethanol. The HPLCꢀgradient grade acetonitrile (CH3CN) and methanol (CH3OH)
were obtained from VWR. Phosphate buffered saline (PBS, 100 mM phosphate + 150 mM NaCl, pH
7.5) and aq. mobileꢀphases for HPLC were prepared using deionized water purified with a MilliꢀQ
system (purified to 18.2 Mꢂ.cm).
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Instruments and methods: Melting points were recorded on a LEICA VMHB Kofler system at
atmospheric pressure. Optical rotations were measured by using a Perkin Elmer 341 polarimeter.
Infrared (IR) spectra were recorded with a universal ATR sampling accessory on a Perkin Elmer FTꢀIR
Spectrum 100 spectrometer. The elemental analyses were carried out with a Flash 2000 Organic
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Elemental Analyzer (Thermo Scientific). H and C NMR spectra (C13APT or C13CPD experiments)
and recorded either on a Bruker AC 200 or on a Bruker DPX 300 spectrometer working at 200 and 300
MHz respectively. Chemical shifts are expressed in parts per million (ppm) from the residual nonꢀ
deuterated solvent signal CDCl3 (δΗ = 7.26, δC = 77.00) or DMSOꢀd6 (δΗ = 2.50, δC = 39.43).19 J values
are in hertz (Hz). Analytical HPLC was performed on a Thermo Scientific Surveyor Plus instrument
equipped with a PDA detector. Semiꢀpreparative HPLC was performed on a Thermo Scientific
SPECTRASYSTEM liquid chromatography system (P4000) equipped with a UVꢀvisible 2000 detector.
Lowꢀresolution mass spectra were obtained with a Finnigan LCQ Advantage MAX (ion trap) apparatus
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equipped with an electrospray (ESI) source. Highꢀresolution mass spectra were recorded on a LCT
Premier XE benchtop orthogonal acceleration timeꢀofꢀflight (oaꢀTOF) mass spectrometer (Waters
Micromass) equipped with an ESI source and in the positive mode. The absorption spectra were
recorded on a Varian Cary 50 scan spectrophotometer by using a rectangular quartz cell (Varian,
standard cell, Open Top, 10 × 10 mm, 3.5 mL). The emission/excitation spectra were recorded with a
Varian Cary Eclipse spectrophotometer after excitation/emission at the suitable wavelength (excitation
and emission filters: auto, excitation and excitation and emission slit: 5 nm) with a semiꢀmicro quartz
fluorescence cell (Hellma, 104FꢀQS, 10 × 4 mm, 1400 ꢃL). All fluorescence spectra were corrected.
The following equation was used to determine the relative fluorescence quantum yield:
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ФF(X) = (AS/AX)(FX/FS)(nX/nS)2 ФF(S)20
Where A is the absorbance at the excitation wavelength. (in the range 0.01ꢀ0.1 A.U.), F is the area
under the corrected emission curve, n is the refractive index of the solvents (at 25 °C) used in
measurements, and the subscripts S and X represent standard and unknown, respectively. The following
standards were used: 7ꢀhydroxycoumarin in PBS (ФF = 76%) and fluorescein in aq. 0.1 M NaOH (ФF
= 90%).
RPꢀHPLC separations: The following system was used for the analytical experiments: column
Thermo Hypersil GOLD C18 (5 ꢃm, 4.6 × 150 mm) with CH3CN and aq. 0.1% trifluoroacetic acid (aq.
TFA 0.1%, pH 2.0) as eluents [100% TFA (1 min), then linear gradient from 0 to 80% (45 min) of
CH3CN]. UV detection at 254 nm. The following chromatographic system was used for the purification
step: column Varian Kromasil C18 (10 ꢃm, 21.2 × 250 mm) with CH3CN and aq. TFA 0.1% as eluents
with the following gradient [100% TFA (5 min), then linear gradient from 20 to 100% (49 min) of
CH3CN] at a flow rate of 20.0 mL/min. Double UV detection was achieved at 368 and 456 nm.
Pyridone (3). This compound was prepared in 96% e.e. according to the Fang modification21 of
Comins’strategy. The e.e. was determined using HPLC equipped with an analytical column Chiralcel
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ODH as chiral stationary phase: eluent system: heptane/isopropanol 9/1; flow: 1 mL/min; UV detection:
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254 nm; retention times: tR1 = 17.94 min (area 1 = 1.95) and tR2 = 22.86 min (area 2 = 98.05).
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2-Chloro-6-hydroxyquinolin-3-carbaldehyde (5). To
a
stirred solution of 2ꢀchloroꢀ6ꢀ
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methoxyquinolinꢀ3ꢀcarbaldehyde (500 mg, 2.26 mmol) in dry CH2Cl2 (15 mL) cooled at ꢀ78 °C was
added BBr3 (0.75 mL, 7.91 mmol, 3.5 equiv.) dropwise. After stirring for 30 min at ꢀ78 °C, the mixture
was warmed to rt and stirred for further 2 h. Thereafter, the reaction mixture was cooled to 0 °C and
quenched by adding ice and cold deionised water. The resulting mixture was extracted with ethyl acetate
(2 × 50 mL). The combined organic layers were washed with brine, dried over anhydrous MgSO4 and
concentrated under reduced pressure. The resulting residue was triturated with CH2Cl2 to afford a
yellow solid (380 mg, 1.83 mmol, yield 82%). Mp 210 °C (decomposition). IR 3278, 1693, 1579, 1500,
1219 cmꢀ1. 1H NMR (300 MHz, DMSOꢀd6) δ10.50 (s, 1H, OH), 10.35 (s, 1H), 8.79 (s, 1H), 7.90 (d, J =
9.0 Hz, 1H), 7.52 (dd, J = 2.4, 9.0 Hz, 1H), 7.43 (d, J = 2.4 Hz, 1H). 13C NMR (75.5 MHz, DMSOꢀd6) δ
189.4, 156.7, 145.3, 143.6, 139.3, 129.2, 127.7, 126.0, 125.9, 110.2. HPLC tR = 16.1 min, purity > 96%.
MS(ESI+):m/z (%): 208 (100) [M + H]+, calcd for C10H6ClNO2: 208.
