Antifolate and antiproliferative activity of 6,8,10-triazaspiro[4.5]deca-6,8-dienes and 1,3,5-triazaspiro[5.5]undeca-1,3-dienes

Article abstract of DOI:10.1016/j.bmc.2009.11.065

Two series of triazaspiroalkanedienes, bearing a substituted phenoxy propyloxy side chain, were identified as potent mammalian DHFR inhibitors. One series has a 6,5-spiro bicyclic ring system and the other series has a 6,6-spiro bicyclic system. Both seri

Full text of DOI:10.1016/j.bmc.2009.11.065

Bioorganic & Medicinal Chemistry 18 (2010) 737–743
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antifolate and antiproliferative activity of 6,8,10-triazaspiro[4.5]deca-6,8-dienes
and 1,3,5-triazaspiro[5.5]undeca-1,3-dienes
a,b,
Xiang Ma a,c, , Wai-Keung Chui
*
*
a Department of Pharmacy, Faculty of Science, 18 Science Drive 4, National University of Singapore, Singapore 117543, Singapore
b Medicinal Chemistry Programme, Office of Life Science, 18 Science Drive 4, National University of Singapore, Singapore 117543, Singapore
c State Key Lab of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, 1111 North Zhongshan No.1 Rd, Shanghai 200437, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of triazaspiroalkanedienes, bearing a substituted phenoxy propyloxy side chain, were identi-
fied as potent mammalian DHFR inhibitors. One series has a 6,5-spiro bicyclic ring system and the other
series has a 6,6-spiro bicyclic system. Both series were synthesized and tested for in vitro mammalian
DHFR inhibitory activity and antiproliferative activity against A549 human lung-cancer cells. Compound
3c showed the highest antiproliferative activity against A549 cells with an IC50 value of 27.1 nM. Rescue
experiment confirmed its antifolate antiproliferative mechanism. The excellent antifolate and antiprolif-
erative activity of selected analogues presented in this study warrants further investigation as potential
leads in the anticancer drug discovery.
Received 27 October 2009
Revised 25 November 2009
Accepted 26 November 2009
Available online 6 December 2009
Keywords:
DHFR inhibitors
Antiproliferative activity
1,3,5-Triazines
Spiro rings
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
inhibitors.9 Among them, 7,9-diamino-10-(30-phenoxypropyloxy)-
6,8,10-triazaspiro[4.5]deca-6,8-diene hydrochloride (3a) and 2,4-
Dihydrofolate reductase (DHFR), a ubiquitous enzyme in all
eukaryotic and prokaryotic cells, is of pivotal importance in bio-
chemistry and medicinal chemistry. DHFR catalyzes the biotrans-
formation of dihydrofolate (DHF) to tetrahydrofolate (THF), using
NADPH as a coenzyme. THF is required for the de novo synthesis
of purines and thymidylate (TMP).1 Therefore, inhibition of DHFR
activity in the absence of salvage could shut off the supply of
TMP for DNA biosynthesis, and eventually leads to cell death (thy-
mineless death). This effect hence forms one of the important bases
in cancer chemotherapy.2
While literally thousands of anticancer DHFR inhibitors were
prepared over the years, most of these DHFR inhibitors are com-
pounds possessing a nucleus that is made up from fused heterocy-
clic rings substituted with amino groups.3–8 For instance,
methotrexate (MTX, 1) contains a 6,6-fused bicyclic ring system;
and a new generation multi-targeted antifolate drug pemetrexed
(PTX, AlimtaÒ, 2) contains a 6,5-fused bicyclic ring system, respec-
tively (Fig. 1). In contrast, the inhibitory action of 6,6- or 6,5-spiro
bicyclic ring systems against DHFR has not been extensively inves-
tigated. In our previous study, we described the discovery of com-
pounds with 6,6- or 6,5-spiro bicyclic ring systems as potent DHFR
diamino-5-(30-phenoxypropyloxy)-1,3,5-triazaspiro[5.5]undeca-
1,3-diene hydrochloride (4a) were identified as key lead structures
with excellent activity in DHFR inhibitory assay (IC50 = 2.3 nM and
6.9 nM, respectively) (Fig. 1).9 Herein, in this paper we report the
structure activity relationship (SAR) study of the leads with the
objective of developing potent and selective antifolate anticancer
agents.
The lead compounds 3a and 4a generally consist of three parts:
a phenyl ring, a 1,3-dioxyalkane linker and a core moiety bearing a
1,3,5-triazine spiro cycloalkane ring. In the previous work, we have
explored the linker length and spiro ring size variations.9 Thus, in
the present report we explored 3a and 4a analogues modified at
4 position of phenyl ring using a Craig plot guided optimization.10
Analogues with a range of electronic properties, shapes and hydro-
phobicity were selected according to all four Craig plot quadrants.
These compounds were synthesized and tested in vitro to demon-
strate whether these properties could be a factor in affecting en-
zyme inhibition and antiproliferative activity against human
cancer cells.
2. Results and discussion
2.1. Chemistry
* Tel.: +65 6516 2657; fax: +65 6779 1554 (X.M.); tel.: +65 6516 2933; fax: +65
6779 1554 (W.C.).
Target compounds 3b–3i and 4b–4j (Table 1) were conveniently
E-mail addresses: maxwellcn@yahoo.com (X. Ma), phacwk@nus.edu.sg (W.-K.
prepared via a Williamson type etherification sequence as de-
Chui).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.065

738
X. Ma, W.-K. Chui / Bioorg. Med. Chem. 18 (2010) 737–743
O
COOH
NH2
N
NH2
N
O
O
OH
n
NH2
N
N
a,b,c
N
H
N
.
HCl
N
N
COOH
H2N
N
R
H2N
N
HCl
n
H2N
N
O
N
n=1, 5a
n=2, 5b
n=1, 3b-3i
n=2, 4b-4j
MTX, 1
Scheme 1. Synthesis of 7,9-diamino-10-(30-aryloxypropyloxy)-6,8,10-triazaspi-
ro[4.5]deca-6,8-diene hydrochlorides (3b–3i) and 2,4-diamino-5-(30-phenoxypro-
pyloxy)-1,3,5-triazaspiro[5.5]undeca-1,3-diene hydrochlorides (4b–4j). Reagents
and conditions: (a) NaOH, methanol; (b) DMF, aryloxy propyl bromide rt; (c)
HCl, pH 2.
H
COOH
HN
H2N
N
O
N
N
H
COOH
Pemetrexed, 2
ophilic substitution to furnish the ether followed by an acidificat-
ion to form target compounds in 44.3–71.4% yields.
