Coronarin D conjugated to methylene homologues of chlorambucil: Synthesis and evaluation of their cytotoxicity

Article abstract of DOI:10.3184/174751912X13362343090928

Methylene homologues of chlorambucil were synthesised and conjugated to the labdane diterpene coronarin D. The products were evaluated for their in vitro cytotoxicity, and were found to exhibit selective activity against MOLT-3 cell line. Two homologues o

Full text of DOI:10.3184/174751912X13362343090928

374 RESEARCH PAPER
JUNE, 374–378
JOURNAL OF CHEMICAL RESEARCH 2012
Coronarin D conjugated to methylene homologues of chlorambucil:
synthesis and evaluation of their cytotoxicity
Nisachon Khunnawutmanotham,a Nitirat Chimnoi,a Wattanachai Champathong,b Pradit
Lerdsirisuk,b Theeraphon Khotmora and Supanna Techasakula,b*
aChulabhorn Research Institute, Vibhavadee-Rangsit Highway, Lak Si, Bangkok 10210, Thailand
bDepartment of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Kasetsart University, Bangkok 10900,
Thailand
Methylene homologues of chlorambucil were synthesised and conjugated to the labdane diterpene coronarin D. The
products were evaluated for their in vitro cytotoxicity, and were found to exhibit selective activity against MOLT-3 cell
line. Two homologues of chlorambucil showed a comparable cytotoxic effect to their parent. However, as compared
with the non-derivatised chlorambucil and its homologues, their conjugation with coronarin D through ester linkage
did not enhance in vitro cytotoxicity against the tested cancer cell lines.
Keywords: chlorambucil, methylene homologues, coronarin D, cytotoxicity
Chlorambucil (1, Fig. 1), 4-[4-(N,N-bis(2-chloroethyl)amino)-
phenyl]butyric acid is an aromatic nitrogen mustard alkylating
agent. It was developed by Everett et al.,1 and is still used as
a chemotherapy for the treatment of chronic lymphocytic
leukaemia. The anti-cancer effects of chlorambucil result from
its interference with DNA replication and RNA transcription
through the alkylation and cross-linking of DNA strands,
leading to DNA damage and cellular death.2 Coronarin D (2,
Fig. 1) is a labdane diterpene that is easily isolated in high
yield from the rhizomes of Hedychium coronarium, and
displays significant cytotoxicity against various cancer cell
lines.3−5 Recently, Kunnumakkara et al.6 reported the anti-
inflammatory mechanism of coronarin D. They found that
coronarin D inhibited both constitutive and inducible nuclear
factor-κB pathway activation. They hypothesised that the anti-
inflammatory and cytotoxic effects of coronarin D were due
to its ability to inhibit NF-κB and NF-κB-regulated gene
expression. They also found that coronarin D potentiated the
cytotoxic effect of different chemotherapeutic agents against
different tumor cell types. The goal of the current study was to
evaluate the cytotoxicity of coronarin D linked as an ester to
chlorambucil and homologues, with a varied methylene chain
length. The masking of the ionizable carboxylic group of the
chlorambucil and homologues, through an ester linkage with a
labdane moiety, should increase the overall lipophilicity of the
nitrogen mustard and possibly enhance the ability of the com-
pound to traverse the cell membrane. Furthermore, the dual
cytotoxic mechanism of conjugated compounds, DNA damag-
ing effects of the nitrogen mustards and the anti-inflammatory
effect of coronarin D, may induce some unique cytotoxicity.
Varying the chain length of methylene of chlorambucil
also allowed an examination of the effect of spacer arm
between coronarin D and the nitrogen mustard moiety on their
cytotoxicity.
Results and discussion
Chemistry
The syntheses of methylene homologues of chlorambucil,
containing zero to two methylene groups (n=0,1,2), are out-
lined in Scheme 1. 4-(N,N-Bis(2-chloroethyl)amino)benzalde
hyde (4),7 the precursor of all homologues, was obtained in
good yield by the treatment of N-phenyldiethanolamine (3)
with phosphoryl chloride in dimethylformamide.8 Oxidation
of 4 with potassium permanganate in pyridine gave 4-(N,N-
bis(2-chloroethyl)amino)benzoic acid (5, n=0) in moderate
yield.7 To prepare 4-(N,N-bis(2-chloroethyl)amino)phenylacet
ic acid (8, n=1),1 4 was subjected to a Wittig reaction with (m
ethoxymethyl)triphenylphosphonium chloride9 in the presence
of 1.0 M sodium bis(trimethylsilyl)amide in tetrahydrofuran to
form the enol ether 6 as an inseparable mixture of E and Z
isomers. The ratio of E and Z isomers, as deduced from the
integration of vinylic proton signals, is approximately 1:1.25.
