Synthesis and biological evaluation of O-alkylated tropolones and related α-ketohydroxy derivatives as ribonucleotide reductase inhibitors

Article abstract of DOI:10.1016/S0223-5234(01)01249-1

A series of O-alkylated tropolones and related α-ketohydroxy compounds were evaluated for their biological activities and were shown to present an expected ribonucleotide reductase inhibition and cytotoxicity against some cancer cell lines but no antitubulin activity. Pharmacomodulation studies were realised to understand and enhance the observed activities. These original benzylic, heterocyclic and allylic compounds have been synthesised by a phase-transfer catalysed O-alkylation developed in our laboratories.

Full text of DOI:10.1016/S0223-5234(01)01249-1

Eur. J. Med. Chem. 36 (2001) 561–568
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(01)01249-1/SCO
Short communication
Synthesis and biological evaluation of O-alkylated tropolones and related
a-ketohydroxy derivatives as ribonucleotide reductase inhibitors
Isabelle Tamburlin-Thumina, Michel P. Crozeta,*, Jean-Claude Barrie`reb, Michel Barreaub ,
Jean-Franc¸ois Rioub, Franc¸ois Lavelleb
aLCMO, UMR 6517, CNRS Universite´s d’Aix-Marseille 1 et 3, Av. Escadrille Normandie-Niemen, BP 562,
F-13397 Marseille Cedex 20, France
bCentre de Recherche de Vitry-Alfortville, Aventis Pharma, 13 Quai Jules Guesde, BP 14,
F-94403 Vitry sur Seine Cedex, France
Received 29 January 2001; revised 11 June 2001; accepted 12 June 2001
Abstract – A series of O-alkylated tropolones and related a-ketohydroxy compounds were evaluated for their biological activities
and were shown to present an expected ribonucleotide reductase inhibition and cytotoxicity against some cancer cell lines but no
antitubulin activity. Pharmacomodulation studies were realised to understand and enhance the observed activities. These original
benzylic, heterocyclic and allylic compounds have been synthesised by a phase-transfer catalysed O-alkylation developed in our
´
laboratories. © 2001 Editions scientifiques et me´dicales Elsevier SAS
O-alkylated a-ketohydroxy compounds / phase-transfer O-alkylation / tropolones / anticancer / ribonucleotide reductase inhibitors
1. Introduction
nalise these observations by working on tropolone
derivatives and related a-ketohydroxy compounds
The ribonucleotide reductases play an important
substituted by allylic, benzylic or heterocyclic moi-
role in cell growth by supplying the 2%-deoxynucle-
eties. The synthesis and the biological evaluation of
otides required for DNA synthesis [1, 2]. Inhibition of
these products are discussed here.
these reductases interferes with the replication of ge-
netic material required for cancer cell division or viral
genome biosynthesis. Because of its importance in
DNA synthesis, ribonucleotide reductase is a poten-
tial target for drug design. Few inhibitors, as hydrox-
yurea or tropolone derivatives [3–10], are known to
block the cellular ribonucleotide reductase and are of
great interest as inhibitors of human immunodefi-
ciency virus-type 1 replication [3–10] or in cancer
chemotherapy [11].
Yamato has shown that some tropolone derivatives
could exhibit antitumoural activity probably related
to iron chelation in the active site of ribonucleotide
reductase [3–10]. These results prompted us to ratio-
2. Chemistry
To explore the structure–activity relationships of
various tropolone derivatives and a-ketohydroxy-re-
lated compounds, we decided to focus our work on
alkylation reaction. Due to the tropolone moiety
structure, we could imagine both C- or O-alkylation.
Literature reports only one direct method to obtain
C-alkylated tropolone analogues via palladium cross-
coupling [12, 13] between a bromotropolone and a
boronic acid derivative. A few other C-alkylated tro-
polones are described but resulted from a thermal
Claisen-type rearrangement [14–17]. For O-alkylated
tropolone synthesis [14–18], some methods are de-
scribed, but all present drawbacks and a lack of
generality.
* Correspondence and reprints
E-mail address: michel.crozet@lcmo.u-3mrs.fr
(M.P. Crozet).