NH2
O
O
2.2. Biological activity
N
N
H2N
N
2.2.1. DHFR inhibitory activity
.
HCl
The screening of the target compounds was carried out spectro-
scopically using an enzyme inhibitory assay against bovine liver
DHFR, which was a type of commercial available model DHFRs from
mammalian sources and showed relatively small differences to hu-
man DHFR.11 The inhibitory activity of the tested compounds com-
pared with that of MTX was expressed in terms of IC50 values
3a
NH2
O
O
N
N
tabulated in Table 1. Guided by the Craig plot, R = F, Cl, NO2 (+
), R = OCH3 (ꢀ , ꢀ ), R = CH3 and tert-butyl (+ , ꢀ ) and
R = CN, SO2NH2 and COCH3 (ꢀ , +
) were selected for this study.10
p,
+r
p
r
p
r
H2N
N
.
HCl
4a
Figure 1. Structures of selected DHFR inhibitors.
p
r
As shown in Table 1, no increases in inhibitory activity were ob-
served with all 40-substituted compounds 3b–3i. Among them,
the bulky substituent introduced by tert-butyl group at 40-position
of phenyl ring led to a 10-fold loss of activity as compared to that of
the non-substituted 3a. Compounds with other substituents, no
matter electron-withdrawing or electron-donating, did not exhibit
better inhibitory activity as compared to the lead 3a. This observa-
tion indicated that substitution at 40-position on the phenyl ring
may present negative steric hindrance on the interaction to the ac-
tive site of the enzyme. Analogues of 4a with different substitutions
on the phenyl ring showed a slightly different activity pattern from
the 3a analogues. The electron-withdrawing and electron-donating
Table 1
IC50 values of antifolate activity against DHFR and antiproliferative activity against
A549
a
b
Compd
R
n
DHFR, IC50 (nM)
A549, IC50 (nM)
3a
3b
3c
3d
3e
3f
3g
3h
3i
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
F
Cl
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2.3c
10.1
7.2
8.4
9.5
17.0
11.2
7.6
40.2 (2.1)
58.8 (1.1)
27.1 (0.8)
65.8 (3.2)
48.1 (2.2)
159.9 (2.0)
59.1 (4.0)
60.7 (3.1)
59.8 (1.2)
69.7 (3.0)
59.2 (1.1)
49.6 (4.1)
329.3 (12.5)
83.3 (1.7)
495.6 (20.1)
51.6 (3.2)
116.4 (7.1)
144.8 (5.6)
166.4 (11.2)
37.4 (2.1)
substituents with +
tory activity; and 4f with a bulky tert-butyl group was the least ac-
tive compound with IC50 of 21.5 nM. Compounds with
p hydrophobicity exhibited decreased inhibi-
NO2
Me
t-Bu
MeO
CN
ꢀp
hydrophobicity substituents showed a varied inhibitory activity.
Compounds having an electro-donating substituent MeO or an elec-
tron-withdrawing substituent SO2NH2 were compatible with high
inhibitory activity. Cyano-substituted compound 4h showed de-
creased activity with the IC50 value up into 10–20 nM range.
CH3CO
H
9.2
6.9c
15.4
13.8
14.7
17.5
21.5
6.1
F
Cl
NO2
Me
t-Bu
MeO
CN
2.2.2. Antiproliferative activity
The potent DHFR inhibitory activity of both series suggests that
they may exhibit good antiproliferative activity against human
cancer cells. To this end, the antiproliferative activity against
A549, an estrogen receptor negative cell line akined to non-small
cell lung carcinoma, was investigated in vitro using a semiauto-
mated fluorometric microculture cytotoxicity assay (FMCA).12,13
The IC50 values of 3a–3i and 4a–4j compared with that of MTX
are being listed in Table 1. Both series showed potent activity
against A459 cells with IC50 values ranged from 27.1 nM to
495.6 nM. 40-Chloro substituted 3c showed the best antiprolifera-
tive activity, and the activity was even better than the positive con-
trol, MTX. Compound 3f with a bulky tert-butyl group led to a great
decrease in the antiproliferative activity. Antiproliferative activity
improvement in 4a series occurred from the first quadrant of
Craig0s plot where the lipophilic and electron-withdrawing groups
were present, such as Cl and F. One exception as shown in 4d
9.7
13.3
5.6
CH3CO
SO2NH2
4j
MTX
3.4
a
b
c
Values are means of three experiments.
Values are means (SEM), n = 3.
Ref. 9.
scribed in Scheme 1. The starting materials 5a and 5b were pre-
pared according to our previous report.9 1-(para-Substituted phe-
nyloxy)-3-bromoalkanes were synthesized via an alkylation of
commercial available para-substituted-phenols with dibromopro-
pane in the presence of potassium carbonate. The target com-
pounds were then obtained from the above two fragments via a
one-pot reaction, where 5a or 5b were deprotonated using sodium
hydroxide; and the produced N-hydroxide anion underwent nucle-

X. Ma, W.-K. Chui / Bioorg. Med. Chem. 18 (2010) 737–743
739
exhibited decreased activity, and it may due to electronic property
no addn
Hx
120
100
80
60
40
20
0
of the NO2 group, which is highly electron-withdrawing and
relatively bulky in size. Electron-donating methoxy group was
observed to improve the inhibitory activity, while bulky electron-
donating and lipophilic t-butyl group reduced the antiproliferative
activity, suggesting an unfavorable effect of the bulky substitution.
All the substitution in the fourth quadrant of Craig0s plot led to a
decrease in antiproliferative effects.
TdR
Hx+TdR
3. Salvage rescue effects
The antifolate mechanism of action with respect to the affected
target enzymes can be identified by employing the salvage route
for nucleotide synthesis in whole cells.14 It was known as a rescue
experiment in which the antifolate effect could be overridden by
the introduction of the relevant metabolites. The nature of these
relevant metabolites provides clues about the metabolic pathway
affected by test compounds. Several of these affected enzymes
are involved in nucleotide biosynthesis metabolic pathway,
namely, DHFR, thymidylate synthase (TS), glycinamide ribonucleo-
tide formyltransferase (GARFT) and aminoimidazole carboxamide
ribonucleotide formyltransferase (AICARFT).15 In cancer cells, for
example, the cytotoxic effect due to the inhibition of TS can be alle-
viated by the supply of thymidine (TdR), which is metabolically
converted to TdR monophosphate.16 Likewise, the cytotoxic effect
due to the inhibition of GARFT or AICARFT can be alleviated by
the introduction of hypoxanthine (Hx), which is metabolized to
inosine monophosphate, an end product of purine biosynthetic
pathway.17 DHFR inhibitors, which affect both TMP and purine
nucleotide synthesis, required the combination of TdR and Hx for
the revival of normal metabolism. In this study, rescue experiment
of 3c, the best inhibitor against A549 cells in both series, was car-
ried out to investigate the end product protection profile and to
gain further insight into the mechanism of action. In comparison,
MTX (a primarily DHFR inhibitor),18 and PTX (a multi-targeted
antifolate against TS, DHFR, GARFT and AICARFT)15 were involved
in the investigation. During the rescue experiment, purine and
TMP were depleted in the cancer cells in the presence of a DHFR
inhibitor. If the main mechanism was due to the DHFR inhibition,
cells supplied with exogenous Hx and TdR would be revived. If
the mechanism other than the DHFR inhibition was involved, no
complete reversal of growth would be observed. We hypothesized
that 3c may have the same mechanism of action to MTX, while it
may exhibit a different mechanism from PTX.