Hydrolysis of the resulting 6 in acidic condition produced
aldehyde 7, which was oxidized with sodium chlorite10 to yield
the desired carboxylic acid 8. Preparation of 3-[4-(N,N-bis(2-
chloroethyl)amino)phenyl]propanoic acid (11, n=2)1 was
accomplished in three steps. First, olefination of 4 with triethyl
phosphonoacetate11 in the presence of sodium hydride yielded
the E isomer of ethyl aminophenylprop-2-enoate 9. Catalytic
hydrogenation of 9 over Pd/C catalyst then provided the ester
10.1 Finally, acidic hydrolysis of 10 gave the desired carbox-
ylic acid 11. Hydrolysis of 10 under basic condition (1M aq.
NaOH) produced only a low yield of 11, together with many
by-products, probably due to the nucleophilicity of hydroxide
ion from base.
Coronarin D was esterified by the chlorambucil homologues
using dicyclohexylcarbodiimide in the presence of 4-(dime-
thylamino)pyridine in dichloromethane (Scheme 2). The con-
jugated products, coronarin D N,N-bis(2-cholroethyl)-4-ami-
nobenzoate (12), coronarin D N,N-bis(2-cholroethyl)-4-amin-
ophenylacetate (13), coronarin D N,N-bis(2-chloroethyl)-
4-aminophenylpropanoate (14), and coronarin D N,N-bis(2-
chloroethyl)-4-aminophenylbutanoate (15), were obtained in
43%, 56%, 54%, and 42% yields, respectively. The sensitive
lactol group of coronarin D might undergo tautomerization in
the reaction solution. This may cause undesired side reactions
leading to the moderate yields of the conjugated products.
Cytotoxicity studies
The cytotoxic activity of all synthesised coronarin D-linked
chlorambucil homologues (12−15), as well as chlorambucil
(1) and its homologues (5, 8, and 11) against T-lymphoblast:
acute lymphoblastic leukaemia (MOLT-3), hepatocarcinoma
Fig. 1 Structures of chlorambucil (1) and coronarin D (2).
* Correspondent. E-mail: fscispt@ku.ac.th; supanna@cri.or.th

JOURNAL OF CHEMICAL RESEARCH 2012 375
Scheme 1 Regagents and conditions: (a) POCl3, DMF, ClCH2CH2Cl, reflux, 3h (94%); (b) KMnO4, H2O, pyridine, rt, 20h (50%);
(c) ClPPh3CH2OCH3, NaHMDS, THF, 0 °C, 0.5h (99%); (d) 2N HCl, THF, reflux, 15 min (52%); (e) NaClO2, NaH2PO4,
2-methyl-2-butene, t-BuOH, rt, 1h (64%); (f) (EtO)2P(O)CH2CO2Et, NaH, THF, rt, 1.5h (99%); (g) H2/Pd(C), EtOAc, rt, 1.5h (84%);
(h) Conc.HCl, 100 °C, 4h (98%).
Scheme 2 Synthesis of coronarin D-linked homologues of chlorambucil.

376 JOURNAL OF CHEMICAL RESEARCH 2012
(HepG2), cholangiocarcinoma (HuCCA-1) and lung carci-
noma (A549) cell lines, is shown in Table 1. All these com-
pounds exhibited selective activity against MOLT-3, but were
inactive against other tested cell lines. Among the homologues
of chlorambucil, homologues 8 and 11 displayed a comparable
cytotoxicity to chlorambucil (1) (IC50 = 1.49, 1.18 and 1.42 µM,
for 8, 11, and 1, respectively), whereas homologue 5 showed
the weakest cytotoxicity against MOLT-3 with IC50 of 5.98 µM.
