Synthesis and characterization of novel natural product-Gd(III) MRI contrast agent conjugates

Article abstract of DOI:10.1016/j.bmcl.2008.10.027

Several novel gadolinium chelates conjugated with paclitaxel, colchicine and thyroxine have been prepared as MRI contrast agents targeted to tubulin and thyroxine-binding globulin, respectively.

Full text of DOI:10.1016/j.bmcl.2008.10.027

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6058–6061
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and characterization of novel natural product-Gd(III) MRI
contrast agent conjugates
Eleni K. Efthimiadou a, Maria E. Katsarou a, Michael Fardis b, Christos Zikos a, Emmanuel N. Pitsinos a,
,
*
Athanasios Kazantzis a, Leondios Leondiadis c, Marina Sagnou d, Dionisios Vourloumis a,
*
a Chemical Biology Program, Institute of Physical Chemistry, NCSR ‘DEMOKRITOS’, PO Box 60228, GR-153 10 Aghia, Paraskevi, Greece
b Institute of Material Sciences, NCSR ‘DEMOKRITOS’, Greece
c Mass Spectrometry and Dioxin Analysis, Institute of Radiodiagnostics and Radiopharmaceuticals, NCSR ‘DEMOKRITOS’, Greece
d NMR Studies of Biomolecules and Pharmaceuticals, Institute of Biology, NCSR ‘D’, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Several novel gadolinium chelates conjugated with paclitaxel, colchicine and thyroxine have been pre-
pared as MRI contrast agents targeted to tubulin and thyroxine-binding globulin, respectively.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 19 September 2008
Revised 7 October 2008
Accepted 7 October 2008
Available online 11 October 2008
Keywords:
MRI contrast agents
Gd(III) complexes
Tubulin
Thyroxine binding globulin
Magnetic resonance imaging (MRI) is a non-invasive imaging
technique, extensively used in the clinic, which allows for high-
definition pictures of a whole-organism to be obtained over ex-
tended periods of time. The technique is often enhanced by the
use of contrast agents (CAs). Among them, paramagnetic gadolin-
ium chelates hold a prominent position.1 In general, these agents
are injected systemically and serve to passively and non-specifi-
cally enhance the signal by increasing the longitudinal relaxation
rate (1/T1) of surrounding water molecules. Furthermore, binding
to a macromolecule increases concentration and retention of the
CA at the binding site and affords an increase in relaxivity, result-
pared, with the aim to guide the gadolinium label towards the bio-
logical target of the natural product, to increase the lipophilicity of
the CA or to facilitate pharmacokinetic studies. In this context, it
should be noted that the delivery of sufficient quantity of the para-
magnetic label and/or the ability of the conjugates to cross cellular
membranes is a challenge.8
In the frame of a research program aiming at the development
of novel, targeted MRI contrast agents, we have designed conju-
gates of paclitaxel, colchicine and thyroxine with the known, and
approved for clinical use as MRI-CA, Gd(III) complexes with diethy-
lenetriaminepentacetic acid bis-methyl amide ([Gd(DTPA-BMA)],
OmniscanTM) and/or 1,4,7,10-tetraazacyclododecane-N,N0,N00,N000-
tetraacetic acid ([Gd-DOTA], DotaremTM) (Fig. 1). We report herein
ing from a decrease in the rotational correlation time (s
r).2 As the
contrast gained is still limited, relatively high doses of CA are re-
quired for sufficient contrast enhancement. There are two major
ways to decrease the unwanted and potentially toxic high CA
administered dose: (i) by employing new Gd(III) complexes en-
dowed with much higher relaxivities; (ii) by improvement of the
biodistribution of CAs. Recently, the development of ‘smart’ CAs
that will accumulate at a specific tissue, or sense a particular bio-
logical event, has attracted considerable attention.3 Thus, gadolin-
ium chelates have been tagged with various targeting elements,
including natural products. Conjugates with peptides,4 steroids,5
carbohydrates,6 and an anticancer anthracycline7 have been pre-
O
O
N
O
N
N
O
N
Gd
N
N
O
OH2
O
N
Gd
O
O
MeHN
NHMe
O
O
O
O
O
O
O
O
O
H
H
[Gd(DOTA)(H2O)] -
(DotaremTM
[Gd(DTPA-BMA)(H2O)]
)
(OmniscanTM
)
* Corresponding authors. Tel.: +30 2106503624; fax: +30 2106511766.