3c
PTX
MTX
Figure 2. TdR and Hx rescue from antifolates growth inhibition in A549 Cells.
was 90.0% reversed by a combination of TdR and of Hx. Only partial
protection was afforded with only TdR added, while addition of Hx
alone was devoid of protection for 3c. It would appear that in the
absence of Hx the inhibition of purine synthesis is indirect and
incomplete. These results were similar to those obtained for MTX
and PTX, thus implicating DHFR as their primary intracellular tar-
get. It also suggested the action of DHFR inhibitors, including 3c,
MTX and PTX, has attributed mainly to the induction of a thymine-
less state in A549 cells. The protective degree for 3c (90.0%) was
slightly lower than that of MTX (96.6%) and PTX (99.3%), suggested
that the effect of 3c on cell growth might not be exclusively due to
the purine and TMP depletion; and other mechanisms independent
of antifolate pathways might involve in the cytotoxic effects. The
concentrations of TdR and Hx in the current experiment were
higher than what was normally found in normal human plasma;
however, significant variations were found in different tissues
and the concentrations in tumors may be greater due to the release
of nucleosides from dead and dying cells. Moreover, tumor cells
in situ would receive continuous supply of circulating salvageable
nucleotides and bases rather than the finite supply, which was
consumed during growth inhibition studies in vitro. Thus,
nucleoside and base salvage may limit the efficacy of these antifo-
lates in cancer patients. Compound 3c seems to have another
mechanism to partially block the rescue by TdR and Hx. The prop-
erty may make 3c potentially more efficacious compared to MTX
and PTX.
To compare the effects of the end product rescue and their inhi-
bition, the cells were exposed to equitoxic levels, their ten times
respective IC50 values. The concentrations used in this study were
therefore selected as: 270 nM for 3c, 370 nM for MTX, and 6.4
for PTX. The effect on the A549 cells growth inhibition in the pres-
ent of 20 M TdR and/or 100 M Hx together with 3c and controls
for an exposure of 72 h was assessed using FMCA. As indicated in
Table 2 and Figure 2, the relative growth control of 3c, MTX and
PTX at 10 ꢁ IC50 were ranged from 11.7% to 15.2% without addition
of rescue agents. The inhibition of 3c on the growth of A549 cells
lM
4. Conclusion
l
l
In conclusion, a number of potent antifolates have been synthe-
sized. All compounds from both series exhibited antiproliferative
effects against A549 lung-cancer cells. Compound 3c showed the
highest antiproliferative activity against A549 cells with an IC50 va-
lue of 27.1 nM. Rescue experiment on 3c demonstrated antifolate
profile of antiproliferative effect on A549 cancer cells. Further re-
sults from our continuous efforts towards the identification of
antifolates with an improved antiproliferative activity will be re-
ported in due course.
Table 2
Protection of A549 from antifolates growth inhibitory effects by 100 lM Hx, 20 lM
TdR, or the combination of 100
l
M Hx + 20
lM TdR
Compd
Relative growtha (% of the control)
w/o
Hx
TdR
Hx
Rescue
agent
(100
lM) (20
l
M)
(100 lM) + TdR(20 lM)
5. Experimental section
3c
MTX
15.2 1.1
14.3 3.0
14.3 1.2 68.0 0.8
16.0 0.8 77.3 11.2 96.6 0.8
12.6 0.0 84.5 2.3 99.3 1.5
90.0 4.3
5.1. Reagents and general method
Pemetrexed 11.7 0.5
All reactions were performed in oven-dried glassware with
magnetic stirring. All reagents were purchased from Sigma–Aldrich
a
Values are means SEM of three experiments.

740
X. Ma, W.-K. Chui / Bioorg. Med. Chem. 18 (2010) 737–743
and used as received unless otherwise noted. TLC analysis was per-
formed using Merck precoated Silica Gel 60 F254 plates. Reactions
were monitored by TLC on silica gel, with detection by UV light
(254 nm). Flash column chromatography was performed with
230–400 mesh silica gel. Melting points were determined on a Gal-
lenkamp Melting Point Apparatus and were uncorrected. Infrared
spectra (using potassium bromide discs) were recorded with a Jas-
co FT/IR-430 Fourier Transform Infrared Spectrometer and re-
ported in reciprocal centimeters (cmꢀ1). The 1H NMR spectra
were recorded on a Bruker ACF 300 MHz NMR Spectrometer. The
chemical shift (d) values are expressed in parts per million (ppm)
relative to tetramethylsilane as an internal standard: s = singlet,
d = doublet, dd = double doublet, m = multiplet. Maximum UV
absorption wavelengths of the compounds were determined on
the UV-160A UV–vis recording spectrophotometer. Elemental
analyses were performed using the Perkin–Elmer 2400 Elemental
Analyzer Series II and all the values are within 0.4% of calculated
values.
5.2.3. 7,9-Diamino-10-(30-(400-nitro-phenoxy)-propyloxy)-
6,8,10-triaza-spiro[4.5]deca-6,8-diene (3d)
From
7,9-diamino-6,8,10-triaza-spiro[4.5]deca-7,9-dien-6-ol
hydrochloride (1 g, 4.6 mM) and 1-(3-Bromo-propyloxy)-4-nitro-
benzene (1.4 g, 5.5 mM), 3d was obtained as a white solid (1.2 g)
in 62.6% yield. Mp: 210–211 °C; 1H NMR (DMSO-d6): d 1.54–1.75
(br m, 6H), 1.97 (br s, 2H), 2.21 (m, 2H, J = 5.7 Hz), 4.10 (t, 2H,
J = 5.7 Hz), 4.23 (t, 2H, J = 5.7 Hz), 7.00 (br s, 1H, ex) 7.16–7.19 (d,
2H, J = 9.0 Hz), 7.81 (br s, 1H, ex), 8.15 (s, 1H, ex), 8.20–8.25 (d,
2H, J = 9.0 Hz), 8.67 (s, 1H, ex), 9.22 (s, 1H, ex); 13C NMR (DMSO-
d6): d 164.71, 163.00, 157.52, 141.94, 126.96, 116.04, 82.28,
75.07, 66.13, 34.36, 27.85, 23.29; ESI-MS: m/z = 363.2 (M+1)+. Anal.