A possible explanation is that the presence of electron-withdra-
wing carboxyl group attached at the para position of the
aromatic ring diminished the electron density on nitrogen,
which affected the formation of the aziridinium ion, the active
intermediate for alkylating nitrogen mustards. This result con-
curred with previously reported data.12−13 Among the coronarin
D-chlorambucil homologues conjugates, conjugates 13 (n=1)
and 14 (n=2) showed strong cytotoxicity against MOLT-3,
whereas conjugate 15 exhibited moderate cytotoxicity, and
conjugate 12 demonstrated only weak cytotoxicity. The weak
cytotoxicities of compounds 5 and 12 could be attributed to
poor aziridinium formation. Comparing the cytotoxicities of
the non-derivatised nitrogen mustards with their correspon-
ding conjugates, 1 (IC50 = 1.42 µM) showed better cytotoxicity
than 15 (IC50 = 4.92 µM). Likewise, 5 (IC50 = 5.98 µM) showed
better cytotoxicity than 12 (IC50 = 22.65 µM). Compound 8
(IC50 = 1.49 µM) exhibited comparable cytotoxicity to 13
(IC50 = 1.41 µM), and 11 (IC50 = 1.18 µM) demonstrated
slightly better cytotoxicity than 14 (IC50 = 1.81 µM). The con-
jugation of chlorambucil and its homologues to coronarin D
through ester linkage apparently did not improve their in vitro
cytotoxicity toward the tested cancer cell lines. Calculated log
P (CLOGP; partition coefficient octanol/water) values of all
compounds (Table 1) were used as a rough estimate of lipophi-
licity. The CLOGP values of the corresponding conjugated
compounds increased more than twice compared with the
non-conjugated compounds. However, their cytotoxicities did
notimprovesignificantly.Theresultindicatedthattheincreased
lipophilicity had no effect on their cytotoxicity, and no correla-
tion between CLOGP and cytotoxicity was observed for this
case. On the other hand, there was a significant correlation
between the calculated amine pKa (Table 1) and the cytotoxic-
ity of the compounds. Compounds 5 and 12 had low calculated
amine pKa values of nitrogen atoms (-0.08 and 0.14, respec-
tively), indicating weak basicity, which resulted in poor aziri-
dinium ion formation and then weak cytotoxicity. In addition,
when the calculated amine pKa values of compounds 1, 8, 11,
and 13−15 were similar (1.69−1.72), their cytotoxicities were
comparable (IC50 = 1.18−1.81 µM), except for compound 15
(IC50 = 4.92 µM). Therefore, the calculated amine pKa values
might serve as a useful tool for the prediction of in vitro cyto-
toxicity against MOLT-3 of the alkylating mustards. Finally,
the cytotoxicities of coronarin D-chlorambucil homologues
conjugates against MOLT-3 cell line were higher than that
of coronarin D (except for compound 12), but comparable to
those of their corresponding non-derivatised nitrogen mus-
tards. Therefore, it could be postulated that the cytotoxicity
of the conjugated compounds is due mainly to the nitrogen
mustards.
Conclusion
A series of chlorambucil homologues and their ester-linked
conjugates with coronarin D were synthesised and evaluated
for their cytotoxicity. The result showed that the conjugation of
chlorambucil and its homologues to coronarin D through ester
linkage did not enhance in vitro cytotoxicity against the tested
cancer cell lines compared with the non-derivatised nitrogen
mustards. Regarding the comparable cytotoxicities of the con-
jugates and their corresponding nitrogen mustards, it could be
postulated that the cytotoxicity of the conjugates is due mainly
to the nitrogen mustards, not coronarin D.
Experimental
1H NMR and 13C NMR spectra were recorded on a Varian Gemini
1
2000 spectrometer (200 MHz for H and 50 MHz for 13C) and on a
Bruker AVANCE 300 (300 MHz for 1H and 75 MHz for 13C). Chemi-
cal shifts were reported as δ values in ppm relative to tetramethylsi-
lane. Mass spectra were obtained on a Finnigan Polaris GCQ mass
spectrometer, whereas accurate masses (HRMS) were obtained using
a Bruker Micro TOF in APCI positive mode. The IR spectra were
recorded in terms of cm−1 on a Perkin Elmer Spectrum One FT-IR
spectrometer. Melting points were determined on a SMP3 melting
point apparatus and reported in °C. Column chromatography was
performed on Merck silica gel 60 (70–230 mesh). Chlorambucil was
purchased from Fluka. Coronarin D was isolated from the rhizomes
of Hedychium coronarium according to the previous report4 and its
structure was confirmed by the spectroscopic methods (1H and 13C
NMR, MS, FTIR).
4-(N,N-Bis(2-chloroethyl)amino)benzaldehyde (4): POCl3 (7.50 mL,
80.46 mmol) was added dropwise to a solution of N,N-dimethylform-
amide (7.50 mL, 96.87 mmol) in 1,2-dichloroethane (30 mL) at 0 °C,
and the mixture was stirred at 0 °C for 10 min. A solution of N-phenyl-
diethanolamine (3.0 g, 16.57 mmol) in N,N-dimethylformamide
(7.50 mL, 96.87 mmol) was added to the above mixture at 0 °C, and
the mixture was refluxed for 3 h. The reaction mixture was poured
onto ice and saturated aqueous potassium acetate was added to adjust
the pH to 6, and the mixture was extracted with CH2Cl2. The
Table 1 Cytotoxicity against cancer cell lines
CLOGPb
Amine pKac
Compound
IC50/µMa
MOLT-3
HepG2
HuCCA-1
A549
1
1.42 0.13
8.71 1.95
5.98 0.31
1.49 0.15
1.18 0.03
22.65 0.86
1.41 0.05
1.81 0.08
4.92 0.30
0.04 0.01
–
%C=19.5d
60.79 6.54
%C=11.2
%C=4.7
%C=25.9
%C=38.3
%C=26.4
%C=0.0
%C=42.0
29.37 1.67
%C=26.0
%C=45.0
33.02 2.22
%C=26.0
%C=34.0
%C=22.0
%C=18.0
%C=11.0
%C=16.0
%C=0.0
–
3.81
4.41
1.72
1.60
1.66
6.91
6.95
7.10
7.28
ND
1.72
NDe
–0.08
1.68
1.72
0.14
1.69
1.72
1.72
ND
2
5
8
154.00 4.06
%C=30.0
%C=0.0
%C=47.0
%C=0.0
%C=4.0
–
11
12
13
14
15
%C=4.5
19.11 3.01
0.34 0.03
Etoposide
Doxorubicin
0.74 0.18
0.38 0.05
ND
ND
a Results are expressed as mean standard error; average of three independent experiments.