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figure 2. Only the 4-isopropyl derivatives 18 and 19
As part of our work on tropolone derivatives, we
(table II) were obtained pure in sufficient quantities to
be tested. Commercially available purpurogallin 5 was
methylated by using dimethylsulphate to give a 1.5:1
mixture of tri- and tetramethylated products 20 and
20%, as shown in figure 3. Maltol derivative 22 was
converted in pyridinone analogue 24 by treatment
with aqueous ammonia (figure 4). The alkylating
agents are commercially available or have been easily
prepared in our laboratory. The desired compounds
were obtained in good to excellent yields and were
exclusively O-alkylated. The tropolonyl esters 25–28
were prepared by classical condensation of tropolones
3 and 4 with the appropriate acyl chlorides in reflux-
ing methylene chloride in the presence of anhydrous
pyridine as described in Section 5 and table IV.
have recently described [19] a new general method for
the synthesis of tropolone ethers. This method is a
phase-transfer alkylation, as shown in figure 1, and
had led us to synthesise various tropolone derivatives
by reaction with benzyl, heterocyclic and allyl halides.
This method was first applied to tropolone 3 and also
extended to substituted tropolone 4 and a-ketohy-
droxy-related compounds like purpurogallin 5 and
maltol 6.
O-Alkylation of 4-isopropyl-tropolone 4 led to a
1:1 mixture of 4- and 6-isopropyl derivatives due to
the possible isomerisation of the anion, as shown in
3. Biological results and discussion
Biological evaluation of the allylic compounds (7–
9) was first investigated in vitro on ribonucleotide
reductase. As all products exhibit in vitro activity on
ribonucleotide reductase comparable to that obtained
with hydroxyurea as a reference compound, their
cytotoxicity was evaluated on P388 cell lines as al-
ready described for Taxotere® [20]. Results were
shown in table I.
Figure 1. General method for O-alkylation of tropolones and
related a-ketohydroxy derivatives, purpurogallin and maltol.
As allylic derivatives (7–9) did not present any
cytotoxicity on cancer cell lines, we turned our atten-
tion to the heterocyclic series (10, 11). In the same
way, they exhibit in vitro inhibition of ribonucleotide
reductase but no cytotoxicity at all, as shown in table
I.
Figure 2. O-Alkylation of 4-isopropyltropolone.
As the same results were obtained with the hetero-
cyclic derivatives (10, 11), we finally chose the ben-
zylic series (12–17). As before, all tested compounds
exhibit in vitro inhibition of ribonucleotide reductase.
In that series, products 13, 15 and 17 did not exhibit
any cytotoxicity but compounds 12 and 16 showed
some cytotoxicity. It was interesting to note that
potency seemed to be related to the substituents of the
benzylic moiety: electron-withdrawing substituent
(13) did not lead to cytotoxicity, on the contrary no
substituent (12) or well-oriented electron-donating
substituent (16) improved the cytotoxicity, as shown
in table I.
Figure 3. Preparation of trimethylpurpurogallin 20.
After modifications to enhance the observed cyto-
toxicity, we retained the 3,4,5-trimethoxybenzyl moi-
ety and made some variations on the tropolone part.
Figure 4. Conversion of O-alkylated maltol 22 in pyridinone
analogue 24.

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563
Table I. Biological activities of compounds 7–17 on ribonucle-
otide reductase and P388 cancer cell line.
or a pyridinone one (24). Results are shown in table
II. All modifications (18, 20, 22) resulted in a com-
plete loss of cytotoxicity except in the case of the
pyridinone derivative 24 which retains some
cytotoxicity.
Finally, we decided to replace the ether link by an
ester one (25–28) (figure 5). In those cases, cytotoxic-
ity was retained only if an isopropyl substituent is
present on the tropolone ring (27, 28), as shown in
table III.
Table II. Biological activities of 3,4,5-trimethoxybenzyl and
2-bromoallyl analogues 18–24 on ribonucleotide reductase and
KB cancer cell line.
First, we tried to change the binding properties by
enhancing the steric hindrance of the substrate part.
This was realised by adding an isopropyl substituent
on the tropolone ring (18). On the contrary, the steric
hindrance was decreased by fusing the two moieties
and keeping the hydroxyl function free (20). Finally,
we retained the 3,4,5-trimethoxybenzyl moiety and
the tropolone ring was replaced by a maltol one (22)