Calcd for C16H22N6O2ꢂ1.8HCl: C, 44.90; H, 5.60; N, 19.63. Found: C,
44.82; H, 5.33; N, 19.39.
5.2.4. 7,9-Diamino-10-(30-(400-tolyloxy)-propyloxy)-6,8,10-
triaza-spiro[4.5]deca-6,8-diene (3e)
From
7,9-diamino-6,8,10-triaza-spiro[4.5]deca-7,9-dien-6-ol
hydrochloride (1 g, 4.6 mM) and 1-(3-bromo-propyloxy)-4-
methyl-benzene (1.3 g, 5.5 mM), 3e was obtained as a white solid
(1.0 g) in 61.3% yield. Mp: 192–193 °C; 1H NMR (DMSO-d6): d
1.54–1.73 (br m, 6H), 2.11 (br s, 2H), 2.13 (t, 2H, J = 6.0), 2.23 (s,
3H), 4.00–4.09 (m, 4H), 6.83 (d, 2H, J = 8.7 Hz), 7.09 (d, 2H,
J = 8.7 Hz), 7.13 (br s, 1H, ex), 7.79 (br s, 1H, ex), 8.12 (s, 1H, ex),
8.65 (s, 1H, ex), 9.47 (s, 1H, ex); 13C NMR (DMSO-d6): d 162.98,
157.63, 157.23, 130.87, 130.32, 115.23, 82.24, 75.29, 64.73, 34.36,
28.11, 23.20, 21.12; ESI-MS: m/z = 332.2 (M+1)+. Anal. Calcd for
C16H22N6O2ꢂHCl: C, 52.91; H, 7.31; N, 18.15. Found: C, 52.86; H,
7.42; N, 18.53.
5.2. General procedure for the synthesis of 3b–3i and 4b–4j
series
1-Hydroxy-1,2-dihydro-1,3,5-triazine (1 g) was dissolved in
methanol (20 mL). One molar equivalent of NaOH was added into
the solution and the mixture was refluxed for 30 min. After being
cooled down to room temperature, methanol was evaporated
using vacuum. The dry white residue was then dissolved in DMF
(5 mL), and 1.2 mol equiv of 3-aryloxypropyl bromide was added
to this solution. The mixture was stirred at room temperature
and TLC was used to monitor the reaction. After the reaction was
completed, the solution was adjusted to pH 2 using concentrated
HCl, followed by evaporating of DMF at room temperature. The res-
idue was filtered and washed using water, followed by recrystalli-
zation in 90% EtOH. In this manner, following compounds were
prepared.
5.2.5. 7,9-Diamino-10-(30-(400-tert-butyl-phenoxy)-propyloxy)-
6,8,10-triaza-spiro[4.5]deca-6,8-diene (3f)
From
7,9-diamino-6,8,10-triaza-spiro[4.5]deca-7,9-dien-6-ol
hydrochloride (1 g, 4.6 mM) and 1-(3-bromo-propyloxy)-4-tert-
butyl-benzene (1.5 g, 5.5 mM), 3f was obtained as a white solid
(1.2 g) in 62.7% yield. Mp: 197–198 °C; 1H NMR (DMSO-d6): d
1.25 (s, 9H), 1.51–1.72 (br m, 6H), 1.96 (br s, 2H), 2.14 (m, 2H),
4.01–4.09 (m, 4H), 6.85 (d, 2H, J = 8.6 Hz), 7.13 (br s, 1H, ex), 7.29
(d, 2H, J = 8.6 Hz), 7.79 (br s, 1H, ex), 8.12 (s, 1H, ex), 8.65 (s, 1H,
ex), 9.53 (s, 1H, ex); 13C NMR (DMSO-d6): d 162.99, 157.65,
157.11, 143.87, 127.10, 114.90, 82.22, 75.33, 64.77, 34.79, 34.31,
32.38, 28.12, 23.09; ESI-MS: m/z = 374.3 (M+1)+. Anal. Calcd for
C20H31N5O2ꢂHClꢂ0.8H2O: C, 56.61; H, 7.98; N, 16.50. Found: C,
56.62; H, 7.85; N, 16.40.
5.2.1. 7,9-Diamino-10-(30-(400-fluoro-phenoxy)-propyloxy)-
6,8,10-triaza-spiro[4.5]deca-6,8-diene (3b)
From
7,9-diamino-6,8,10-triaza-spiro[4.5]deca-7,9-dien-6-ol
hydrochloride (1 g, 4.6 mM) and 1-(3-bromo-propyloxy)-4-flu-
oro-benzene (1.3 g, 5.5 mM), 3b was obtained as a white solid
(1.1 g) in 63.6% yield. Mp: 182–183 °C; 1H NMR (DMSO-d6): d
1.51–1.73 (br m, 6H), 1.97 (br s, 2H), 2.15 (m, 2H, J = 5.6 Hz),
4.02–4.10 (m, 4H), 6.83–6.99 (m, 2H), 6.09–7.18 (m, 2H), 7.81
(br s, 1H, ex), 8.14 (s, 1H, ex), 8.66 (s, 1H, ex), 9.47 (s, 1H, ex);
13C NMR (DMSO-d6): d 162.98, 157.61, 156.00, 155.71, 117.04,
116.73, 116.71, 116.59, 82.24, 75.20, 65.36, 34.34, 28.06, 23.20;
ESI-MS: m/z = 336.2 (M+1)+. Anal. Calcd for C16H22FN5O2ꢂHCl: C,
49.29; H, 6.46; N, 17.96. Found: C, 48.93; H, 5.99; N, 18.19.