b CLOGP = calculated log P [Marvin 5.6.0.0, 2011, ChemAxon (http://www.chemaxon.com)]
c Calculated amine pKa [Marvin 5.6.0.0, 2011, ChemAxon (http://www.chemaxon.com)]
d Inactive (IC50≥50 µg mL−1); reported in %cytotoxicity at the substance concentration of 50 µg mL−1.
e Not determined

JOURNAL OF CHEMICAL RESEARCH 2012 377
combined organic layer was washed with water, dried over Na2SO4,
and concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (15% EtOAc/hexane) to produce 4
(3.83 g, 94%) as a white solid; m.p. 87–88 °C (lit.7 87–88 °C); FTIR
(KBr), νmax: 1667, 1589, 1522, 1404, 1361, 1165, 714 cm−1; 1H NMR
(200 MHz, CDCl3): δ 9.81 (s, 1H, CHO), 7.78 (d, J = 8.8 Hz, 2H,
CHaro), 6.74 (d, J = 8.8 Hz, 2H, CHaro), 3.65–3.89 (m, 8H,
2×NCH2CH2Cl); 13C NMR (50 MHz, CDCl3): δ 190.1, 151.0, 132.3,
126.8, 111.3, 53.3, 40.0 ppm; MS (EI), m/z (relative intensity):
245(M+, 45), 196(100), 132(32), 117(13), 91(5), 77(14), 63(6), 51(10);
HRMS (APCI) Calcd for C11H14NOCl2 [M+H]+ 246.0447; found
246.0435.
4-(N,N-Bis(2-chloroethyl)amino)benzoic acid (5): A solution of
KMnO4 (0.71 g, 4.50 mmol) in water (15 mL) was added dropwise
to a refluxing, stirred solution of 4 (0.46 g, 1.88 mmol) in pyridine
(7.50 mL). The mixture was then stirred at room temperature for 20 h.
The MnO2 was removed by filtration and washed with hot water. The
filtrate was concentrated under reduced pressure. The residue was
then added with water and cooled in an ice bath and neutralised with
10% aq. HCl to pH 6. The brown precipitate acid was collected by
filtration and purified by silica gel column chromatography (20%
EtOAc/hexane) to provide 5 as a white solid (0.30 g, 50%); m.p. 166–
168 °C, lit.7 m.p. (168–169 °C); FTIR (KBr), νmax: 1667, 1599, 1183,
833, 720 cm−1; 1H NMR (200 MHz, acetone-d6): δ 7.79 (d, J = 8.8 Hz,
2H, CHaro), 6.90 (d, J = 8.8 Hz, 2H, CHaro), 4.00–3.70 (m, 8H,
2×NCH2CH2Cl); 13C NMR (50 MHz, acetone-d6): δ 168.0, 151.3,
132.6, 119.5, 112.1, 53.6, 41.4 ppm; MS (EI), m/z (relative intensity):
261(M+, 37), 212(100), 148(12), 132(18), 117(10), 104(4), 77(6),
63(5); HRMS (APCI) Calcd for C11H14NO2Cl2 [M+H]+ 262.0396;
found 262.0389.
(E)- and (Z)-N,N-Bis(2-chloroethyl)-4-(2-methoxyvinyl)aniline (6):
Sodium hexamethyldisilazide (1.0 M in tetrahydrofuran, 14.7 mL,
14.7 mmol) was added dropwise to a 0 °C cooled suspension of (met
hoxymethyl)triphenylphosphonium chloride (4.20 g, 12.25 mmol) in
anhydrous tetrahydrofuran (20 mL). The deep red mixture was stirred
for 20 min at that temperature to ensure complete ylid formation.
A solution of 4 (2.00 g, 8.16 mmol) in anhydrous tetrahydrofuran
(15 mL) was then added dropwise to the red ylid solution, and the
mixture was stirred for another 1 h under N2 atmosphere at 0 °C. Water
was added to the reaction mixture, and the tetrahydrofuran was
removed under reduced pressure. The residue was extracted with
CH2Cl2. The combined organic layer was washed with water, dried
over Na2SO4, and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (5% EtOAc/hexane)
to provide an inseparable mixture of E- and Z-isomers of enol ether 6
m/z (relative intensity): 259(M+, 18), 230(28), 210(100), 181(6),
146(6), 132(9), 118(27), 75(6); HRMS (APCI) Calcd for C12H16NOCl2
[M+H]+ 260.0603; found 260.0600.