E-mail address: vourloumis@chem.demokritos.gr (D. Vourloumis).
Figure 1. Structures of some clinically used Gd-chelates.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.027

E. K. Efthimiadou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6058–6061
6059
the synthesis of the targeted conjugates along with their relevant
toxicities and magnetic properties.
Microtubules are long stiff polymers formed from molecules of
tubulin (heterodimer of a- and b-form). They are critical for inter-
O
S
MeO
MeO
O
N
NH
O
H
N
O
O
N
Gd
nal cell organelle trafficking and vital for mitotic spindle formation
during cell division.9 Paclitaxel, a natural product clinically used
for the therapy of several carcinoma lines (TaxolTM), binds to micro-
tubules with high affinity (Kd ꢀ 10ꢁ8 M) and a maximum stoichi-
ometry of one ligand per heterodimer, promoting tubulin
polymerization.10 On the other hand, each molecule of the alkaloid
colchicine, which is clinically utilized for the treatment of autoin-
flammatory diseases and gout, binds tightly to one tubulin mole-
cule and prevents its polymerization.11 The obvious
complementarity of the two has inspired the synthesis of several
paclitaxel12 and colchicine derivatives,11 to study intracellular drug
distribution and their interference with the, otherwise, finely bal-
anced assembly/disassembly dynamics of microtubules. Based on
the above mentioned literature evidence, paclitaxel and colchicine
conjugates are expected to target the CA to tissues of high tubulin
content, such as sites of actively propagating tumours. In addition,
they may facilitate the prescription of these clinically used drugs
on a personalized medicine basis, or the identification of any pos-
sible, previously undetected, preference in their distribution in the
body.
Thyroxine (T4) is one of the hormones produced by the thyroid
gland. Only a minute fraction (<0.03%) of T4 circulating in the blood
is free and thus available to tissues. Most of it is present in the bio-
logically inactive state, bound to transport proteins, mainly thyrox-
ine-binding globulin (TBG) but also transthyretin (TTR) and
albumin (HSA).13 These proteins sequester one (TBG, TTR) or sev-
eral (HSA) hormone molecules. Although albumin is present in
higher concentration (40,000 mg/L vs 16 mg/L of TBG), TBG has
the highest affinity (Ka = 1 ꢂ 1010 Mꢁ1) and carries most (75%) of
the serum’s T4.14 Binding of T4 by these proteins safeguards the
body from the effects of abrupt fluctuations in hormone secretion.
Interestingly, in the case of TBG, selective hormone release at sites
of inflammation has also been reported.15 Consequently, thyroxine
conjugates are expected to exhibit increased retention time in the
blood-stream, compared with the untagged CA, and thus improved
performance in cardiovascular MRI. Furthermore, T4 conjugates
might allow selective MRI of inflammation sites.
For the synthesis of colchicine-Gd(III) conjugates the natural
product 1 was deacetylated, as previously described, to yield amine
2 in 40% overall yield (Scheme 1).11b In order to address the possi-
bility of the gadolinium-complex interfering with binding of the
natural product to its biological target, a short, 4-atom linker,
was introduced through a coupling reaction with 3 mediated by
DIC, followed by liberation of the corresponding amine through
acidic treatment. In parallel, complexation of GdCl3 with commer-
cially available p-SCN-Bn-DOTA was accomplished under carefully
controlled pH conditions,5b producing complexed isothiocyanate 6
in 75% yield, spectroscopically identical to the one reported previ-
ously.5b Thus, no further attempts for the determination of Gd(III)-
concentration in the complex were performed. Mixing of the two
colchicine analogues 2 and 4 with 6 in DMSO, in the presence of
triethylamine, furnished the corresponding conjugates 7 and 8 in
60% yield, after HPLC purification.