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4. Conclusion
All tested compounds present an in vitro inhibition
of the ribonucleotide reductase, but only few present
cytotoxic properties on P388 or KB cell lines. In order
to increase the potency observed for the best com-
pound (16), we made some structural variations but,
at this stage, we are not able to explain clearly which
substituents are really involved in the interaction with
the enzyme.
Figure 5. Preparation of ester derivatives 25–28.
Table III. Biological activities of polymethoxybenzyl ana-
logues in ester series 25–28 on ribonucleotide reductase and
P388 cancer cell line.
5. Experimental
5.1. Chemistry
Melting points were determined on a Buchi capillary
melting point apparatus and are uncorrected. Elemental
analyses were performed by the Centre de Microanaly-
1
ses of the University of Aix-Marseille 3. Both H- and
13C-NMR spectra were determined on a Bruker AC 200
1
spectrometer. The H-NMR shifts were reported in ppm
downfield from Me4Si and the 13C-NMR shifts were
referenced to the solvent peaks (CDCl3; 76.9 ppm or
DMSO-d6; 39.6 ppm). Silicagel 60 (Merck, 0.063–0.200
mm, 70–230 mesh ASTM) was used for LC. TLC were
performed on 5×10 cm aluminium plates coated with
silicagel 60 F-254 (Merck) in an appropriate solvent. All
commercial alkylating agents, purpurogallin 5, and mal-
tol 6 were purchased from Aldrich Chemical Co. Com-
mercial products were of the highest purity available
and were used as received. Substituted benzyl chlorides
were prepared from the corresponding aldehydes by
reduction with NaBH4 and subsequent chlorination.
Tropolone 3 [21], 4-isopropyl-tropolone 4 [21, 22],
trimethylpurpurogallin 20 [23], 6-chloromethyl-1,3-
dimethyl-1H,3H-pyrimidine-2,4-dione [24], and 2-
chloromethylimidazo[1,2-a]pyridine [25] were prepared
from known procedures.
Interestingly, when the same variations were made
on compound 9, which did not present any cytotoxic-
ity, the introduction of an isopropyl group on the
tropolone ring (19) or the fusion of a trime-
thoxyphenyl moiety (21) resulted in the apparition of
poor cytotoxicity, not observed in the case of the
maltol-related compound 23.
5.1.1. Alkylation of tropolones and related compounds;
general procedures [19]
5.1.1.1. Stoichiometric method
m
The appropriate tropolone (6.07 mmol) was treated
In addition, compounds were tested for inhibition
of tubulin polymerisation. Only compound 19 slightly
inhibits tubulin polymerisation at high concentration
(100 mg ml−1). This activity disappears at lower con-
centration (10 mg ml−1).
with Bu4NOH (40% in water, 3.82 ml, 6.07 mmol) under
an inert atmosphere at r.t. for 1 h. A solution of the
appropriate alkylating agent (6.07 mmol) in CH2Cl2 (2.5
ml mmol−1) was added slowly. The reaction was con-
ducted at r.t. or at reflux depending on the alkylating