2-(4-(N,N-Bis(2-chloroethyl)amino)phenyl)acetic acid (8): A solu-
tion of sodium chlorite (384 mg, 4.25 mmol) in NaH2PO4 pH 3.5
buffer (3.4 mL) was added dropwise to a stirred solution of 7 (880 mg,
3.4 mmol) and 2-methyl-2-butene (3.6 mL, 34 mmol) in tert-butanol
(15 mL) at room temperature and the mixture was stirred for 1 h. Then
the mixture was basified to pH 10 with 6N NaOH, and the tert-butanol
was removed under reduced pressure. The residue was dissolved in
water and extracted with hexane. The aqueous layer was acidified to
pH 3 with 10% HCl and extracted with diethyl ether. The organic
layer was washed with water, dried over Na2SO4, filtered and concen-
trated under reduced pressure. The residue was purified by silica gel
column chromatography (50% EtOAc/hexane) to provide 8 (600 mg,
64%) as a white solid, m.p. 103.5−104.5 °C (lit.8 105 °C); FTIR
(KBr), νmax: 1704, 1616, 1520, 1354, 1249, 1181, 805 cm−1; 1H NMR
(300 MHz, CDCl3): δ 7.17 (d, J = 8.7 Hz, 2H, CHaro), 6.65 (d, J =
8.7 Hz, 2H, CHaro), 3.74–3.59 (m, 8H, 2×NCH2CH2Cl), 3.55 (s, 2H,
CH2COOH); 13C NMR (75 MHz, CDCl3): δ 177.5, 145.3, 130.7,
122.2, 112.1, 53.5, 40.4, 39.8 ppm; MS (EI), m/z (relative intensity):
275(M+,8), 226(100), 144(8), 132(8), 118(45), 90(9), 77(7), 63(12);
HRMS (APCI) Calcd for C12H16NO2Cl2 [M+H]+ 276.0553; found
276.0553.
Ethyl
3-[4-(N,N-bis(2-chloroethyl)amino)phenyl]prop-2-enoate
(9): Triethyl phosphonoacetate (0.88 mL, 4.41 mmol) was slowly
added to a 0 °C cooled stirred suspension of NaH (60% oil dispersion,
0.26 g, 6.61 mmol) in dry tetrahydrofuran (5 mL), and the reaction
mixture was stirred at 0 °C for 30 min under N2 atmosphere. A solu-
tion of 4 (0.90 g, 3.67 mmol) in dry tetrahydrofuran (10 mL) was then
added, and the reaction mixture was stirred at room temperature for an
additional 1.5 h. The reaction mixture was added with water and
extracted with ethyl acetate. The combined organic layer was washed
with water, dried over Na2SO4, and concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatography
(20% EtOAc/hexane) to yield 9 (1.15 g, 99.3%) as a pale yellow solid;
m.p. 78–79 °C; FTIR (KBr), νmax: 1698, 1597, 1518, 1153, 813 cm−1;
1H NMR (200 MHz, CDCl3): δ 7.61 (d, J = 16.0 Hz, 1H, CH=CH),
7.44 (d, J = 8.8 Hz, 2H, CHaro), 6.68 (d, J = 8.8 Hz, 2H, CHaro), 6.26
(d, J = 16.0 Hz, 1H, CH=CH), 4.25 (q, J = 7.3 Hz, 2H, CH2CH3),
3.60–3.80 (m, 8H, 2×NCH2CH2Cl), 1.33 (t, J = 7.3 Hz, 3H, CH2CH3);
13C NMR (50 MHz, CDCl3): δ 167.5, 147.6, 144.2, 130.0, 124.0,
114.1, 111.9, 60.2, 53.3, 40.2, 14.3 ppm; MS (EI), m/z (relative inten-
sity): 315(M+, 84), 266(100), 157(4); HRMS (APCI) Calcd for
C15H20NO2Cl2 [M+H]+ 316.0866; found 316.0872.
in a ratio of 1:1.25 (2.20 g, 98%) as colourless oil. FTIR (KBr), νmax
:
Ethyl 3-[4-(N,N-bis(2-chloroethyl)amino)phenyl]propanoate (10):
A mixture of 9 (1.00 g, 4.08 mmol) and 10% Pd/C (0.01 g) in ethyl
acetate (5.0 mL) was evacuated and flushed thrice with hydrogen gas.