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6-(2-Azidoethoxy)-2-chloro-quinolin-3-carbaldehyde (6).To a stirred solution of 5 (100 mg, 0.48
mmol) in dried DMF (1.5 mL), under atmosphere of Argon, at rt was added anhydrous solid K2CO3
(333 mg, 2.4 mmol, 5 equiv.). The reaction mixture turned red immediately. After 5 min stirring at room
temperature, the azide (126 mg, 0.60 mmol, 1.25 equiv.) was added to the solution. The reaction
mixture was heated at 50 °C for 12 h. The mixture was cooled to room temperature, solubilized in a
minimum of water/DMSO (1:1) and directly filtered over silica gel (EtOAc/cyclohexane 1:1) to afford
the 2ꢀazidoethyl ether 6 (143 mg, 0.52 mmol, 63%) as a yellow solid. Mp: 124ꢀ126 °C. IR 2928, 2118,
1690, 1577, 1497, 1236 cmꢀ1. 1H NMR (200 MHz, DMSOꢀd6) δ 10.33 (s, 1H), 8.74 (s, 1H), 7.91 (d, J =
9.0 Hz, 1H), 7.65 (d, J = 2.6, 1H), 7.57 (dd, J = 2.6, 9.0, 1H), 4.30 (t, J = 4.6 Hz, 2H), 3.76 (t, J = 4.6
Hz, 2H). 13C NMR (50 MHz, DMSOꢀd6) δ 189.3, 156.7, 146.5, 144.5, 139.5, 129.2, 127.4, 126.2, 125.9,
108.5, 67.2, 49.2. HPLC: tR = 24.9 min, purity > 92%. MS(ESI+): m/z (%): 277 (100) [M + H]+, calcd
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for C12H10ClN4O2: 277. Elemental analysis (%) calcd: C,52.09; H, 3.28; N, 20.25; found: C, 52.18; H,
3.27; N, 20.24.
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(6-(2-Azidoethoxy)-2-chloroquinolin-3-yl)methanol (7). To a stirred solution of 6 (164 mg, 0.59
mmol) in 4 mL of CH3OH/CH2Cl2 (1:1) at 0 °C was added solid NaBH4 (22 mg, 0.59 mmol, 1 equiv.).
The reaction mixture was warmed to rt and stirred for 3 h. Then the solution was quenched with a
solution of sat. aq. NH4Cl (20 mL) and extracted with EtOAc (3 × 40 mL). The combined organic layers
were washed with brine (20 mL), dried over anhydrous MgSO4 and filtered, and the solvent was
removed under reduced pressure. The resulting residue was dissolved in a minimum of DMSO and
purified by chromatography over silica gel (CH2Cl2 then cyclohexane/EtOAc 1:1) to afford the alcohol
7 (123 mg, 0.44 mmol, 74%) as a yellow solid. Mp: 150ꢀ152°C. IR 3383, 2928, 2107, 1621, 1497, 1221
cmꢀ1. 1H NMR (300 MHz, DMSOꢀd6) δ 8.31 (s, 1H), 7.83 (d, J = 9.0 Hz, 1H), 7.48 (s, 1H), 7.37 (d, J =
9,0, 1H), 5.72 (s, 1H), 4.66 (s, 2H), 4.29 (s, 2H), 3.74 (s, 2H). 13C NMR (75.5 MHz, DMSOꢀd6) δ 156.3,
145.8, 141.8, 134.6, 134.0, 129.0, 128.2, 122.1, 106.8, 67.0, 59.8, 49.4. HPLC: tR = 20.9 min, purity >
93%. MS(ESI+): m/z (%): 279.13 (100) [M + H]+, calcd for C12H12ClN4O2: 279. Elemental analysis (%)
calcd: C, 51.72; H, 3.98; N, 20.10; found: C, 51.55; H, 4.07; N, 19.70
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6-(2-Azidoethoxy)-3-(bromomethyl)-2-chloroquinoline (2). To a stirred solution of 7 (121 mg, 0.44
mmol) in dry CH2Cl2 (4 mL) cooled at 0 °C was added PBr3 (45 ꢃL, 0.47 mmol, 1.1 equiv.). The
reaction was stirred for 12 h at rt. Next, the solution was cooled to 0°C and quenched by sequential
addition of CH3OH (200 ꢃL) and sat. aq. NaHCO3 (20 mL), extracted with dichloromethane (3 × 30
mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous MgSO4,
filtered and the solvent was removed under reduced pressure. The resulting residue was purified by
chromatography over silica gel (cyclohexane/EtOAc 9:1) to afford 2 (97 mg, 0.28 mmol, 65% yield) as
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a white solid. IR 2931, 2107, 1621, 1494, 1234, 829 cmꢀ1. H NMR (300 MHz, CDCl3) δ 8.08 (s, 1H),
7.89 (d, J = 9.0, 1H), 7.39 (dd, J = 2.1, 9.0 Hz, 1H), 7.03 (d, J = 2.1 Hz, 1H), 4.67 (s, 2H), 4.23 (t, J =
4.8 Hz, 2H), 3.67 (t, J = 4.8 Hz, 2H). 13C NMR (75.5 MHz, CDCl3) δ 156.9, 147.5, 143.4, 137.9, 129.8,
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129.7, 128.0, 123.5, 105.8, 67.2, 49.8, 29.8. HPLC: tR = 29.7 min, purity > 98%. HRMS (ESI+): Calc.
for C12H11BrClN4O: 342.9784; found: 342.9784.
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(S)-7-(6-(2-Azidoethoxy)-2-chloroquinolin-3-methyl)-4-ethyl-4-hydroxy-1,7-dihydro-4H-
pyranol[3,4-c]pyridine-3,8-dione (8). To a stirred solution of 3 (54 mg, 0.26 mmol) in dry DME (4
mL) at 0 °C was added anhydrous tBuOK (32 mg, 0.28 mmol, 1.1 equiv.). The resulting mixture was
stirred at 0 °C for 15 min and at rt for further 15 min. A solution of the benzyl bromide 2 (97 mg, 0.28
mmol, 1.1 equiv.) in dry DME (2 mL) was added to the previous solution, and the resulting reaction
mixture was stirred for 12 h at rt and 1 h under reflux (for a complete conversion). Thereafter, the
mixture was cooled to rt and concentrated. The residue was filtered over a silica gel pad
(cyclohexane/EtOAc 1:1 then EtOAc) to afford 8 (73 mg, 0.15 mmol, 60%) as a white solid; Mp: 82ꢀ
84°C. IR 3371, 2940, 2109, 1743, 1657, 1599, 1228 cmꢀ1.1H NMR (300 MHz, CDCl3) δ 7.98 (s, 1H),
7.89 (d, J = 9.0 Hz, 1H), 7.62 (d, J = 7.2 Hz, 1H), 7.40 (dd, J = 9.3; 2.4, 1H), 7.05 (d, J 2.4, 1H), 6.58 (d,
J 7.2, 1H), 5.57 (d, J 16.2, 1H), 5.45 (d, J = 15.3 Hz, 1H), 5.24 (d, J = 15.3 Hz, 1H), 5.16 (d, J = 16.2
Hz, 1H), 4.22 (t, J = 4.8, 2H), 3.78 (s, 1H), 3.66 (t, J = 5.1 Hz, 2H), 1.83 ꢀ 1.76 (m, 2H), 0.97 (t, J = 7.2
Hz, 3H). 13C NMR (75.5 MHz, CDCl3) δ 173.6, 158.7, 156.9, 149.0, 146.9, 143.3, 138.2, 138.0, 129.7,
127.9, 127.1, 123.8, 119.1, 105.9, 103.2, 72.1, 67.2, 66.3, 50.4, 49.8, 31.3, 7.6. HPLC: tR = 23.3 min,
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purity > 86%. [α]22 +27° (c 2.7, CHCl3). HRMS (ESI+): Calc. for C22H21ClN5O5: 470.1231; found:
D
470.1237.