5.2.6. 7,9-Diamino-10-(30-(400-methoxy-phenoxy)-propyloxy)-
6,8,10-triaza-spiro[4.5]deca-6,8-diene (3g)
From
7,9-diamino-6,8,10-triaza-spiro[4.5]deca-7,9-dien-6-ol
hydrochloride (1 g, 4.6 mM) and 1-(3-bromo-propyloxy)-4-meth-
oxy-benzene (1.3 g, 5.5 mM), 3g was obtained as a white solid
(1.1 g) in 58.0% yield. Mp: 187–188 °C; 1H NMR (DMSO-d6): d
1.54–1.73 (br m, 6H), 1.98 (br s, 2H), 2.12 (m, 2H, J = 5.6 Hz),
3.69 (s, 3H), 3.99 (t, 2H, J = 6.0 Hz), 4.07 (t, 2H, J = 6.0 Hz), 6.87 (s,
4H), 7.10 (br s, 1H, ex), 7.64 (br s, 1H, ex), 8.12 (s, 1H, ex), 8.65
(s, 1H, ex), 9.44 (s, 1H, ex); 13C NMR (DMSO-d6): d 162.98,
157.62, 154.46, 153.33, 116.29, 115.65, 82.24, 75.31, 65.21, 56.40,
34.36, 28.16, 23.21; ESI-MS: m/z = 348.2 (M+1)+. Anal. Calcd for
C17H25N5O3ꢂHClꢂ0.8H2O: C, 51.27; H, 6.98; N, 17.58. Found: C,
51.22; H, 6.61; N, 17.61.
5.2.2. 7,9-Diamino-10-(30-(400-chloro-phenoxy)-propyloxy)-
6,8,10-triaza-spiro[4.5]deca-6,8-diene (3c)
From
7,9-diamino-6,8,10-triaza-spiro[4.5]deca-7,9-dien-6-ol
hydrochloride (1 g, 4.6 mM) and 1-(3-bromo-propyloxy)-4-
chloro-benzene (1.4 g, 5.5 mM), 3c was obtained as a white solid
(1.3 g) in 67.1% yield. Mp: 199–200 °C; 1H NMR (DMSO-d6): d
1.53–1.74 (br m, 6H), 1.97 (br s, 2H), 2.15 (m, 2H, J = 5.7 Hz),
4.06 (m, 4H), 6.96–6.99 (d, 2H, J = 9.0 Hz), 7.32–7.35 (m, 2H,
J = 9.0 Hz), 7.79 (br s, 1H, ex), 8.13 (s, 1H, ex), 8.66 (s,
1H, ex), 9.36 (s, 1H, ex); 13C NMR (DMSO-d6): d 162.98, 158.22,
157.53, 130.30, 125.38, 117.18, 82.26, 75.16, 65.21, 34.35,
27.98, 23.26; ESI-MS: m/z = 352.2 (M+1)+. Anal. Calcd for
C16H22ClN5O2ꢂ1.7HCl: C, 46.44; H, 5.77; N, 16.92. Found: C,
46.37; H, 5.57; N, 16.90.
5.2.7. 400-(30-(7,9-Diamino-6,8,10-triaza-spiro[4.5]deca-7,9-
dien-6-yloxy)-propyloxy)-benzonitrile (3h)
From
7,9-diamino-6,8,10-triaza-spiro[4.5]deca-7,9-dien-6-ol
hydrochloride (1 g, 4.6 mM) and 4-(3-bromo-propyloxy)-benzoni-
trile (1.3 g, 5.5 mM), 3h was obtained as a white solid (1.2 g) in
66.6% yield. Mp: 202–203 °C; 1H NMR (DMSO-d6): d 1.49–1.73

X. Ma, W.-K. Chui / Bioorg. Med. Chem. 18 (2010) 737–743
741
(br m, 6H), 1.95 (br s, 2H), 2.18 (m, 2H, J = 5.6 Hz), 2.52 (s, 3H), 4.05
(t, 2H, J = 6.0 Hz), 4.09 (t, 2H, J = 6.0 Hz), 7.13 (q, 2H, J = 7.1 Hz,
J = 2.3 Hz), 7.79 (d, 2H, J = 7.1 Hz, J = 2.3 Hz), 8.13 (s, 1H, ex), 8.65
(s, 1H, ex), 9.53 (s, 1H, ex); 13C NMR (DMSO-d6): d 162.96,
162.86, 157.65, 135.28, 120.15, 116.55, 103.98, 82.22, 75.06,
65.55, 34.31, 27.84, 23.16; ESI-MS: m/z = 343.2 (M+1)+. Anal. Calcd
for C17H26N6O2ꢂHClꢂH2O: C, 51.45; H, 6.35; N, 21.18. Found: C,
51.42; H, 6.02; N, 21.25.
5.2.12. 2,4-Diamino-5-(30-(400-tolyloxy)-propyloxy)-1,3,5-triaza-
spiro[5.5]undeca-1,3-diene hydrochloride (4e)
From 2,4-diamino-1,3,5-triaza-spiro[5.5]undeca-2,4-dien-1-ol
hydrochloride (1 g, 4.3 mM) and 1-(3-bromo-propyloxy)-4-
methyl-benzene (1.2 g, 5.1 mM), 4e was obtained as a white solid
(1.0 g) in 61.4% yield. Mp: 214–215 °C; 1H NMR (DMSO-d6): d
0.80 (br s, 1H), 1.44–1.69 (br t, 9H), 2.14 (br s, 2H), 2.23 (s, 3H),
4.04 (m, 4H), 6.84 (d, 2H, J = 8.7 Hz), 7.09 (d, 2H, J = 8.3 Hz), 7.33
(br s, 1H, ex), 7.81 (br s, 1H, ex), 8.06 (s, 1H, ex), 8.59 (s, 1H, ex),
9.21 (s, 1H, ex); 13C NMR (DMSO-d6): d 162.16, 157.54, 157.25,
130.86, 130.32, 115.21, 75.23, 64.63, 28.06, 24.84, 21.77, 21.11;
ESI-MS: m/z = 346.3 (M+1)+. Anal. Calcd for C18H27N5O2ꢂHCl: C,
56.61; H, 7.39; N, 18.34. Found: C, 56.47; H, 7.45; N, 18.60.
