Cyclopenta[g]quinazoline-based antifolates: The effect of the chirality at the 6-position on the inhibition of thymidylate synthase (TS)

Article abstract of DOI:10.1016/S0960-894X(01)00612-6

Cyclopenta[g]quinazoline-based inhibitors of thymidylate synthase (TS) possess a chiral centre at the 6-position of the molecule. The effect of this chirality on the inhibition of TS was investigated by synthesising compounds 6S-1a-c, 6R-1a-c. It was shown, in particular with the diastereoisomers 6S-1c, 6R-1c, that the inhibitory activity against TS is mainly due to the 6S diastereoisomer rather than the 6R diastereoisomer, which is virtually inactive.

Full text of DOI:10.1016/S0960-894X(01)00612-6

Bioorganic & Medicinal Chemistry Letters 11 (2001) 3015–3017
Cyclopenta[g]quinazoline-Based Antifolates: The Effect of the
Chirality at the 6-Position on the Inhibition of
Thymidylate Synthase (TS)
V. Bavetsias,* J. H. Marriott, D. S. Theti, J. C. Melin, S. C. Wilson and A. L. Jackman
CRC Centre for Cancer Therapeutics at The Institute of Cancer Research, Chemistry Department, CRC Laboratory,
15 Cotswold Road, Sutton, Surrey SM2 5NG, UK
Received 5July 2001; revised 24 August 2001; accepted 5September 2001
Abstract—Cyclopenta[g]quinazoline-based inhibitors of thymidylate synthase (TS) possess a chiral centre at the 6-position of the
molecule. The effect of this chirality on the inhibition of TS was investigated by synthesising compounds 6S-1a–c, 6R-1a–c. It was
shown, in particular with the diastereoisomers 6S-1c, 6R-1c, that the inhibitory activity against TS is mainly due to the 6S dia-
stereoisomer rather than the 6R diastereoisomer, which is virtually inactive. # 2001 Elsevier Science Ltd. All rights reserved.
The thymidylate synthase (TS) enzyme that catalyses
the conversion of 20-deoxyuridine 50-monophosphate
(dUMP) to thymidine 50-monophosphate (TMP) has
been an attractive target in cancer chemotherapy for sev-
eral years. A number of structurally diverse molecules have
been clinically evaluated and raltitrexed (Tomudex) has
been approved for the treatment of colorectal cancer.1
or 6R-2 followed by the removal of the protecting
groups (Scheme 1).2 The acids 6S-2 and 6R-2 were pre-
pared by the enzymatic hydrolysis of N-(4-{N-[(6RS)-2-
methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quin-
azolin-6-yl]-N-(prop-2-ynyl)amino}benzoyl)-l-glutamic
acid as previously described, and the enantiomeric pur-
ity was determined by chiral HPLC.3 The absolute ste-
reochemistry of 6S-2 was established by carrying out an
X-ray crystal structure determination on a compound
derived from 6S-2.3 Compounds 6S-3d, 6R-3d were
prepared from the corresponding acids and 2d via di-
ethyl phosphorocyanidate (DEPC) carboxyl activation.
To synthesise 6S-3e, 6R-3e, 6S-3f, 6R-3f, the acids 6S-2
and 6R-2 were first converted into their penta-
fluorophenyl esters which then reacted with the appro-
priate ligand 2e, 2f. Alkaline or acidic hydrolysis of the
ester protecting groups afforded the final products 6S-
1a–c, 6R-1a–c. These compounds were analysed by
chiral HPLC (ASTEC Cyclobond I column, ASTEC
Cyclobond II, or ASTEC Chirobiotic T column) and, as
expected, the stereochemical integrity for each of these
molecules was correlated with that of the starting mat-
erials (i.e., the acids 6S-2 and 6R-2). This meant that
compounds 6R-1a–c were contaminated with ꢁ10% of
the corresponding 6S-diastereoisomer. However, in the
case of 6R-1c, it was possible to remove the undesired
6S-diastereoisomer by semipreparative HPLC (Chiro-
biotic T column (25cm ꢂ10 mm); mobile phase: MeOH
containing 0.1% AcOH and 0.1% Et3N; flow=5mL/
min, l=230 nm). So, this compound (6R-1c) was
obtained in a pure form regarding the stereochemistry
at the 6-position.
Cyclopenta[g]quinazoline-based antifolates constitute a
new and promising class of inhibitors of thymidylate
synthase that display a high inhibitory activity against
this enzyme.2ꢀ4
The presence of the cyclopenta[g]quinazoline ring
introduces a chiral centre at the 6-position of the mole-
cule (Fig. 1). From the two diastereoisomers with regard
to the stereochemistry at the 6-position, it was thought
that the 6S-diastereoisomer is mainly responsible for the
inhibition of TS since this stereochemistry shapes the
molecule into a conformation that is favourable for
binding to the enzyme.5 To investigate this, it was
necessary to prepare pure 6S and 6R-diastereoisomers
for some of the most potent inhibitors of the enzyme
that have been originally reported as a mixture of dia-
stereoisomers (i.e., compounds 1a–c, Fig. 1).2
The synthesis of 6S-1a–c, 6R-1a–c was achieved by
coupling the appropriately protected ligand 2d–f to 6S-2
*Corresponding author. Fax: +44-20-8770-7899; e-mail: vassilio@icr.
ac.uk
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00612-6