(S)-7-(6-(2-tert-Butyl
ethoxycarbamate)-2-chloroquinolin-3-methyl)-4-ethyl-4-hydroxy-1,7-
dihydro-4H-pyranol[3,4-c]pyridine-3,8-dione (9).The azide derivative 8 (26 mg, 0.05 mmol) was
dissolved in a mixture of EtOAcꢀCH3OH (2 : 1, v/v, 3 mL). Diꢀtertꢀbutyl dicarbonate (24 mg, 0.11
mmol, 2 equiv.) and Lindlar catalyst (7 mg, 5% by wt.) were sequentially added. The reaction flask was
purged and flushed with Ar gas then with H2 gas. The resulting reaction mixture was stirred at rt under
an H2 atmosphere for 12 h. Thereafter, the reaction mixture was filtered through a Celite® 545 pad, the
filter cake was washed with EtOAc (5 mL), and the combined filtrates were concentrated under reduced
pressure. The resulting residue was filtered on silica gel pad by using a step gradient of EtOAc (20ꢀ
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80%) in cyclohexane as the mobile phase affording 9 as a white solid (28 mg, 0.05 mmol, yield 93%);
Mp: 88ꢀ90°C. IR 3347, 2977, 1746, 1703, 1658, 1497, 1164 cmꢀ1.1H NMR (200 MHz, CDCl3) δ 7.95 (s,
1H), 7.86 (d, J = 9.2 Hz, 1H), 7.61 (d, J = 7.2 Hz, 1H), 7.34 (dd, J = 2.4, 9.2 Hz, 1H), 7.02 (d, J = 2.4
Hz, 1H), 6.59 (d, J = 7.2 Hz, 1H), 5.58 (d, J = 16.1 Hz, 1H), 5.42 (d, J = 15.2 Hz, 1H), 5.24 (d, J = 15.6
Hz, 1H) 5.15 (d, J = 16.6 Hz, 1H), 5.07ꢀ5.02 (m, 1H), 4.09 (t, J = 5.2 Hz, 2H), 3.79 (s, 1H), 3.56 (t, J =
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5.2 Hz, 2H), 1.86ꢀ1.75 (m, 2H), 1.43 (s, 9H), 0.97 (t, J = 7.4 Hz, 3H). C NMR (50 MHz, CDCl3) δ
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173.6, 158.7, 157.3, 155.7, 149.1, 146.7, 143.2, 138.1, 138.0, 129.6, 128.0, 127.1, 123.7, 119.2, 105.9,
103.2, 79.6, 72.1, 67.5, 66.3, 50.3, 39.8, 31.4, 28.3, 7.6. HPLC: tR = 24.7 min, purity > 96%. [α]22D +27°
(c 2, CHCl3). HRMS (ESI+): Calc. for C27H31ClN3O7: 544.1851; found: 544.1857.
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10-(2-tert-Butyl ethoxycarbamate)-(S)-camptothecin(10). In an ovenꢀdried reaction glass tube, a
solution of 9 (18 mg, 0.03 mmol) in dry CH3CN (1 mL) and KOAc (8 mg, 0.08 mmol, 2.5 equiv.) were
mixed. Ar gas was bubbled through the reaction mixture for 15 min. Then, (PPh3)2Pd(OAc)2 (4.9 mg,
0.006 mmol, 0.2 equiv.) was added and Ar gas was bubbled again through the reaction mixture for
further 15 min. The flask was sealed and the reaction mixture was stirred at 100 °C for 18 h. Thereafter,
the mixture was allowed to cool at rt and then directly filtered over a silica gel pad by using a step
gradient of EtOAc (50ꢀ100%) in cyclohexane as the mobile phase to afford the desired fused
pentacyclic alkaloid derivative 10 as a yellow light solid (12 mg, 0.02 mmol, yield 71%). IR 3345,
2977, 1755, 1656, 1502, 1238 cmꢀ1. H NMR (300 MHz, CDCl3) δ 8.19 (s, 1H), 8.04 (d, J = 9.3, 1H),
1
7.61 (s, 1H), 7.36 (dd, J = 2.1, 9.3 Hz, 1H), 7.06 (d, J = 2.1 Hz, 1H), 5.71 (d, J = 16.2 Hz, 1H), 5.29ꢀ
5.20 (m, 4H), 4.22 (s, 1H), 4.16 (t, J = 4.8 Hz, 2H), 3.66ꢀ3.62 (m, 2H), 1.94ꢀ1.83 (m, 2H), 1.47 (s, 9H),
1.01 (t, J = 7.2 Hz, 3H). 13C NMR (75.5 MHz, CDCl3) δ 173.8, 157.6, 157.5, 155.9, 150.1, 149.9, 146.3,
144.7, 131.0, 129.5, 129.2, 129.0, 123.3, 118.0, 106.0, 97.4, 79.7, 72.7, 67.5, 66.1, 50.0, 39.9, 31.4,
28.3, 7.8. HPLC: tR = 23.5 min, purity > 93%. HRMS (ESI+): Calc. for C27H31ClN3O7 : 508.2084;
found: 508.2090.
Fluorescein-isonipecotic acid derivative (4). This fluorophore carboxylic acid was prepared
according to a procedure recently reported by us22
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Fluorescein-labeled camptothecin (1).
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(a) Removal of the Boc group. To a solution of 10 (12 mg, 0.02 mmol) in CH2Cl2 (1 mL) cooled at 0
°C was added TFA (0.4 mL, 2.4 mmol, 118 equiv.) dropwise and the resulting mixture was stirred at rt
for 2 h. Thereafter, the reaction mixture was concentrated, and the residue was triturated with preꢀcooled
Et2O to afford the corresponding TFA salt of aminoꢀcamptothecin derivative 11. MS (ESI+): m/z (%):
408 (100) [M+H]+, calcd for C22H22N3O5: 408.