After stirring under H2 atmosphere for 1.5 h, the Pd/C was removed by
filtration through Celite and washed with ethyl acetate. The filtrate
was concentrated under reduced pressure, and the residue was purified
by silica gel column chromatography (5% EtOAc/hexane) to provide
10 (0.84 g, 83.5%) as a pale yellow oil (lit.8 40.5 °C); FTIR (KBr),
νmax: 1731, 1519, 1353, 1180, 811 cm−1; 1H NMR (200 MHz, CDCl3):
δ 7.09 (d, J = 8.8 Hz, 2H, CHaro), 6.67 (d, J = 8.8 Hz, 2H, CHaro), 4.13
(q, J = 7.3 Hz, 2H, CH2CH3), 3.79–3.55 (m, 8H, 2×NCH2CH2Cl), 2.87
(t, J = 7.3 Hz, 2H, CH2CH2), 2.57 (t, J = 7.3 Hz, 2H, CH2CH2), 1.24
(t, J = 7.3 Hz, 3H, CH2CH3); 13C NMR (50 MHz, CDCl3): δ 173.0,
144.0, 129.6, 122.4, 112.7, 60.4, 53.8, 40.2, 36.1, 29.9, 14.2 ppm; MS
(EI), m/z (relative intensity): 317(M+, 33), 268(100), 240(10), 230(5),
179(3), 132(6), 118(7), 77(3); HRMS (APCI) Calcd for C15H22NO2Cl2
[M+H]+ 318.1022; found 318.1017.
3-[4-N,N-Bis(2-chloroethyl)amino)phenyl]propanoic acid (11): A
solution of 10 (100 mg, 0.32 mmol) in conc. HCl (5 mL) was heated
at 100 °C for 4 h. After cooling to room temperature, the reaction
mixture was poured into ice water and neutralised to pH 6 with satu-
rated aqueous NaHCO3. The reaction mixture was extracted with ethyl
acetate. The combined organic layer was washed with water, dried
over Na2SO4, and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (50% EtOAc/
hexane) to yield 11 (89 mg, 97.8%) as a white solid, m.p. 113.5−114.5
°C, lit.8 114−115 °C); FTIR (KBr), νmax: 1710, 1524, 1210, 816 cm−1;
1H NMR (200 MHz, CDCl3): δ 7.14 (d, J = 8.4 Hz, 2H, CHaro), 6.62
(d, J = 8.4 Hz, 2H, CHaro), 3.80–3.54 (m, 8H, 2×NCH2CH2Cl), 2.89 (t,
J = 7.4 Hz, 2H, CH2CH2), 2.64 (t, J = 7.4 Hz, 2H, CH2CH2); 13C NMR
1641, 1608, 1516, 1352, 1240, 1177, 1093, 823 cm−1; 1H NMR
(200 MHz, CDCl3): 7.49 (d, J = 8.8 Hz, 2H, CHaro of Z isomer), 7.14
(d, J = 8.8 Hz, 2H, CHaro of E isomer), 6.91 (d, J = 12.8 Hz, 1H,
CH=CH of E isomer), 6.62 (d, J = 8.8 Hz, 2H, CHaro of Z isomer), 6.61
(d, J = 8.8 Hz, 2H, CHaro of E isomer), 6.03 (d, J = 7.0 Hz, 1H, CH=CH
of Z isomer), 5.75 (d, J = 12.8 Hz, 1H, CH=CH of E isomer), 5.15 (d,
J=7.0Hz,1H,CH=CHofZisomer),3.75–3.57(m,8H,2×NCH2CH2Cl
of E and Z isomers), 3.74 (s, 3H, OCH3 of Z isomer), 3.66 (s, 3H,
OCH3 of E isomer); 13C NMR (50 MHz, CDCl3): δ 147.0E, 145.9Z,
144.2E, 143.9Z, 129.6Z, 126.5E, 126.2E, 126.0Z, 112.6E, 112.0Z, 105.3Z,
104.7E, 60.4Z, 56.5Z, 53.6E and Z, 40.5E and Z ppm; MS (EI), m/z (relative
intensity): 273(M+, 22), 224(100), 210(9), 161(33), 146(18), 118(31),
91(31), 63(10); HRMS (APCI) Calcd for C13H18NOCl2 [M+H]+
274.0760; found 274.0758.