MeO
N
O
O
N
8
OMe
O
O
g. 6
60%
O
a. (Boc)2O
b. MeONa
c. 20% TFA
MeO
MeO
MeO
NH2
O
NH
MeO
40% overall
MeO
MeO
2
1
O
OMe
OMe
O
d. DIC,DMAP
e. 20%T FA
78% overall
HO
NHBoc
5: p-SCN-Bn-DOTA
3
H2N
f. GdCl3
75%
pH=6.5
MeO
O
O
O
NH
6: p-SCN-Bn-DOTA-Gd
O
MeO
MeO
N
N
4
O
O
Gd
N
O
O
NCS
N
OMe
g. 6
60%
O
O
O
S
O
N
N
H
HN
N
O
O
O
Gd
N
O
MeO
MeO
O
O
N
NH
O
7
MeO
O
O
OMe
Scheme 1. Synthesis of colchicine derivatives 7 and 8. Reagents and conditions: (a)
(Boc)2O (5.5 equiv), DMAP (1.1 equiv), Et3N (11.0 equiv), 12 h, 0 °C, 60%; (b) NaOMe
(3.5 equiv), MeOH (0.20 M), 30 min, 0 °C, 75%; (c) 20% TFA, CH2Cl2, pentamethyl-
benzene (4.0 equiv), 8 h, 40 °C, 90%; (d) 3 (2.1 equiv), DIC (1.5 equiv), DMAP (0.01
equiv), 3 h, 0 ? 25 °C, 86%; (e) 20% TFA, CH2Cl2, pentamethylbenzene (4.0 equiv),
8 h, 40 °C, 90%; (f) GdCl3 (1.0 equiv), phosphate buffer, pH 6.5, 30 min, 75%; (g) 6,
Et3N (1.5 equiv), DMSO (0.05 M), 24 h, 25 °C, 60% for 7 and 8. Boc = t-butoxycar-
bonyl; DIC = N,N0-diisopropylcarbodiimide; DMAP = 4-(dimethylamino)pyridine;
TFA = trifluoroacetic acid; DMSO = dimethyl sulfoxide.
For the synthesis of thyroxine conjugates, peracetylation of the
natural product was required to allow the selective coupling of the
carboxyl-moiety with an appropriately protected diamine-linker.
Subsequent saponification of the acetate ester followed by removal
of the Boc protective group, furnished amine 16 in 75% overall
yield. Attachment of the Gd(III) complex 6 was performed as be-
fore, yielding conjugate 17 in quantitative yield. Reaction of 16
with commercially available dianhydride 18 produced, after com-
plexation with GdCl3 in pyridine, dimeric compound 19 in 65%
overall yield (Scheme 3).
The effect of compounds 7, 8, 14, 15, 17, and 19 on the viability
of two human cancer cell lines, HeLa and MCF-7 was assessed,
using the MTT assay.16 Complex 6 was also tested in various con-
centrations (in the lM range). The IC50 values were estimated from
the % viability versus concentration plots, using sigmoidal fit and
Similarly, taking advantage of the differential reactivity of the
hydroxyls at 20- and 7-positions, paclitaxel was selectively pro-
tected in the form of a Cbz-carbonate (Scheme 2).12a Introduction
of an attachment linker was performed by DCC mediated coupling
of protected c- or d-aminoacids to furnish, upon subsequent simul-
taneous removal of the Cbz protecting groups by catalytic hydroge-
nation, 12 or 13 in 95% yield. Attachment of the complexed
isothiocyanate 6 was performed as before, yielding conjugates 14
and 15 in 60% and 65% yield, respectively.