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(b) Amide coupling reaction. The TFA salt was dissolved in dry DMF (0.6 mL) and the mixture was
cooled to 0 °C. Fluorescein carboxylic acid 4 (12.2 mg, 0.03 mmol, 1.2 equiv.), DIEA (20 ꢃL, 0.16
mmol, 7 equiv.), HOBt monohydrate (3.6 mg, 0.03 mmol, 1.2 equiv.), and DCC (6 mg, 0.03 mmol, 1.2
equiv.) were sequentially added. The resulting reaction mixture was stirred at rt for 12 h and then
evaporated almost to dryness. The resulting residue was purified by semiꢀpreparative RPꢀHPLC (tR=
26.91ꢀ29.76 min). The productꢀcontaining fractions were lyophilized to afford the desired fluorescent
bioconjugate 1 as an orange amorphous powder (7.7 mg, 0.01 mmol, overall yield 40% for the two
steps). IR 3353, 2940, 1748, 1594, 1453, 1173 cmꢀ1.1H NMR (300 MHz, DMSOꢀd6) δ 8.50 (s, 1H),
8.10ꢀ8.04 (m, 2H), 7.70ꢀ7.68 (m, 2H), 7.62ꢀ7.60 (m, 1H), 7.52ꢀ7.46 (m, 3H), 7.27 (s, 1H), 7.14ꢀ7.11 (m,
2H), 6.78ꢀ6.76 (m, 3H), 5.41 (s, 2H), 5.24 (s, 2H), 4.16ꢀ4.12 (m, 2H), 4.05ꢀ4.02 (m, 1H), 3.52ꢀ3.44 (m,
1H), 3.50ꢀ3.45 (m, 2H), 2.36ꢀ2.26 (m, 2H), 1.86ꢀ1.84 (m, 2H), 1.60ꢀ1.56 (m, 2H), 1.22ꢀ1.15 (m, 3H),
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0.88 (t, J = 6.9 Hz, 3H). C NMR (75.5 MHz, DMSOꢀd6) δ 173.9, 172.4, 166.3, 157.2, 156.7, 156.6,
150.1, 149.9, 145.6, 143.9, 135.8, 131.6, 130.5, 130.4, 130.2, 130.1, 129.9, 129.6, 129.3, 129.3, 127.1,
123.0, 120.8, 118.3, 115.2, 106.9, 102.8, 96.0, 72.3, 66.8, 65.2, 50.1, 45.6, 41.0, 37.9, 30.2, 28.4, 27.9,
8.5, 7.7. HPLC: tR = 21.2 min, purity > 95%. HRMS (ESI+): Calc. for C48H41N4O10: 833.2823; found:
833.2844.
Compounds, cell lines and culture conditions
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NIH3T3 mouse fibroblasts cells (obtained from America Type Culture Collection) were cultured in
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high glucose DMEM supplemented with 10% fetal calf serum (FBS), 2 mM Lꢀglutamine and 100 U/ml
penicillin/streptomycin at 37 °C in a humidified 5% CO2 incubator. Human cell lines from glioblastoma
U87 and T98 were obtained from the American Type Culture Collection. The T98 cells were maintained
in HAMꢀF12 and U87 cells were maintained in DMEM (Invitrogen, Cergy Pontoise, France) containing
10% FBS and 1% sodium pyruvate at 37 °C in a humidified atmosphere containing 5% CO2. Culture
media were replaced every three days.
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Topoisomerase I relaxation assay. Topo I inhibitory activity of CPTꢀFI versus CPT was assayed by
supercoiled DNA relaxation assay. Supercoiled DNA (TopoGEN, FL, USA) was incubated at 37°C for
30 min with purified human Topo I (TopoGEN, FL, USA) which was preꢀmixed with increasing
concentrations CPTꢀFI (50ꢀ200 ꢃM) or CPT (as control, 50 ꢃMꢀ1 mM), then terminated with proteinase
K/SDS stop buffer, and loaded on to 2% agarose gel. The standard reaction system contained: 0.25 ꢁg
DNA, 10 mM TrisꢀHCl, pH 7.9, 1 mM EDTA, 150 mM NaCl, 0.1% bovine serum albumin, 0.1 mM
spermidine, and 1 U Topo I extracted from T98 cell lines. The samples were electrophoresed in a
horizontal 2% agarose gel in TAE (tris base, acetic acid and EDTA) buffer at 1.5 V/cm during 2 h at
room temperature. DMSO concentrations in each reaction were maintained at 1 % by the addition of
serially diluted drug stocks so as not to produce solvent mediated inhibition of Topo I activity. The gels
were only postꢀstained with ethidium bromide, destained in water and photographed under UV
illumination.
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Cell growth assay
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T98 cells were plated in quadruplicates in 12ꢀwell plates (Costar, 15 × 103 cells/well for 24 h before
treatment in HAMꢀF12 (for T98 cells) and DMEM (for U87 cells), supplemented with 10% FBS. Cells
were then incubated in the absence or presence of graded concentrations of CPT (TCI, Zwijndrecht,
Belgium) or CPTꢀFI (0.05 to 5 ꢃM) for 24, 48, and 72 h, respectively. Controls were determined by
using a DMSO or FI concentration corresponding to the dilution of CPT used for 5 ꢃM concentration
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tested. After treatment, cells were harvested, rinsed by PBS, detached by accutase (Millipore,
Molsheim, France), and cellular density was measured by direct cell counting in tampon ZꢀtriꢀPak
(Beckman, Lognes, France) with an electronic cell counter (Z2, Coulter Beckmann).
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Cell cycle analysis
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T98 cell lines were plated in 75 cm2 flasks (Techno plastic products AG, Trasadingen, Switzerland).
When cells reached 80% confluence, they were incubated in the absence or presence of CPT and CPTꢀ
FI (50 nM and 0.5 ꢃM) for 24 h or 48 h. Cells treated with DMSO corresponding to CPT concentrations
were used as control. Cell cycle analysis was done by DNA staining with propidium iodide by flow
cytometry. Briefly, after treatment, cells were trypsinized, harvested in PBS, and fixed in ice cold 70%
ethanol for 2 h. Staining was done with 0.6 mg/mL RNase (30 min, RT, SigmaꢀAldrich) and 50 ꢃg/mL
propidium iodide (30 min, RT, SigmaꢀAldrich). Cell cycle status was quantified by FacsCalibur flow
cytometer (BD Biosciences) using CellQuest analysis software (Becton Dickinson Immunocytometry
Systems) using an excitation laser set at 480 nm and a detection wavelength of 670 nm (FL3), to avoid
detection of FI excitation.
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Imaging of camptothecin-FI incorporation in living cells
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Glioblastoma T98 cell line cultured in glass bottom microwell dishes 35ꢀmm size (MatTek, Ashland,
USA) containing growth medium, and maintained in a live cell chamber at a fixed temperature of 37°C.
T98 cellular uptake of CPT and CPTꢀFI 1 (5 ꢃM) was monitored using a confocal laserꢀscanning
microscope TSC SP5 by video timeꢀlapse microscopy at the gain and offset (Leica, Heidelberg,
Germany) equipped with a AOBS system and a white laser (excitation flexibility).
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The cellular uptake of CPT and CPTꢀFI was also examined by Flow cytometry. T98 cells were plated
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in 75 cm2 culture flasks containing the growth medium 24 h before treatments with CPT and CPTꢀFI
during 5, 15 and 30 min or 24 and 48 h treatments with CPT and CPTꢀFI (0.5 ꢃM each). The
quantitative fluorescence was observed with a FacsCalibur flow cytometer (BD Biosciences) using
CellQuest analysis software (Becton Dickinson Immunocytometry Systems) using an excitation laser set
at 480 nm and a detection wavelength of 515 nm, to allow detection of FI excitation.