2-(4-(N,N-Bis(2-chloroethyl)amino)phenyl)acetaldehyde (7): 2N
HCl (1 mL) was added to a solution of 6 (1.0 g, 66.4 mmol) in tetra-
hydrofuran (10 mL), and the solution was refluxed for 15 min. The
mixture was cooled to room temperature, and water was added. The
mixture was concentrated under reduce pressure to remove tetrahy-
drofuran, and the residue was extracted with dichloromethane. The
combined organic layer was washed with saturated aqueous NaHCO3
and water, dried over Na2SO4, filtered and concentrated under reduced
pressure. The residue was purified by silica gel column chromatogra-
phy (10% EtOAc/hexane) to provide 7 (500 mg, 52%) as a colourless
oil. FTIR (KBr), νmax: 1718, 1614, 1518, 1353, 1180, 810, 737 cm−1;
1H NMR (300 MHz, CDCl3): δ 9.71 (t, J = 2.3 Hz, 1H, CHO), 7.10 (d,
J = 8.7 Hz, 2H, CHaro), 6.69 (d, J = 8.7 Hz, 2H, CHaro), 3.76–3.59 (m,
10H, 2×NCH2CH2Cl and CH2CHO); 13C NMR (75 MHz, CDCl3):
δ 199.7, 145.4, 130.9, 120.5, 112.4, 53.4, 49.5, 40.4 ppm; MS (EI),

378 JOURNAL OF CHEMICAL RESEARCH 2012
(50 MHz, CDCl3): δ 178.7, 144.7, 129.5, 129.4, 112.4, 53.6, 40.5,
35.8, 29.5 ppm; MS (EI), m/z (relative intensity): 289(M+, 27),
240(100), 180(10), 144(6), 132(6), 118(17), 91(5), 77(6); HRMS
(APCI) Calcd for C13H18NO2Cl2 [M+H]+ 290.0709; found 290.0704.
144.1, 130.1, 129.7, 122.2, 112.7, 107.5/107.4, 92.3, 56.1, 55.4, 53.8,
42.0, 40.4, 39.5, 39.3, 37.8, 33.8, 33.6, 33.3, 32.0, 31.9, 26.2, 25.7,
24.1, 21.7, 19.3, 14.4 ppm; MS (EI), m/z (relative intensity):
603(M+,12), 554(46), 302(32), 286(100), 254(87), 238(38), 230(22),
224(35); HRMS (APCI) Calcd for C34H48NO4Cl2 [M+H]+ 604.2955;
found 604.2948.
Synthesis of coronarin D linked mustard agents (12−15); general
procedure
Cytotoxicity studies
A solution of the nitrogen mustard agent (1 mmol) and dimethylami-
nopyridine (0.3 mmol) in CH2Cl2 (5 mL) was added to a solution of
coronarin D (1 mmol) in CH2Cl2 (5 mL). The mixture was stirred at
room temperature for 5 min with guard tube; a solution of dicyclohex-
ylcarbodiimide (1 mmol) in CH2Cl2 (10 mL) was then added, and the
reaction mixture was stirred at room temperature for a further 5 h.
Water was then added, and the reaction mixture was extracted with
CH2Cl2. The combined organic layer was washed with water, dried
over Na2SO4, and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography to provide the
desired coronarin D linked mustard agent.
The cytotoxic activity of compounds 1, 5, 8, and 11−15 was evaluated
against a panel of cancer cell lines, namely, MOLT-3, HepG2, HuCCA-
1, and A549, using previously reported procedures.14 Briefly, the cells
in the logarithmic growth phase were seeded in 96-well plates (Costar,
3599, USA) at a density of 10,000–15,000 cells/well, and incubated at
37 °C in a humidified atmosphere with 95% air and 5% CO2. After
24 h, an additional medium containing either the test compounds
or the vehicle was added at the desired final concentrations, and the
cultures were further incubated for 48 h. Afterward, the cell mono-
layer was washed with phosphate-buffered saline PBS (pH 7.2), fixed
with 95% ethanol, stained with crystal violet solution, and lysed with
0.1 N HCl in methanol. The absorbance of the lysate was measured at
540 nm on an automatic microtiter plate reader (Multiskan Ascent,
Labsystem). All tests were carried out in triplicate, and the mean value
was calculated. The activity was expressed as IC50 (concentration of
substances that inhibits 50% of cell growth) using etoposide and
doxorubicin (Sigma Aldrich) as the standard drugs.
Coronarin D N,N-bis(2-chloroethyl)-4-aminobenzoate (12): FTIR
(KBr), νmax: 2930, 1768, 1720, 1604, 1521, 1270, 1178, 1096,
1
957 cm−1; H NMR (200 MHz, CDCl3): δ 7.90 (d, J = 8.8 Hz, 2H),
6.90–6.80 (m, 2H), 6.65 (d, J = 8.8 Hz, 2H), one set (1H) of 4.84
(s)/4.41 (s) and one set (1H) of 4.83 (s)/4.35 (s), 3.84–3.62 (m, 8H),
3.30–3.10 (m, 1H), 3.00–2.85 (m, 1H), 0.88 (s, 3H), 0.81 (s, 3H), 0.72
(s, 3H);13CNMR(50MHz, CDCl3):δ168.9, 164.5, 150.4, 148.1/148.0,
144.5/144.4, 132.3, 122.6, 117.2, 110.9, 107.4, 92.9, 56.1, 55.4, 53.2,
42.0, 40.0, 39.5, 39.3, 37.8, 33.5, 32.3, 32.2, 25.7, 24.1, 21.7, 19.3,
14.3 ppm; MS (EI), m/z (relative intensity): 486(25), 394(27), 358(15),
290(100), 277(16), 215(31), 164(9), 152(28); HRMS (APCI) Calcd
for C31H42NO4Cl2 [M+H]+ 562.2485; found 562.2481.