are presented in Table 1. In general, no significant cytotoxicity

6060
E. K. Efthimiadou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6058–6061
a. Cbz-Cl, pyridine
O
b. CbzNH(CH2)2COOH,
O
O
or
AcO
OH
O
AcO
HO
O
O
OH
CbzNH(CH2)3COOH,
n=2,3
NHCbz
NH
2'
O
Ph
Ph
NH O
Ph
DCC, DMAP
7
Ph
O
86% for 2 steps
O
O
O
OH
OCbz
H
OBz
9
H
OBz
10: n=2
11: n=3
OAc
OAc
O
O
O
N
N
c. H2,10%,Pd/C
quantitative
H
N
O
Gd
O
N
S
N
O
O
O
O
O
O
NH
AcO
HO
O
AcO
O
O
O
n=2,3
NH2
n=2,3
Ph
NH O
Ph
Ph
NH
O
O
2'
d. 6, Et3N
O
Ph
O
O
60% - 65%
O
OH
OH
H
OBz
12: n=2
13: n=3
H
HO
OAc
OAc
OBz
14: n=2
15: n=3
Scheme 2. Reagents and conditions: (a) Cbz-Cl (30.0 equiv), CH2Cl2 (0.02 M), pyridine (100 equiv), 25 °C, 24 h, 90%; (b) CbzNH-CH2CH2COOH or CbzNH-CH2CH2CH2COOH
(4.4 equiv), DCC (4.4 equiv), DMAP (1.0 equiv), 23 °C, 18 h, 95% for 10 and 95% for 11; (c) H2, 10% Pd/C (5% wt), MeOH (0.015 M), 23 °C, 2 h, quantitative for 12 and 13; (d) p-
SCN-Bn-DOTA-Gd, 6 (1.08 equiv), Et3N (1.5 equiv), DMSO (0.05 M), 23 °C, 24 h, 60% for 14 and 65% 15. CbzCl = benzyl chloroformate; DCC = N, N0- dicyclohexylcarbodiimide.
a
. Ac2O, Pyridine
b. EDC, iPr2NEt,
I
O
I
O
NHBoc
HO
I
I
(CH2)4NH2
H2N
HO
I
I
N
H
OH
c. NaOMe, MeOH
d. 20% TFA
NH2
NHAc
O
O
I 16: Thyr(CH2)4NH2
L
-Thyroxine
I
O
O
O
e. p-SCN-Bn-DOTA-Gd
6
N
Gd
N
O
N
N
O
17: Gd(DOTA-Thyr)
I
O
O
O
I
I
S
NHAc
H
O
N
N
H
N
H
OH
O
I
N
f. 16, DMF, Et3N
g. GdCl3, Pyridine
O
O
N
N
O
Gd
O
Thyr(H2C)4HN
NH(CH2)4Thyr
O
O
O
O
O
O
N
N
O
O
N
O
O
19: DTPA-Thyr-Gd
18
COOH
i
Scheme 3. Synthesis of thyroxine derivatives 17 and 19. Reagents and conditions: (a) Ac2O (2.5 equiv), pyridine (0.3 M), 0 ? 23 °C, 12 h; (b) EDC (1.1 equiv), Pr2NEt (1.2
equiv), CH2Cl2/DMF (1/1, 0.04 M), 0 ? 23 °C, 12 h; (c). NaOMe (0.1 equiv), MeOH (0.05 M), 0 ? 23 °C, 12 h, 75% over three steps; (d) 20% TFA in CH2Cl2, 30 min, 0 ? 23 °C,
100%; (e) p-SCN-Bn-DOTA-Gd, 6 (1.08 equiv), Et3N (1.5 equiv), DMSO (0.02 M), 23 °C, 24 h, 100%; (f) 16 (2.0 equiv), DMF (0.05 M), Et3N (6.0 equiv), 23 °C, 24 h, 96%; (g)
GdCl3ꢃ6H2O (1.1 equiv, 1.0 M in H2O), pyridine (0.04 M), 70 °C, 4 h, 68%. Ac = acetyl; EDC = N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide; THF = tetrahydrofuran;
DMF = dimethyl formamide.