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Heterotopic tumoral growth study
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Swiss nude mice (Charles River Laboratory, L’Arbresle, France) were kept in a temperatureꢀ
controlled room (21 ± 1 °C), under an established photoperiod (lights on 07.00ꢀ19.00 h) with free access
to food and tap water. The work described in this article was carried out in accordance with the
Directive 2010/63/EU of the european parliament and of the council of 22th September 2010 on the
protection of animals used for scientific purposes, published in the Official Journal of the European
Union L276/33 (20.10.2010) and authorized by the French Ethical Committee. These experiments were
conducted under the supervision of authorized investigators (H. Castel; authorization no. 76.98 from the
Ministry of Food, Agriculture and Fisheries.
For intratumoral experiments, the tumorigenic NIH3T3 cell lines were used to monitor the direct
intratumoral penetration of the CPTꢀFI. Thus, a cohort of 6ꢀ to 7ꢀweekꢀold male Swiss Nude mice
(Charles River Laboratory, L’Arbresle, France) was sedated by isoflurane inhalation (Aerane, Baxter)
and implanted subcutaneously (s.c.) into right flanks with 1 × 106 NIH3T3 cells/100 ꢃL PBS. A stock
solution of CPTꢀFI in DMSO (10ꢀ2 M) was prepared and stored at ꢀ20°C. Aliquots of CPTꢀFI at 40
mg/kg, were freshly prepared in DMSO/NaCl 0.9%, for each day injection. When tumors reached a
volume of 700 mm3, 10 ꢃL of CPTꢀFI or vehicle at the corresponding concentration were injected in the
tumor. Tumors sizes were measured every two days by the use of a caliper (Bioblock, 0.01mm
precision, Fisher Scientific SAS, Illkirsh, France) and volume was calculated as V = (1/2) a X b2 (a,
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length of the apparent tumoral mass; b, width of the apparent tumoral mass). Mice weight was also
controlled every two days. Animals were sacrificed when tumors reached the maximal ethical volume of
2000 mm3. Tumors were immediately collected, frozen in isopentane and maintained at ꢀ80°C until
immunohistochemistry experiments.
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For intravenous administration of FITC or CPTꢀFI, and tumor distribution, a cohort of 6ꢀ to 7ꢀweekꢀ
old male Swiss Nude mice (Charles River Laboratory, L’Arbresle, France) was sedated by isoflurane
inhalation (Aerane, Baxter) and implanted subcutaneously (s.c.) into right flanks with 1 × 106
glioblastoma cell line U87 cells/100 ꢃL PBS. A stock solution of FITC or CPTꢀFI in DMSO (10ꢀ2 M)
was prepared and stored at ꢀ20 °C. Aliquots of CPTꢀFI, were freshly prepared in DMSO/NaCl 0.9%, for
each injection. When tumors reached a volume of 300 mm3, 100 ꢃL of FITC (66 mg/kg; 0.5 ꢃmol) or
CPTꢀFI (66 mg/Kg) were injected via the tail vein. Animals were sacrificed 4 or 8 hours after bolus
administration of the fluorescent molecules. Tumors were immediately collected, frozen in isopentane
and maintained at ꢀ80 °C until immunohistochemical analysis.
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NIH3T3 Tumor sections (60 ꢃm thick) or U87 tumor sections (60 ꢃm thick) resulting from
intravenous exposure to FITC or CPTꢀFI, were cut in a Cryostat (Cryostat CM1950, Leica
microsystèmes, Nanterre, France), mounted on gelatinꢀcoated slides and maintained at ꢀ20 °C until
experiment. Briefly, slides were first rinsed in PBS, fixed in a paraformaldehyde (PFA) solution (4%, 20
min, RT) and washed three times in PBS. Sections were permeabilized and preꢀincubated in a PBS,
BSA 0.1%, NDS, 1:50, Triton X100 0.3%. Slides were then incubated with 4',6ꢀdiamidinoꢀ2ꢀ
phenylindole dihydrochloride (DAPI, 1/1000e, 10 min, RT, SigmaꢀAldrich, Saint Quentin Fallavier,
France), washed again and mounted in Mowiol 4ꢀ88 (Calbiochem, Meudon, France). U87 tumors were
coꢀimmunostained with an antiꢀCollagen IV antibody (Santa Cruz Biotechnology, Le Perrayꢀenꢀ
Yvelines, France), in order to visualize hyperplastic tumor vessels. Slices were then examined using a
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confocal conventional microscope (Leica TCS SP2 AOBS, Nanterre, France) and a macroconfocal
miscroscope (Leica TCS LSI). Analyses from the confocal acquisitions were achieved by using the
ImageJ 1.44. software.
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Statistical analysis
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All statistical analyses were conducted using the GraphPad Prism 4.00 software (GraphPad
Software). Among the growth tumoral cell experiments, nonꢀparametric statistical tests were used, i.e.
KruskalꢀWallis ANOVA and MannꢀWhitney tests for dual comparisons. P < 0.05 was considered as
significant.
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Results and Discussion
To illustrate that proofꢀofꢀconcept of possible fluorescent modifications of chemotherapy for
investigation of biodistribution and pharmacokinetic, the most classical camptothecinoid, 20(S)ꢀ
camptothecin, was selected as bioactive model compound, along with the wellꢀestablished fluorescein as
the green emitting fluorescent label.23 Although nearꢀinfrared probes are ordinary used for in vivo
imaging, green fluorescence dyes have been preferred and widely used to image tumors and superficial
tissues on small animals such as mice, mainly because of the short penetration distance of green light in
tissue. 24
To be viable and rapidly extended to a wide range of CPT analogues used as cancer therapy in
patients, a late stage modular synthetic approach to the fluorescent conjugate was investigated. Also, it
is noteworthy that most of the biologically active analogues are AB and/or E ring modified CPT.
Consequently, the synthetic route chosen for the construction of the fluorescent conjugate involves the
sequential and late stage assembly of three distinct molecular units: the AB ring system 2 with DE
pyridinoneꢀbased heterocycle system 3 (described by Comins)25 and finally the subsequent peptideꢀtype
coupling reaction between the pentacyclic alkaloid formed and the fluorophore 4 (Fig. 2). The use of an
unusual fluorescein derivative bearing a tertiary amide of isonipecotic acid at the 2’ꢀposition instead of
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the current carboxyl group was preferred to provide steric protection and so to improve the
photostability of the fluorescein core.26 Furthermore, this is the most common way to avoid the
undesired cyclization equilibrium leading to the formation of the colorless and nonꢀfluorescent
spirolactone (especially at pH < 6). This fluorescein derivative exhibits valuable fluorescence properties
in the green spectral region and was attached at the C10 position of the Aꢀring of the bioactive
compound to retain the biological activity. Indeed, substitution on rings C,D and E are usually not well
tolerated,13 and introduction of an electronꢀrich function at C10 position does not alter the biological
activity of CPT, as illustrated by the C10 phenol substitution found in the topotecan structure (Figure 1).