Financial support from the Center of Excellence for Innova-
tion in Chemistry (PERCH-CIC), Commission on Higher
Education, Ministry of Education is gratefully acknowledged.
We also thank P. Intachote, Laboratory of Immunology,
S. Sengsai, and B. Saimanee, the Integrated Research Unit, the
Chulabhorn Research Institute, for the cytotoxicity test.
Coronarin D N,N-bis(2-chloroethyl)-4-aminophenylethanoate (13):
1
FTIR (KBr), νmax: 2931, 1770, 1520, 1131, 973, 960 cm−1; H NMR
(300 MHz, CDCl3): δ 7.14 (d, J = 8.2 Hz, 2H), 6.85–6.75 (m, 1H),
6.70–6.60 (m, 3H), one set (1H) of 4.84 (s)/4.38 (s) and one set (1H)
of 4.82 (s)/4.33 (s), 3.74–3.52 (m, 10H), 3.20–3.05 (m, 1H), 2.85–2.75
(m, 1H), 0.89 (s, 3H), 0.82 (s, 3H), 0.72 (s, 3H); 13C NMR (75 MHz,
CDCl3): δ 170.3, 168.7, 148.0, 145.3, 144.8/144.7, 130.5, 122.2,
121.6, 112.1, 107.4/107.3, 92.7, 56.1, 55.3, 53.4, 42.0/41.9, 40.4,
39.9/39.8, 39.4, 39.3/39.2, 37.7, 33.5, 32.0, 31.9, 25.7, 24.0, 21.7,
19.3/19.2, 14.3 ppm; MS (EI), m/z (relative intensity): 575(M+, 4),
526(10), 230(100), 226(52), 118(52), 81(43), 69(72), 55(50); HRMS
(APCI) Calcd for C32H44NO4Cl2 [M+H]+ 576.2642; found 576.2660.
Coronarin-D N,N-bis(2-chloroethyl)-4-aminophenylpropanoate (14):
FTIR (KBr), νmax: 2929, 1764, 1519, 1134, 958 cm−1; 1H NMR
(200 MHz, CDCl3): δ 7.09 (d, J = 8.8 Hz, 2H), 6.86–6.77 (m, 1H),
6.69–6.67 (m, 1H), 6.62 (d, J = 8.8 Hz, 2H), one set (1H) of 4.83
(s)/4.38 (s) and one set (1H) of 4.82 (s)/4.32 (s), 3.75–3.57 (m, 8H),
3.20–3.02 (m, 1H), 2.90–2.56 (m, 5H), 0.89 (s, 3H), 0.82 (s, 3H), 0.72
(s, 3H); 13C NMR (50 MHz, CDCl3): δ 171.2, 168.7, 148.1, 144.7,
144.6, 129.5, 129.1, 122.2, 112.4, 107.5, 92.3, 56.2, 55.4, 53.6, 42.0,
40.5, 39.5, 39.4, 37.8, 36.0, 33.6, 32.0, 31.9, 29.4, 25.8, 24.1, 21.7,
19.3, 14.4 ppm; MS (EI), m/z (relative intensity): 589(M+, 85),
553(14), 540(100), 498(4), 337(9), 289(5), 240(18), 91(6); HRMS
(APCI) Calcd for C33H46NO4Cl2 [M+H]+ 590.2798; found 590.2789.
Coronarin D N,N-bis(2-chloroethyl)-4-aminophenylbutanoate (15):
FTIR (KBr), νmax: 2932, 1762, 1519, 1387, 1134, 959 cm−1; 1H NMR
(200 MHz, CDCl3): δ 7.05 (d, J = 8.8 Hz, 2H), 6.88–6.75 (m, 1H),
6.70–6.60 (m, 3H), one set (1H) of 4.82 (s)/4.37 (s) and one set (1H)
of 4.82 (s)/4.32 (s), 3.75–3.57 (m, 8H), 3.20–3.02 (m, 1H), 2.86–2.70
(m, 1H), 2.62–2.50 (m, 2H), 0.88 (s, 3H), 0.81 (s, 3H), 0.71 (s, 3H);
13C NMR (50 MHz, CDCl3): δ 171.7, 168.7, 148.0, 144.7/144.6,
Received 12 March 2012; accepted 31 March 2012
Paper 1201207 doi: 10.3184/174751912X13362343090928
Published online: 4 June 2012
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