was observed for almost all the compounds tested (micromolar
range). This is highly encouraging since we are aiming the develop-
ment of relatively non-toxic bioconjugates with CA properties to
ensure the systemic safety of the imaging process. From all com-
pounds tested, 15 exerted relatively enhanced toxicity against both
cell lines. Also compound 7 had a cytotoxic effect against MCF-7
relaxation times with and without CA, respectively, C is the con-
centration of CA in mM and r1 is the relaxivity expressed in
mMꢁ1 sꢁ1. Gadolinium complex solutions of at least three different
concentrations, ranging from 0.5 to 4.0 mM, were employed. Meth-
anol or DMSO was added as co-solvent (50%) in order to facilitate
solubilization of the complexes. The results are presented in Table
1. Compounds 8, 14, and 15 exhibit comparable relaxivities in the
expected range (r = 3.5–5).1,2,6c The higher values observed for 7,
17, and 19 may be due to the hydrophobic nature of colchicine
and thyroxine molecules, rendering the corresponding complexes
amphiphilic enough to aggregate, resulting in an increase in the
rotational correlation time, and consequently an increase in
relaxivity.5a,5b
cells with an IC50 value of 90
lM. Interestingly, compound 17 de-
creased the viability of human breast cancer line, MCF-7
(IC50 = 114 lM), a fact that may be related to the hormone-depen-
dent nature of the cells.17 All other compounds tested did not have
any significant effect on the viability of either HeLa or MCF-7 cells.
The relaxivities of the new CAs prepared were measured based
on the equation (1/T1s) = (1/T1c) + r1[C], where T1s and T1c are the

E. K. Efthimiadou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6058–6061
6061
Table 1
this article can be found, in the online version, at doi:10.1016/
j.bmcl.2008.10.027.
Cytotoxic effects and relaxivities of new contrast agents
Relaxivityb,c (mMꢁ1 sꢁ1
)
a
a
Compound
HeLa IC50
(lM)
MCF-7 IC50
(
lM)
References and notes
6
7
8
14
15
17
19
>400
>400
>400
>400
37
>400
90
N/A
13
3.8
3.7
4.7
13
1. (a) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Chem. Rev. 1999, 99,
>400
110
117
114
>400
ˇ
2293; b Hermann, P.; Kotek, J.; Kubícek, V.; Lukeš, I. Dalton Trans. 2008, 3027.
2. Aime, S.; Botta, M.; Fasano, M.; Terreno, E. Acc. Chem. Res. 1999, 32, 941.
3. (a) Artemov, D. J. Cell. Biochem. 2003, 90, 518; (b) Morawski, A. M.; Lanza, G. A.;
Wickline, S. A. Curr. Opin. Biotechnol. 2005, 16, 89; (c) Angelovski, G.; Fouskova,
P.; Mamedov, I.; Canals, S.; Toth, E.; Logothetis, N. K. Chem. Bio. Chem. 2008, 9,
1729 (and references cited therein).
4. De León-Rodríguez, L. M.; Kovacs, Z. Bioconjug. Chem. 2008, 19, 391.
5. (a) Lattuada, L.; Lux, G. Tetrahedron Lett. 2003, 44, 3893; (b) Lee, J.; Zylika, M. J.;
Anderson, D. J.; Burdette, J. E.; Woodruff, T. K.; Meade, T. J. J. Am. Chem. Soc.
2005, 127, 13164; (c) Guananathan, C.; Pais, A.; Furman-Haran, E.; Seger, D.;
Eyal, E.; Mukhopadhyay, S.; Ben-David, Y.; Leitus, G.; Cohen, H.; Vilan, A.;
Degani, H.; Milstein, D. Bioconjug. Chem. 2007, 18, 1361; (d) Lee, J.; Burdette, J.
E.; MacRenaris, K. W.; Mustafi, D.; Woodruff, T. K.; Meade, T. J. Chem. Biol. 2007,
14, 824; (e) Chong, H.-S.; Song, H. A.; Lim, S.; MacRenaris, K.; Ma, X.; Lee, H.;
Bui, P.; Meade, T. Bioorg. Med. Chem. Lett. 2008, 18, 2505.