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The DE ring fragment 3 was prepared from 2ꢀmethoxypyridine according to the Fang modification of
Comins’strategy in 96% e.e (Scheme 1, part 1).21 The AB building block 2 was synthesized in four steps
from commercially available 2ꢀchloroꢀ6ꢀmethoxyꢀ3ꢀquinolinecarboxaldehyde (Scheme 1, part 2). First,
deprotection of the aromatic methyl ether was achieved with BBr3 in dry CH2Cl2 to afford 5 in 82%
yield. Next, the resulting phenol derivative was reacted with 2ꢀazidoethyl tosylate27 in a standard SN2
reaction to give the corresponding ether 6. It is noteworthy that the azido group has proven to be a
particularly useful amine precursor due to its good stability under both acidic and basic conditions
required for the next steps of the synthesis. The aldehyde 6 was readily reduced with NaBH4 in CH2Cl2ꢀ
MeOH leading to the corresponding alcohol 7 in a good 74% yield. Final treatment of the primary
alcohol with phosphorus tribromide in dry CH2Cl2 afforded the AB fragment 2 with 65% yield.
The two fragments 2 and 3 were joined as described, on treatment with tꢀBuOK in 1,2ꢀ
dimethoxyethane (DME) to provide the tetracycle 8 (Scheme 2). However, the Heck ringꢀclosure
strategy failed to form the C ring system, whatever the reaction conditions tested, probably due to the
presence of the azido functionality. However, the use of Lindlar catalyst along with (Boc)2O directly
and chemoselectively reduced azido 8 into the corresponding NꢀBocꢀprotected amine 9 in almost
quantitative yield (93%), without dehalogenation of the aromatic chloride.28 Gratifyingly, the Heck
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reaction proceeded to afford the corresponding pentacyclic alkaloid 10 in a good 71% yield. Next, the
Boc group was removed with trifluoroacetic acid (TFA) in CH2Cl2 to furnish the corresponding primary
amine 11. The amineꢀreactive labeling reagent 4 derived from fluorescein was prepared using an
optimized (twoꢀstep) procedure inspired by the work of Gao et al. (Scheme 3).26 Amidification of
fluorescein with tertꢀbutyl isonipecotate was readily achieved by treatment with DCCꢀHOBt in dry
DMF. Thereafter, the tertꢀbutyl ester was removed by TFA in CH2Cl2. Due to the high polarity of the
resulting carboxylic acid derivative, a purification by semiꢀpreparative RPꢀHPLC was found more
convenient than standard chromatography over silica gel. Thus, fluorescein 4 was obtained with a good
43% overall yield. Compared to the synthetic protocol reported by Gao et al.26 which involves the
chromatographic isolation of the Nꢀhydroxysuccinimidyl ester (NHS ester) of fluorescein prior to its
aminolysis with an alkyl isonipecotate derivative, our synthetic alternative significantly improved the
yield and shortened the overall duration of the synthetic process.22 Finally, this fluorescent carboxylic
acid was coupled to the primary amino group of 11 under standard peptideꢀcoupling conditions. In our
case, DCCꢀHOBt in the presence of DIEA was preferred to a phosphonium or an uronium salt (such as
BOP or TSTU) because these latter coupling reagents are prone to react with the phenol group of
fluorescein to give undesired sideꢀproducts leading to a dramatic drop in the isolated yield for the
targeted fluorescent compound. The formation of the CPTꢀfluorescein conjugate 1 (CPTꢀFI) was
carefully checked by analytical RPꢀHPLC to avoid overꢀcoupling reaction between the product 1 and
the unreacted fluorescein 4. Upon completion of the conjugation reaction, the crude mixture was
directly purified by semiꢀpreparative RPꢀHPLC to isolate pure compound 1 in a satisfying 40% yield
over 2 steps. Its structure was confirmed by various spectroscopic data and its high purity was checked
by RPꢀHPLC (see Supporting Information). Analysis of 1 using ESI mass spectrometry indicated the
covalent attachment of the fluorescein label and confirmed the integrity of the pentacyclic scaffold of
CPT. Thus, the first synthesis of CPTꢀFI was achieved in 11 steps and 1.4% overall yield from
commercial 2ꢀmethoxypyridine and 15.8 % from the known pyridinone 3.
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The fluorescent probe 1 in hand, its complete photophysical properties were determined, along with
those of 4 and 11 (Table 1 and Figure 3), under simulated physiological conditions (i.e., phosphate
buffered saline (PBS), pH 7.5), to ensure that the spectral properties of the fluorescein core were kept
upon its conjugation to the CPT alkaloid scaffold. As expected, the UVꢀvis absorption spectrum of the
conjugate 1 was the sum of CPT derivative 11 and fluorescein 4 (Fig. 3a), that suggests very weak or no
electronic interaction (e.g., groundꢀstate dimer) between these two fluorophores in the ground state.
Although the fluorescein derivative 1 displays a smaller fluorescence quantum yield of 37% compared
to the parent free xanthene dye 4, satisfyingly, its emission and excitation spectra of 1 are in good
agreement with those recorded for 4 or those generally reported for fluorescein derivatives such as 4
(Figure 3b). Consequently, as anticipated and experimentallyꢀvalidated, the fluorescein part of the
chromophoric pair 1 could be selectively excited at 480 nm demonstrating attractive spectral factures
suitable for further biological imaging applications (Figure 3a). Finally, for informative purpose, the
ability of the CPT part of the probe to transfer energy to the fluorescein moiety was also investigated.
Indeed, as illustrated in Figure 3a, upon excitation at 350 nm (absorption maximum of CPT core), CPTꢀ
FI exhibits a strong green emission band centered at 524 nm and a weak blue emission band at 424 nm,
indicating energy transfer between these two FRET partners with efficiency up to 91% (see Supporting
Information for details about the energy transfer efficiency (E. T. E.) calculation).
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Next, biological properties of the conjugate 1 were investigated and compared with the CPT of
interest, 20(S)ꢀcamptothecin. In order to verify whether CPTꢀFI exhibited a similar CPTꢀinhibitory
activity on Topoisomerase I (TOPO I) activity, we first extracted nuclear proteins from T98 cells and
then evaluated the respective roles of CPT and CPTꢀFI on TOPO I activity. Then, we showed that T98
nucleus lysates converted a majority of supercoiled DNA into relaxed DNA as observed in lanes 2 and 3
(Fig 4A and B). Increasing concentrations of CPT (50 to 1000 ꢃM) and CPTꢀFI (50ꢀ200 ꢃM) prevented
the super coiled DNA from being converted into relaxed DNA (Fig 4A and B), CPTꢀFI being more
efficient than CPT in reproducible experiments. Thus, we demonstrate that CPTꢀFI exhibit a very potent
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inhibitory control of TOPO I from T98 suggesting that the linker modification of CPT to cross FITC
would have even favor CPT affinity for TOPO I.