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565
agent, and was monitored by TLC (CH2Cl2–EtOAc
5.1.3. Preparation of tropolonyl esters; general method
(table IV)
7:3). After dilution with water (20 ml) and vigorous
stirring for 10 min, the aqueous layer was extracted with
CH2Cl2 (3×30 ml). The combined organic layers were
dried (MgSO4), and concentrated under reduced pres-
sure. Purification of the residue was done by chromatog-
raphy on silicagel, eluting with 30% EtOAc in CH2Cl2.
To a solution of the appropriate tropolone (0.5 g, 4.10
mmol) in CH2Cl2 (10 ml) at r.t. under an inert atmo-
sphere were added the appropriate acyl chloride (4.10
mmol) and anhydrous pyridine (1.5 ml, 18.65 mmol).
After stirring under reflux for 12 h the solvents were
removed under reduced pressure and the residue was
diluted with CH2Cl2 (10 ml). The organic layer was
washed with an aqueous saturated solution of Na2CO3,
dried (MgSO4) and concentrated under reduced pres-
sure. The remaining oil was crystallised from EtOH to
give the esters as white crystals [26].
5.1.1.2. Catalytic method
The appropriate tropolone (1 equiv., 8.20 mmol) was
added to 25% aq. KOH (5 ml). After stirring at r.t., a
yellow coloration was observed and H2O was evapo-
rated under 0.005 mbar at r.t. The yellow solid was
recrystallised from acetone to give the potassium salt of
tropolone in 95% yield. The appropriate alkylating
agent (1 equiv.) was added slowly to the above potas-
sium salt of tropolone (1 equiv.) followed by Bu4NBr
(0.1 equiv.) in H2O (0.4 ml mmol−1). The reaction was
conducted at r.t. or reflux depending on the alkylating
agent, and monitored by TLC (CH2Cl2–EtOAc 7:3).
Workup and purification were identical as above.
5.2. Biology
5.2.1. Ribonucleotide Reductase test—principle of the
test
RDR is one of the key enzymes for DNA replication
and catalyses the reduction of ribonucleotides into de-
oxyribonucleotides. Enzymatic activity was correlated to
the quantity of dCDP formed. After reaction, residual
CDP and dCDP formed were, respectively, hydrolysed
into cytidine (C) and deoxycytidine (dC). These two
compounds were separated by chromatography and ra-
dioactive dC amounts were measured.
5.1.2. Conversion of maltol derivative 22 to the
corresponding pyridinone analogue 24
O-Alkylated maltol derivative 22 (2.01 g, 6.57 mmol)
was treated with a mixture of EtOH (26.13 ml), NH4OH
(32% in H2O, 56.28 ml) and NaOH 2 N (10.60 ml)
under an inert atmosphere under reflux for 48 h. After
cooling, the reaction mixture was concentrated under
reduced pressure. The residue was then diluted with
H2O (10 ml) and pH 7 was reached by titration with a
diluted hydrochloric solution. The aqueous layer was
extracted with CH2Cl2 (3×30 ml) and the combined
organic layers were dried (MgSO4) and concentrated
under reduced pressure. Purification of the residue done
by chromatography on silicagel, eluting with 5% MeOH
in CH2Cl2, followed by recrystallisation from acetone
gave the desired pyridinone, 2-methyl-3-(3,4,5-
trimethoxybenzyloxy)-1H-pyridin-4-one (24), as a white
solid in 95% yield (1.90 g). m.p.: 187–188 °C . 1H-
NMR (DMSO-d6) l 2.10 (s, 3H), 3.64 (s, 3H), 3.74 (s,
6H), 4.96 (s, 2H), 6.85 [AB, Dw=1.3: 6.14 (d, 1H,
J=7.3 Hz), 7.46 (d, 1H, J=7.0 Hz)], 6.71 (s, 2H).
13C-NMR (DMSO-d6) l 13.68, 55.78 (2x), 60.00, 71.89,
105.59 (2x), 115.99, 133.63, 134.56, 136.96, 139.06,
152.61 (2x), 161.00, 174.47.
5.2.2. Reagents
All commercially available reagents were purchased
from Sigma–Aldrich except the 14C-labelled CDP pur-
chased from CEA. Except buffers, all the reagents were
prepared on ice and were stored as aliquots at −20 °C.
Apyrase and alcaline phosphatase solutions were stored
at −70 °C after preparation and/or utilisation. Borate
buffer (50 mM, pH 9): a 19.06 g ml−1 solution of
sodium tetraborate was prepared and pH 9 was reached
by adding a 1 N solution of sodium hydroxide. Hepes
buffer (27 mM, pH 7.4)–MgCl2 (32.5 mM)–NaF (25.5
mM): a 1 l solution was prepared by adding Hepes (1
M, 27 ml), MgCl2 (1 M, 32.5 ml) and NaF (1 M, 21.5
ml) and then pH was checked. Hepes buffer (20 mM,
pH 7.4)–MgCl2 (2 mM)–DTT (2 mM): a 1 l solution
was prepared by adding Hepes (1 M, 20 ml), MgCl2 (1
M, 2 ml) and DTT (1 M, 2 ml). Adenosine 5%-triphos-
phate (ATP, 13.5 mM): a 7.437 mg ml−1 solution was
prepared with distilled water and neutralised carefully
by adding 5 ml aliquots of a 1 N sodium hydroxide
solution. DL–dithiothreitol (DTT 27 mM): a 4.16 mg
ml−1 solution was prepared with distilled water. Cy-
tidine 5%-diphosphate (CDP, 0.33 mM): a 0.133 mg ml−1
Anal. Found: C, 62.93; H, 6.20; N, 4.60. Calc. for
C16H19NO5 (305.35): C, 62.94; H, 6.27; N, 4.59%.