>400
>400
8.0
a
All compounds were added as 1:1 DMSO/H2O or EtOH/H2O solutions and were
incubated for 24 h.
b
pH = 7.0; r2 = 0.99–0.96.
Measurements were performed on a Bruker MSL-100 spectrometer, equipped
c
with a superconducting magnet operating at 2.35 Tesla and yielding a 1H NMR
resonance frequency of 100.17 MHz. All measurements have been made at room
temperature (about 25 °C).
6. (a) Rongved, P.; Fritzell, T. H.; Strande, P.; Klaveness, J. Carbohydr. Res. 1996,
287, 77; (b) Laurent, S.; Elst, L. V.; Fu, Y.; Muller, R. N. Bioconjug. Chem. 2004, 15,
99; (c) Duimstra, J. A.; Fermia, F. J.; Meade, T. J. J. Am. Chem. Soc. 2005, 127,
12847; (d) Song, Y.; Kohlmeir, E. K.; Meade, T. J. J. Am. Chem. Soc. 2008, 130,
6662.
7. Frullano, L.; Tejerina, B.; Meade, T. J. Inorg. Chem. 2006, 45, 8489.
8. (a) Allen, M. J.; MacRenaris, K. W.; Venkatasubramanian, P. N.; Meade, T. J.
Chem. Biol. 2004, 11, 301; (b) Burdette, J. E. J. Mol. Endocrinol. 2008, 40, 253.
9. Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J. D. In Molecular
Biology of the Cell; Garland Publishing, Inc.: New York, 1994; pp 803–814.
10. Nicolaou, K. C.; Dai, W.-M.; Guy, R. K. Angew. Chem., Int. Ed. Engl. 1994, 33, 15.
11. (a) Bhattacharyya, B.; Panda, D.; Gupta, S.; Banerjee, M. Med. Res. Rev. 2008, 28,
155; (b) Ducray, P.; Lebeau, L.; Mioskowski, C. Helv. Chim. Acta 1996, 79, 2346.
12. (a) Guy, R. K.; Scott, Z. A.; Sloboda, R. D.; Nicolaou, K. C. Chem. Biol. 1996, 3,
1021; (b) Lillo, M. P.; Cañadas, O.; Dale, R. E.; Acuña, A. U. Biochemistry 2002, 41,
12436.
In conclusion, several novel gadolinium chelates conjugated
with paclitaxel, colchicine and thyroxine have been prepared. They
exhibit useful relaxivities which are expected to improve in the
presence of their biological targets (tubulin or TBG). The cytotoxic-
ities observed for some of the compounds could indicate their abil-
ity to cross the cell membrane under the conditions tested. Further
studies to validate their performance in the presence of their target
proteins and their cellular uptake are underway.
Acknowledgment
Financial support by the General Secretariat of Research and
Technology of the Greek Ministry of Development (program ‘PEP
Attikis 2000–2006’ 1.2, action 1.2.1) is gratefully acknowledged.
13. For previous work targeting HSA see: Caravan, P.; Parigi, G.; Chasse, J. M.;
Cloutier, N. J.; Ellison, J. J.; Lauffer, R. B.; Luchinat, C.; McDermid, S. A.; Spiller,
M.; McMurry, T. J. Inorg. Chem. 2007, 46, 6632.
14. Refetoff,
S.
In
Thyroid
Hormone
Serum
Transport
Proteins,
www.thyroidmanager.org, February 5, 2007.
15. Jirasakuldech, B.; Schussler, G. C.; Yap, M. G.; Drew, H.; Josephson, A.; Michl, J. J.
Clin. Endocrinol. Metab. 2000, 85, 3996.
Supplementary data
16. Mosmann, T. J. Immunol. 1983, 65, 55.
17. Tang, H.-Y.; Lin, H.-Y.; Zhang, S.; Davis, F. B.; Davis, P. J. Endocrinology 2004, 145,
3265.
Detailed synthetic procedures and spectroscopic data for final
products and selected intermediates, cell proliferation assay and
relaxometric measurements. Supplementary data associated with