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The prolonged exposure of proliferating and/or tumoral cells to topoisomerase inhibitors as CPTꢀ
derivatives results from DNA alteration and apoptotic events.29,30,31 Accurate evaluation of the impact of
CPTꢀFI as efficient antiꢀneoplasic compound, passed first through measurement of drug distribution
using in vitro model systems, as a more practical method of determining how efficiently a drug
penetrate cells. Thus, we first compared the kinetics of incorporation of CPT and CPTꢀFI in cultured
human glioblastoma cell line T98. CPTꢀFI (0.5 ꢃM) was shown to be rapidly internalized (short-term,
Figure 5a) in T98 cells after 5 minꢀtreatment, and appeared persistently cellꢀcaptured from 24 to 48 h
(long-term, Figure 5b), yielding a 10ꢀfold increase in intracellular intensity value from 5 min to 48 h
treatment (Figure 5c). We then performed timeꢀlapse confocal microscopy of CPTꢀFI (5 ꢃM)
incorporation into living T98 cells. When extracellularly applied, the fluorescence was detected after 5
min of exposure, as revealed by punctuate fluorescent dots detected around cell nuclei (Figure 5f). As
expected, upon excitation laser set at 480 nm, the autofluorescence of CPT could not be detected neither
at 5 min nor 30 min postꢀincubation as shown in Figure 5e (see Supporting Information). Retention in
tumor cells can be due to binding at the site of lethal activity (usually DNA), although basic drugs can
be sequestered in acidic organelles such as perinuclear endosomes.
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We thus hypothesize that specific labeling of CPT with FI may increase fluorescence intensity and/or
facilitate detection with microscopic available equipments facilitating the detection by optical imaging.
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Then, we compared the impact of CPT and CPTꢀFI analogues on growth of the glioblastoma U87and
T98 human cell lines from 24 to 72 h of treatment. Both cell lines were exposed to increasing
concentrations yielding doseꢀdependent proliferation rate inhibition starting from the 24 h treatment.
Phase contrast microphotographs of nonꢀtreated T98 glioblastoma cell lines, showed an adherent
monolayer of polygonal cells (Figure 6a). In the presence of CPT, and in a lesser extent, CPTꢀFI, there
was a morphological response characterized by cytoplasm retraction and rounding up of the cell body,
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compared to untreated cells (0) (Figure 6a). These effects were accompanied by a modified glioma cell
morphology and integrity, suggestive of a drastic impact of the two CPT analogues on human glioma
growth and apoptosis. Moreover, the strong CPTꢀFI staining observed at 5 ꢃM could be assigned to
their aggregation (c.f. Supporting Information for comparative solubility studies between CPTꢀFI and
CPT). As shown in Figure 5b, the doseꢀresponse curves indicate that CPT efficiently decreased cell
growth from the 0.05 ꢃM concentration. Likewise, CPTꢀFI showed significant cytotoxic effects on the
two cell lines in time and doseꢀdependent manners (Figure 6b and c).
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CPT and CPTꢀFI showed cytotoxicity, causing antiꢀproliferating activity in glioma cell lines in a
dose and timeꢀdependent manner. CPTꢀFI exhibiting more than a 10 fold decrease of the CPT efficiency
in T98 and retaining equivalent activity in U87. These observations show a real interest for a use of the
CPTꢀFI for body distribution and understanding of exposed nonꢀcancerous organs.
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In order to verify whether morphological changes and cell growth inhibition observed after CPTꢀFI
may be associated to topoisomerase and DNA duplication arrest, cell cycle was investigated in T98 in
the absence or the presence of both CPT and its derivatives (Figure 7). The cell cycle distribution of the
T98 glioblastoma cell line was examined via flow cytometry, in the absence or the presence of CPT
(0.05 and 0.5 ꢃM) after 24 h treatment, or CPTꢀFI (0.05 and 0.5 ꢃM) after 24 h and 48 h treatment. At
24 h, CPT induced a doseꢀdependent increase of the G2/M phase from 13% (control) to 24% (0.05 ꢃM)
and 73% (0.5 ꢃM), accompanied by a decrease of cell populations in the S phase of the cell cycle, from
17 to 11% (Figure 7a). In contrast, CPTꢀFI failed to markedly alter cell cycle after 24 h, but evoked a
CPTꢀlike G2/M cell accumulation after 48 h treatment (Control, 15%; 0.05 ꢃM, 19%; 0.5 ꢃM, 43%)
(Figure 7b). CPTꢀFI exhibits inferior cytotoxic effect than CPT partly due to its lower solubility in
physiological pH. Indeed CPTꢀFI markedly precipitates at 5 ꢃM (40%) at while this phenomenon was
observed from 10 ꢃM with CPT (c.f. Supporting Information). This observation suggests that we
compare the effects of 5 ꢃM CPT with those of 3 ꢃM CPTꢀFI on cell cycle and toxicity. Besides it has
been reported that substituents at position 10 of the quinoline part of the CPT derivatives may interact
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with the receptor site with the drug binding site of topoisomerase I cleavage complexes.32,13 While small
substituents at 10 position of the A ring generally increase efficacy in DNA topoisomerase I inhibition,
the presence of bulkier groups should more lead to diminished inhibitory activity. Interestingly, the
modification of CPT presented here to bear a bulky fluorescein derivative, displays a better affinity
toward topoisomerase I than the native CPT. However, we observed in cellulo activity of CPTꢀFI lower
than the unmodified CPT, likely attributed to a less hydrophobic properties and reduced membrane
penetration, to reach the cell nulei. Together these results thus confirm that CPT induced accumulation
in G2/M and T98 glioblastoma cell arrest, and also demonstrate that CPTꢀFI provokes similar effects
but requires longer incubation time and/or higher concentration, establishing the antiꢀcancer efficiency
in vitro of the CPTꢀFI derivative.