5.2.8. 1-(400-(30-(7,9-Diamino-6,8,10-triaza-spiro[4.5]deca-7,9-
dien-6-yloxy)-propyloxy)-phenyl)-ethanone (3i)
From
7,9-diamino-6,8,10-triaza-spiro[4.5]deca-7,9-dien-6-ol
hydrochloride (1 g, 4.6 mM) and 1-[4-(3-bromo-propyloxy)-phe-
nyl]-ethanone (1.4 g, 5.5 mM), 3i was obtained as a white solid
(1.3 g) in 66.9% yield. Mp: 194–195 °C; 1H NMR (DMSO-d6): d
1.54–1.73 (br m, 6H), 1.97 (br s, 2H), 2.19 (m, 2H, J = 5.6 Hz),
2.52 (s, 3H), 4.09 (t, 2H, J = 6.0 Hz), 4.16 (t, 2H, J = 6.0 Hz), 7.05
(d, 2H, J = 5.8 Hz), 7.93 (d, 2H, J = 5.8 Hz), 8.14 (s, 1H, ex), 8.65 (s,
1H, ex), 9.36 (s, 1H, ex). 13C NMR (DMSO-d6): d 197.36, 163.24,
162.98, 157.61, 131.55, 131.03, 115.26, 82.25, 75.13, 65.31, 34.35,
27.95, 27.47, 23.22; ESI-MS: m/z = 360.2 (M+1)+. Anal. Calcd for
C18H25N5O3ꢂHClꢂH2O: C, 52.23; H, 6.82; N, 16.92. Found: C, 51.87;
H, 6.47; N, 16.86.
5.2.13. 2,4-Diamino-5-(30-(400-tert-butylphenoxy)-propyloxy)-
1,3,5-triazaspiro[5.5]undeca-1,3-diene hydrochloride (4f)
From 2,4-diamino-1,3,5-triaza-spiro[5.5]undeca-2,4-dien-1-ol
hydrochloride (1 g, 4.3 mM) and 1-(3-bromo-propyloxy)-4-tert-
butyl-benzene (1.4 g, 5.1 mM), 4f was obtained as a white solid
(0.94 g) in 47.4% yield. Mp: 222–223 °C; 1H NMR (DMSO-d6): d
0.74 (br s, 1H), 1.35 (s, 9H), 1.39–1.68 (br t, 9H), 2.15 (br s, 2H),
4.05 (t, 4H, J = 5.3 Hz, J = 5.7 Hz), 6.87 (d, 2H, J = 8.6 Hz), 7.14 (br
s, 1H, ex), 7.29 (d, 2H, J = 8.7 Hz), 7.84 (br s, 1H, ex), 8.08(s, 1H,
ex), 8.60 (s, 1H, ex), 8.88 (s, 1H, ex).; 13C NMR (DMSO-d6): d
162.19, 157.37, 157.17, 143.86, 127.10, 114.85, 75.18, 75.06,
64.49, 34.78, 32.37, 28.06, 24.72, 21.78; ESI-MS: m/z = 388.3
(M+1)+. Anal. Calcd for C21H33N5O2ꢂ2HCl: C, 54.78; H, 7.66; N,
15.21. Found: C, 54.82; H, 7.46; N, 15.23.
5.2.9. 2,4-Diamino-5-(30-(400-fluorophenoxy)-propyloxy)-1,3,5-
triazaspiro[5.5]undeca-1,3-diene hydrochloride (4b)
From 5-hydroxy-1,3,5-triazaspiro[5.5]undeca-1,3-diene-2,4-dia
mine hydrochloride (1 g, 4.3 mM) and 30-(p-fluorophenoxy) propyl
bromide (1.2 g, 5.1 mM), 4b was obtained as a white solid (1.0 g)
in 62.1% yield. Mp: 211–212 °C; 1H NMR (DMSO-d6): d 0.77 (br s,
1H), 1.48–1.69 (br d, 9H), 2.14 (br s, 2H), 4.04–4.07 (m, 4H), 6.93–
6.99 (m, 2H), 7.09–7.16 (d, 2H), 7.40 (br s, 1H, ex), 7.81 (br s, 1H,
ex), 8.08(s, 1H, ex), 8.60 (s, 1H, ex), 9.28 (s, 1H, ex); 13C NMR
(DMSO-d6): d 162.31, 157.16, 155.54, 117.04, 116.73, 116.60,
116.50, 75.15, 75.03, 65.10, 27.83, 24.56, 21.72; ESI-MS: m/z =
350.3 (M+1)+. Anal. Calcd for C17H24FN5O2ꢂHCl: C, 52.92; H, 6.53;
N, 18.15. Found: C, 52.75; H, 6.56; N, 18.06.
5.2.14. 2,4-Diamino-5-(30-(400-methoxyphenoxy)-propyloxy)-
1,3,5-triazaspiro[5.5]undeca-1,3-diene hydrochloride (4g)
From 2,4-diamino-1,3,5-triaza-spiro[5.5]undeca-2,4-dien-1-ol
hydrochloride (1 g, 4.3 mM) and 30-(p-methoxyphenoxy) propyl
bromide (1.3 g, 5.1 mM), 4g was obtained as
a white solid
(0.93 g) in 51.3% yield. Mp: 202–203 °C; 1H NMR (DMSO-d6): d
0.80 (br s, 1H), 1.45–1.70 (br t, 9H), 2.13 (br s, 2H), 3.69 (s, 3H),
3.99–4.07 (m, 4H), 6.84–6.91 (m, 4H), 7.12 (br s, 1H, ex), 7.84 (br
s, 1H, ex), 8.08(s, 1H, ex), 8.61 (s, 1H, ex), 8.95 (s, 1H, ex); 13C
5.2.10. 2,4-Diamino-5-(30-(400-chlorophenoxy)-propyloxy)-1,3,5-
triazaspiro[5.5]undeca-1,3-diene hydrochloride (4c)
NMR (DMSO-d6): d 162.37, 157.45, 154.49, 153.38, 116.25,
115.68, 75.20, 75.10, 66.95, 56.43, 28.11, 24.82, 21.83; ESI-MS:
m/z = 362.3 (M+1)+. Anal. Calcd for C18H27N5O2ꢂHClꢂ1.2H2O: C,
51.53; H, 7.30; N, 16.69. Found: C, 51.54; H, 6.97; N, 16.47.