3016
V. Bavetsias et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3015–3017
Figure 1. Cyclopenta[g]quinazoline-based inhibitors of TS.
Scheme 1. Reagents and conditions: (i) for 6S-3d, 6R-3d: 2d, DEPC, Et3N, DMF; for 6S-3e, 6R-3e: CF3CO2C6F5, pyridine, DMA, column chro-
matography then 2e, HOBt (cat), DMF; for 6S-3f, 6R-3f: CF3CO2C6F5, pyridine, DMA, column chromatography then 2f, HOBt (cat), Et3N, DMF;
(ii) for 6S-1a, 6R-1a: TFA; for 6S-1b, 6R-1b: TFA–H2O; for 6S-1c, 6R-1c: 1 N NaOH, H2O–MeOH.
Table 1. Inhibition of thymidylate synthase
Regarding inhibition of TS, 6R-1a was ꢁ10-fold less
potent against TS compared with 6S-1a (Table 1).
Likewise compound 6R-1b was approximately ꢁ20-fold
less potent than 6S-1b (Table 1). These results indicated
that indeed the TS activity is mainly due to the 6S-dia-
stereoisomer, since 6R-1a, 6R-1b were contaminated
with ꢁ10% of the corresponding 6S-diastereoisomer.
This was unequivocally shown by comparing the TS
inhibitory activity of 6R-1c (devoid of any 6S-diaster-
eoisomer) with that of 6S-1c. Indeed, 6R-1c was ꢁ1800-
fold less potent an inhibitor of the enzyme than 6S-1c
(Table 1).
Compd
L1210TS Kiapp (nM)
6S-1a
6R-1a
6S-1b
6R-1b
6S-1c
6R-1c
0.33ꢃ0.16
3.5, 3.9
0.17, 0.16
2.1, 4.8
0.71ꢃ0.16
1200, 1460
6R-1a and 6R-1b contaminated with ꢁ10% of 6S-1a and 6S-1b,
respectively. Kiapps normalised to CB3717 (L1210TS Kiapp for
CB3717=20nM).2 L1210TS Kiapp for 6RS-1a=0.42; for 6RS-
1b=0.2; for 6RS-1c=0.78 nM.2
In conclusion, cyclopenta[g]quinazoline-based anti-
folates that possess a chiral centre at the 6-position
constitute a new class of potent inhibitors of TS. The
effect of the stereochemistry at the 6-position on inhi-
biting the TS enzyme was studied by synthesising com-
pounds 6S-1a–c, 6R-1a–c. It was shown, in particular

V. Bavetsias et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3015–3017
3017
with the pair of compounds 6R-1c, 6S-1c that the 6R-
diastereoisomer was virtually inactive compared with its
6S counterpart.
Pharm. Des. 1996, 2, 263. (c) Bavetsias, V.; Jackman, A. L.
Curr. Med. Chem. 1998, 5, 265. (d) Jackman, A. L.; Judson,
I. R. Exp. Opin. Invest. Drugs 1996, 5, 719.
2. Bavetsias, V.; Marriott, J. H.; Melin, C.; Kimbell, R.;
Matusiak, Z. S.; Boyle, F. T.; Jackman, A. L. J. Med. Chem.
2000, 43, 1910.
3. Marriott, J. H.; Neidle, S.; Matusiak, Z.; Bavetsias, V.;
Jackman, A. L.; Melin, C.; Boyle, F. T. J. Chem. Soc., Perkin
Trans. 1 1999, 1495.
4. Boyle, F. T.; Matusiak, Z. S.; Kimbell, R.; Jackman, A. L.
Proc. Am. Assoc. Cancer Res. 1996, 37 (abstr. 2610).
5. For a discussion of the optimum binding conformation of
some quinazolin-4-one-based antifolates to the ‘humanised’
Escherichia coli TS, see: Boyle, F. T.; Stephens, T. C.; Aver-
buch, S. D.; Jackman, A. L. In Anticancer Drug Development
Guide: Antifolate Drugs in Cancer Therapy; Jackman, A. L.
Ed.; Humana: Totowa, NJ, 1999; p 243.
Acknowledgement
This work was supported by grants from the Cancer
Research Campaign (CRC).
References and Notes
1. For recent reviews, see: (a) Jackson, R. C. In Anticancer
Drug Development Guide: Antifolate Drugs in Cancer Therapy;
Jackman, A. L. Ed.; Humana: Totowa, NJ, 1999; p 1. (b)
Gangjee, A.; Elzein, E.; Kothare, M.; Vasudevan, A. Curr.