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Unfortunately, most drugs are not amenable to direct in vivo followꢀup because they lack sufficient
color or fluorescent properties except the clinicallyꢀused drugs doxorubicin, mitoxantrone and
topotecan, which exhibit autofluorescence. But initial evidence for limited penetration through tissue of
the commonly used anticancer drug doxorubicin was obtained by studying the distribution of its
fluorescence in a multicellular layer model.33 Here, to further extend the possible use of our CPTꢀ
fluorescent probe as a cytotoxic drug whose fate can be followed in vivo, intratumoral administrations of
CPTꢀFI (CPTꢀFI, 5 ꢃM, n=4) were performed once daily during four days in xenogafts of NIHꢀ3T3
murine fibroblastic cells in Nude mice (Figure 8a). As compared to intratumoral injections of
PBS/DMSO (sham, n=4, not shown), histological examination of CPTꢀFI injected tumors revealed that
green fluorescence accumulated to the greatest extent at the border of the tumor and near the site of
injection (Figure 8a). At higher magnification, CPTꢀFI appeared to be mainly distributed in lacks area
surrounding apparent viable cells whereas PBS injected tumors failed to show any detectable
autofluorescence (Figure 8b). Deeper inside the tumor, some CPTꢀFI green zones would show
cytotoxicity and diminished cell viability, and colocalization of the CPTꢀFI and DAPI stainings in
isolated cell, illustrates in vivo intranucleus incorporation (Figure 8c). Thus, confocal images of the
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CPTꢀFI distribution in consecutive sections show that only the tumor area exposed to CPTꢀFI injections
is detectable, suggesting limited penetration of the drug. This restricted diffusion into the tumor may be
due i) to rapid uptake of the drug by perivascular cells and ii) mostly by the cellular packing density and
the number of adhesions between cells, as recently proposed.34 Thus, intravenous administration
strategy was employed to monitor the in vivo behavior of CPTꢀFI compared to FITC alone and to
characterize tumor distribution. FITC (100 ꢃl, n=2) or CPTꢀFI (100 ꢃl, n=3) solutions at 66 mg/Kg was
bolus injected through the tail vein into Nude mice bearing U87 glioblastoma tumors. After 4 and 8 h,
tumors were frozen and then analyzed. Confocal microscopic images were acquired 4 h after FITC or
CPTꢀFI injection (Figure 9a). As expected, tumors did not show FITC signal whereas they exhibited
very dense green staining in vascular and perivascular compartments after exposure to systemic CPTꢀFI
(Figure 9a), and was mainly colocalized with collagen IV (Figure 9b). After 8 h, CPTꢀFI was not found
with collagen IV immunostaining, and was more distributed throughout the glioma tumor parenchyma
(Figure 9b). This data clearly indicate that CPTꢀFI can be delivered via intravenous injection, and that 8
h after administration, the labeled chemotherapy penetrated into the tumor parenchyma through the
leaky tumoral vascular network, particularly dense in glioma tumors. Thus, it can be expected that this
type of small taggedꢀantiꢀcancer molecules, may be more widely dedicated to the study and the
evaluation of pharmacokinetic, tumor penetration and nonꢀcancerous sites of deleterious side effects.
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Conclusions
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In summary, we have described for the first time an original synthetic approach enabling the efficient
fluorescent labeling of anticancer drug camptothecin which is known to exhibit a moderate chemical
stability. The key point was the introduction of an azidoꢀterminated linker onto the C10 position of the
CPT quinoline ring, which acts as an optimal protecting group for the primary amino group only
unveiled immediately prior to the final fluorescent labeling based on a peptideꢀtype coupling reaction.
Besides, careful functionalization at the C10 position of the pentacyclic alkaloid has shown to preserve
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the essential biological features of the CPT bioactive alkaloid, although longer incubation periods or
higher concentrations are required. In addition, we demonstrated the feasibility of using a fluorescent
topoisomease inhibitor CPT as a model drug to monitor in realꢀtime cell in vivo delivery by fluorescent
imaging inducing tumor senescence and arrest of cell growth. This new FIꢀtagged chemotherapy with
similar characteristics as the CPT of interest will be very useful for continuous in vivo monitoring of a
pseudoꢀ drug upon externally triggered delivery. But mainly, the present aim was to open a new avenue
of specific design of antiꢀcancer molecules as fluorescent probes, for the understanding of specific
anticancer effects as well as nonꢀdesired effects including anemia, fatigue or cognitive deficits
commonly described in patients after chemotherapy.
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Acknowledgment
This work was supported by the Agence Nationale de la Recherche (Programme PIRIBio 2009, ANR
“CLICKMASSLINK”) for a postdoctoral fellowship to SD, La Région HauteꢀNormandie via the
CRUNCh program (CPER 2007ꢀ2013) and the CNRS. We thank Albert Marcual (COBRA ꢀ CNRS
UMR 6014) for HRMS analyses, Annick Leboisselier (COBRA ꢀ CNRS UMR 6014, INSA de Rouen)
for elemental analyses and Laetitia Bailly (COBRA ꢀ CNRS UMR 6014, INSA de Rouen) for chiral
HPLC analyses. Xavier Brune (Research master trainee, Université de Rouen, JanuaryꢀJune 2012) is
greatly acknowledged for his contribution to the largeꢀscale synthesis of fluoresceinꢀisonipecotic acid
derivative 4.
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Supporting Information.1H and C NMR data for all compounds and their HPLC chromatograph,
and experimental details about photophysical characterization of CPTꢀFI. This material is available free
of charge via the Internet at http://pubs.acs.org.
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References
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ε (dm3 molꢀ1 cmꢀ1)
Cmpd
λabs, max (nm)
λem, max (nm)
Ф F (%)
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47 700
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CPTꢀFI 1
265, 378, 502
499
424, 524
522
37%a
79%a
61%b
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265, 363, 378
29 250, 27 640, 30 740
424
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8
9
a For λabs, max = 502 and 499 nm respectively; and determinated by using fluorescein (Ф = 90% in aq. 0.1 M NaOH) as
F
standard (excitation at 460 nm).b For λabs, max = 363 nm; and determinated by using 7ꢀhydroxycoumarin (ФF = 76% in PBS) as
standard (excitation at 350 nm).
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Table 1: Photophysical properties of the fluorescent conjugate 1 and of its components 11 and 4 in PBS at 25
°C
Scheme 1. Synthesis of AB and CD ring systems 2 and 3
Scheme 2. Synthesis of the fluorescein-labeled CPT 1
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Scheme 3
Figure 1. Camptothecin and its derivatives.
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O
1
2
3
O
O
N
10
H
N
N
N
4
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9
O
O
Et
OH O
O
O
OH
Fluorescein-labelled 20(S)-CPT, 1
O
OH
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N
O
D
O
10
O
HN
Br
O
N3
E
A
B
N
O
Cl
Et
3
OH
O
O
OH
4
2
Figure 2. Fluoresceinꢀlabeled CPT 1: retrosynthetic analysis.
a
b
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Figure 3. Normalized absorption (—), emission [(—), λex 350 nm and (—) λex 480 nm for 1, (—), λex
350 nm for 11 and 460 nm for 4] spectra, and excitation [(—), λem 425 nm for 1, 570 nm for 4 and 560
nm for 11] of a) camptothecinꢀfluorescein conjugate CPTꢀFI 1 in PBS at 25 °C; b) fluoresceinꢀ
isonipecotic acid derivative 4; c) aminated CPT derivative 11.
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