From 2,4-diamino-1,3,5-triaza-spiro[5.5]undeca-2,4-dien-1-ol
hydrochloride (1 g, 4.3 mM) and 30-(p-chlorophenoxy) propyl bro-
mide (1.2 g, 5.1 mM), 4c was obtained as a white solid (1.1 g) in
64.8% yield. Mp: 219–220 °C; 1H NMR (DMSO-d6): d 0.79 (br s,
1H), 1.48–1.68 (br d, 9H), 2.16 (br s, 2H), 4.08 (m, 4H), 6.99 (d,
2H, J = 8.6 Hz), 7.34 (d, 2H, J = 8.7 Hz), 7.37 (br s, 1H, ex), 7.81 (br
s, 1H, ex), 8.08(s, 1H, ex), 8.60 (s, 1H, ex), 9.28 (s, 1H, ex); 13C
5.2.15. 400-(30-(2,4-Diamino-1,3,5-triaza-spiro[5.5]undeca-2,4-
dien-1-yloxy)-propyloxy)-benzonitrile hydrochloride (4h)
From 2,4-diamino-1,3,5-triaza-spiro[5.5]undeca-2,4-dien-1-ol
hydrochloride (1 g, 4.3 mM) and p-(3-bromo-propyloxy)-benzoni-
trile (1.2 g, 5.1 mM), 4h was obtained as a white solid (0.91 g) in
53.4% yield. Mp: 221–222 °C; 1H NMR (DMSO-d6): d 0.76 (br s,
1H), 1.48–1.69 (br d, 9H), 2.20 (br s, 2H), 4.05 (br s, 2H), 4.19 (t,
2H, J = 5.6 Hz, J = 6.0 Hz), 7.15 (d, 2H, J = 9.0 Hz), 7.44 (br s, 1H,
ex), 7.79 (d, 2H, J = 9.1 Hz), 7.79 (br s, 1H, ex), 8.09(s, 1H, ex),
8.60 (s, 1H, ex), 9.32 (s, 1H, ex). 13C NMR (DMSO-d6): d 162.89,
162.09, 157.61, 135.26, 120.15, 116.57, 103.95, 75.26, 74.91,
65.45, 27.80, 24.90, 21.75; ESI-MS: m/z = 357.2 (M+1)+. Anal. Calcd
for C18H24N6O2ꢂHClꢂ0.2H2O: C, 54.53; H, 6.46; N, 21.20. Found: C,
54.60; H, 6.42; N, 21.02.
NMR (DMSO-d6):
d 162.28, 158.12, 157.21, 130.31, 125.43,
117.10, 75.15, 75.01, 64.99, 27.79, 24.62, 21.74; ESI-MS: m/z =
366.1 (M+1)+. Anal. Calcd for C17H24ClN5O2ꢂHCl: C, 50.75; H, 6.26;
N, 17.41. Found: C, 50.49; H, 6.20; N, 17.57.
5.2.11. 2,4-Diamino-5-(30-(400-nitrophenoxy)-propyloxy)-1,3,5-
triazaspiro[5.5]undeca-1,3-diene hydrochloride (4d)
From 2,4-diamino-1,3,5-triaza-spiro[5.5]undeca-2,4-dien-1-ol
hydrochloride (1 g, 4.3 mM) and 1-(3-bromo-propyloxy)-4-nitro-
benzene (1.3 g, 5.1 mM), 4d was obtained as a yellowish solid
(1.1 g) in 60.5% yield. Mp: 212–213 °C; 1H NMR (DMSO-d6): d
0.79 (br s, 1H), 1.45–1.70 (br d, 9H), 2.22 (br s, 2H), 4.06 (br s,
2H), 4.25 (m, 2H, J = 6.0 Hz), 7.17 (br s, 1H, ex), 7.19 (d, 2H,
J = 9.4 Hz), 7.83 (br s, 1H, ex), 8.11 (s, 1H, ex), 8.24 (d, 2H,
J = 9.0 Hz), 8.62 (s, 1H, ex), 8.99 (s, 1H, ex). 13C NMR (DMSO-d6):
d 164.75, 162.15, 157.47, 141.94, 126.95, 116.08, 75.20, 74.96,
66.05, 27.79, 24.84, 21.81; ESI-MS: m/z = 377.2 (M+1)+. Anal. Calcd
for C17H24N6O4ꢂ1.6HCl: C, 46.97; H, 5.93; N, 19.33. Found: C, 46.99;
H, 5.78; N, 19.12.
5.2.16. 1-(400-(30-(2,4-Diamino-1,3,5-triaza-spiro[5.5]undeca-
2,4-dien-1-yloxy)-propyloxy)-phenyl)-ethanone hydrochloride
(4i)
From 2,4-diamino-1,3,5-triaza-spiro[5.5]undeca-2,4-dien-1-ol
hydrochloride (1 g, 4.3 mM) and 1-[4-(3-bromo-propyloxy)-phe-
nyl]-ethanone (1.3 g, 5.1 mM), 4i was obtained as a white solid
(1.2 g) in 67.1% yield. Mp: 206–207 °C; 1H NMR (DMSO-d6): d
0.77 (br s, 1H), 1.47–1.69 (br t, 9H), 2.19 (br s, 2H), 2.54 (s, 3H),

742
X. Ma, W.-K. Chui / Bioorg. Med. Chem. 18 (2010) 737–743
4.05 (t, 2H, J = 5.6 Hz, J = 4.5 Hz), 4.19 (t, 2H, J = 5.6 Hz, J = 6.0 Hz),
7.07 (d, 2H, J = 8.6 Hz), 7.33 (br s, 1H, ex), 7.80 (br s, 1H, ex), 7.94
(d, 2H, J = 8.7 Hz), 8.09(s, 1H, ex), 8.60 (s, 1H, ex), 8.85 (s, 1H, ex),
9.21 (s, 1H, ex); ESI-MS: m/z = 374.2 (M+1)+; 13C NMR (DMSO-
d6): d 197.37, 163.28, 162.15, 157.58, 139.68, 131.55, 131.04,
115.36, 75.24, 75.03, 65.24, 34.63, 27.47, 24.88, 21.78. Anal. Calcd
for C19H27N5O3ꢂHClꢂH2O: C, 53.78; H, 7.03; N, 16.50. Found: C,
53.81; H, 6.88; N, 16.62.
5.4.2. Drugs and reagents
Stock solution were prepared by dissolving compounds using
5% DMSO in phosphate-buffered saline (PBS) at a concentration
of 1.0 mmol/L and stored at ꢀ20 °C. MTX was obtained from
Department of Pharmacy, National University Hospital (NUH) as
solution of 25 mg/2 mL. PTX was kindly supplied by Pharmacy of
Oncology and Hematology department of NUH. The test com-
pounds were diluted to the working concentration with culture
medium before use. A stock solution of fluorescein diacetate
(FDA) was prepared in 10 mg/mL concentration in DMSO, kept fro-
zen (ꢀ20 °C) and protected from light.