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solution was prepared with distilled water. 2%-Deoxycy-
tidine: a 10 mg ml−1 solution was prepared with distilled
water. Apyrase: a 3000 U ml−1 solution was prepared,
corresponding to 666 ml of phosphate buffer (0.01 M,
pH 7). Alcaline phosphatase: a 10 mg ml−1 solution was
prepared from the commercially available form (3 U
mg−1) with phosphate buffer (0.01 M, pH 7). 14C-la-
belled cytidine 5%-phosphate (50 mCi ml−1): to the com-
mercially available form (0.50 ml, 10% in EtOH, 0.054
mCi) was added 500 ml of distilled water.
tion (Beckman Rotor 50Ti ultracentrifugation) of the
obtained suspension (1 h 30 at 40 000 tr min−1), the
floating part was taken with a Pasteur pipette, avoiding
taking the lipidic layer. The obtained suspension was
dyalised at least for 1 h against 100× its volume of
Hepes buffer 20 mM pH 7.4–2 mM MgCl2–2 mM DTT
in the cold room. The cellular extract was split (0.5 ml
per tube), frozen at −70 °C and stored at −70 °C .
Proteins were dosed by the Folin technique and concen-
tration was supposed to be around 25 mg ml−1
.
5.2.4. Reaction
5.2.3. Enzyme preparation
Each mixture was realised at 3 °C . In each 1.5 ml
Eppendorf tube were added 10 ml Hepes buffer 27
mM–MgCl2 32.5 mM–NaF 21.5 mM, 10 ml of DTT 27
mM, 10 ml ATP 13.5 mM, 7.5 ml of CDP 0.33 mM, 2.5
ml of CDP* 50 mCi ml−1, 2.5 ml of tested inhibitor (20
mM, 2 mM) and 10 ml enzyme preparation. After 30
min of incubation at 37 °C , the reaction was quenched
by heating at 100 °C for 2–3 min. Incubation was
Every manipulation was done in an ice-bath. S180
tumour cell-lines were used. Tumours were washed and
necrotic areas were cut. Then tumours were weighed (x
g), roughly cut and added to (0.8 ml×x g) of Hepes
buffer 20 mM pH 7.4–2 mM MgCl2–2 mM DTT in a
tube. The mixture was crushed (Ultraturrax) at least five
times for 10 s at the higher speed with a break between
each crushing to allow a good cooling. After centrifuga-
Table IV. O-Acylated products 25–28 prepared.

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567
continued for 1 h at 37 °C after adding 5 ml apyrase
(3000 U ml−1). Another 1 h of incubation at 37 °C was
done after adding 30 ml borate buffer (50 mM, pH 9.0),
5 ml cytidine (10 mg ml−1), 5 ml deoxycytidine (10 mg
ml−1), 5 ml alcaline phosphatase (10 mg ml−1). The
reaction was quenched by heating at 100 °C for 10 min
and tubes were centrifugated (Eppendorf bench
centrifugation) for 2 min.
(EGTA: 0.5 mM); (MgCl2: 6 mM); (glycerol: 3.4 mM);
(GTP: 1 mM)]. The reaction was started by a tempera-
ture variation between 6 and 37 °C in a thermostat cell
with a 1 cm optical distance, and was monitored the
turbidity measurement at 400 nm (Uvikon 931 Spec-
trophotometer). When a potential inhibitor (100 mg
ml−1) was tested, it was added to tubulin solution just
before the temperature progression.
5.2.5. Chromatographies
Chromatographies were done on 20×20 plastic
support coated with a cellulose sheet with fluorescence
indicator (Sclucher and Schuell). Spots of 5 ml were
spaced at 1.5 cm and dried before elution with a mobile
phase composed of ammonium acetate 5 M pH 9.0 (10
ml), saturated sodium tetraborate (40 ml), EtOH (90 ml)
and EDTA 0.5 M (500 ml). Time separation was ca. 4 h.
Acknowledgements
Financial support by the CNRS and Rhoˆne-Poulenc
Rorer (Aventis Pharma) through a Grant (BDI for
I.T.T.) is gratefully acknowledged.
References
5.2.6. Results
Radioactivity was measured using
analyser.
a
Berthold
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acid 7-oxocyclohepta-1,3,5-trienyl ester (25): Anal. Found: C,
64.52; H, 5.14. Calc. for C17H16O6 (316.31): C, 64.55; H, 5.10.
4-Methoxybenzoic 7-oxocyclohepta-1,3,5-trienyl ester (26):
Anal. Found: C, 70.32; H, 4.70. Calc. for C15H12O4 (256.26): C,
70.31; H, 4.72. 3,4,5-Trimethoxy benzoic acid 3-isopropyl-7-ox-
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(28): Anal. Found C, 72.44; H, 6.05. Calc. for C18H18O4
(298.34): C, 72.47; H, 6.08.
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.