5.2.17. 400-(30-(2,4-Diamino-1,3,5-triaza-spiro[5.5]undeca-2,4-di
en-1-yloxy)-propyloxy)-benzenesulfonamide hydrochloride (4j)
From 2,4-diamino-1,3,5-triaza-spiro[5.5]undeca-2,4-dien-1-ol
hydrochloride (1 g, 4.3 mM) and 4-(3-bromo-propyloxy)-benzene-
sulfonamide (1.5 g, 5.1 mM), 4j was obtained as a white solid
(0.96 g) in 44.3% yield. Mp: 198–199 °C; 1H NMR (DMSO-d6): d
0.79–0.83 (br d, 1H, J = 12.1 Hz), 1.40–1.72 (br t, 9H), 2.20 (br s,
2H), 4.07 (br s, 2H), 4.16–4.20 (t, 2H, J = 6.0 Hz), 7.00 (br s, 1H,
ex), 7.11–7.14 (d, 2H, J = 9.0 Hz), 7.23 (s, 2H), 7.76–7.79 (d, 2H,
J = 8.6 Hz), 7.80 (br s, 1H, ex), 8.12(s, 1H, ex), 8.70 (br s, 2H, ex);
13C NMR (DMSO-d6): d 162.20, 161.78, 157.32, 137.34, 128.74,
115.47, 75.18, 75.07, 65.32, 27.85, 24.77, 21.83; ESI-MS: m/
z = 410.6 (M+1)+. Anal. Calcd for C17H26N6O4Sꢂ2HClꢂH2O: C, 40.72;
H, 6.03; N, 16.76. Found: C, 40.64; H, 5.78; N, 16.66.
5.4.3. Fluorometric microculture cytotoxicity assay (FMCA)
The FMCA is based on measurement of fluorescence generated
from hydrolysis of fluorescein diacetate (FDA) to fluorescein by
cells with intact plasma membranes and has been described in de-
tail previously. The drug cytotoxicity and cell proliferation were
determined using the previously described FMCA12, exponential
cell growth is guaranteed during the whole time of incubation.
Briefly, by utilization of 96-well microtiter plates, 200 lL of a cell
suspension were plated into each well (A549, 1000 cells per well).
The plates were incubated at 37 °C for 24 h in a humidified atmo-
sphere (5.0%, CO2) to allow the cells to adhere to the bottom. The
medium of the plates was then replaced with 200 lL of medium
5.3. DHFR enzyme assay
containing the test compounds. Twelve wells without compounds
were served as controls and twelve wells containing culture med-
ium only served as blanks. After an incubation period of 72 h, the
medium was removed by multichannel pipette. After one wash
The assay was performed at 37 °C in a Hewlett–Packard 8453
Diode Array UV–vis Spectrophotometer with HP 89090A Peltier
Temperature Controller at a detection wavelength of 340 nm and
full scale deflection of 4.0 absorbance units. The assay was carried
out over 6 min with absorbance readings taken every 5 s. A graph
of absorbance readings versus time was plotted and the gradient
over the linear range from 60 to 300 s was taken to be the rate of
the reaction. The assay was conducted by mixing the appropriate
volumes of phosphate buffer, NADPH, DHF, bovine liver DHFR
and inhibitor in the cuvette. The rate of consumption of NADPH
at 340 nm during the conversion of dihydrofolic acid to tetrahydro-
folic acid was monitored. The reduction in absorbance over 6 min
in the absence of the inhibitor was taken as the control. Total inhi-
bition of DHFR was indicated by an insignificant change in the rate
of consumption of NADPH and no inhibition would be seen when
the rate of consumption of NADPH was equal to that of the control.
The percentage activity of each inhibitor was calculated by the fol-
lowing formulae:
with PBS, 200 lL/well PBS containing FDA (2 lg/mL) was added.
Subsequently, the plates were incubated for 45 min at 37 °C and
the fluorescence generated from each well was then read at an
excitation wavelength of 485 nm and an emission wavelength of
535 nm by a scanning fluorometer. In a preliminary study, the fluo-
rescence was found to be proportional to the number of viable cells
in the well. Quality criteria for a technically successful assay in-
cluded a proportion of 70% cells in control wells after 72 h of incu-
bation, a fluorescence signal in control wells of greater than or
equal to five times the mean blank value, and a mean coefficient
of variation in control wells of <30%. The results are presented as
survival index (SI), defined as fluorescence in test wells/fluores-
cence in control wells (blank values subtracted) ꢁ 100. Thus, a
low numerical value indicates high sensitivity to the cytotoxic ef-
fect of the drug. IC50 value was defined as the concentration giving
a SI 50% of the control SI. Compounds were tested in triplicate and
the results are expressed as means SEM for the data combined
from separate experiments.
Slope of inhibited enzyme
(i)
Activity ¼
ꢁ 100%
Slope of uninhibited enzyme
(ii)
Inhibition ¼ 100% ꢀ Activity
5.5. Rescue experiment
FMCA assays were carried out using the method stated above.
Investigations of the effect of rescue agents were conducted using
a fixed concentration of antifolates and new synthesized com-
pound 3c at the 10 ꢁ IC50 concentration determined as above for
A graph of percentage inhibition against the logarithmic con-
centration ( M) was plotted for each pot and the IC50 value was ta-
ken to be the concentration ( M) at which 50% inhibition was
l
l
observed. All the experiment was carried out in triplicate.
A549. Cells in 200 lL of medium were seeded in each well of 96-
well plates. After 24 h of incubation, the medium was replaced
with medium containing 10 ꢁ IC50 concentration of compounds
in the absence of protection, or with the same concentration of
5.4. Antiproliferative assay
5.4.1. Cells and cell culture
drug plus 20 lM TdR, 100 lM Hx, 20 lM TdR and 100 lM Hx in
A549 cells were kindly supplied by Collaboration Labs in Depart-
ment of Pharmacy, NUS. A549 cells were grown in Dulbecco’s mod-
ified Eagle’s medium (DMEM), supplemented with 1% penicillin/
streptomycin and 10% fetal bovine serum under the same condition.
The cell lines were passaged two times weekly after previous treat-
ment with trypsin (0.05%)/ethylenediaminetetraacetic acid (0.02%).
Cell viability of the stock cultures used for subsequent experiments
was always above 95% as assessed by trypan blue solution.
the medium. Twelve wells were used for each test solution and
for the control, which contained the corresponding amount of sol-
vents for dissolving compounds. After the incubation of 72 h, the
medium was removed with multichannel pipette. After one wash
with PBS, 200 lL/well PBS containing FDA (2 lg/mL) was added.
Subsequently, the plates were incubated for 45 min at 37 °C and
the fluorescence generated from each well was then read at an
excitation wavelength of 485 nm and an emission wavelength of

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Acknowledgments
The authors would like to thank the Academic Research Funds
of National University of Singapore (R148-000-052-112) for sup-
porting this piece of work. We gratefully appreciate the excellent
help from Ms. Yang Hong and Mr. Sun Lingyi in generating 13C
NMR spectra.
References and notes
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