The preparation of 2,6-disubstituted pyridinyl phosphine oxides as novel anti-cancer agents

Article abstract of DOI:10.1016/j.bmcl.2009.02.091

A series of 2,6-dimethoxylpyridinyl phosphine oxides have been synthesized and examined for their antitumor activity. 2,6-Dimethoxy-3-phenyl-4-diphenylphosphinoylpyridine 2 has been employed as the lead compound for this study. We found out that the prese

Full text of DOI:10.1016/j.bmcl.2009.02.091

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2266–2269
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The preparation of 2,6-disubstituted pyridinyl phosphine oxides as novel
anti-cancer agents
b
a
a
a
a
Kim Hung Lam a, , Roberto Gambari , Marcus Chun Wah Yuen , Chi Wai Kan , Penni Chan , Lijin Xu ,
*
Weijun Tang a, Chung Hin Chui c, Gregory Yin Ming Cheng c, Raymond Siu Ming Wong c, Fung Yi Lau c,
Cindy Sze Wai Tong c, Andrew Kit Wah Chan c, Paul Bo San Lai d, Stanton Hon Lung Kok a, Chor Hing Cheng a,
Albert Sun Chi Chan a, , Johnny Cheuk On Tang
a,
*
*
a Department of Applied Biology and Chemical Technology, Institute of Textiles and Clothing, Hong Kong Polytechnic University, Hong Kong, China
b BioPharmaNet, Department of Biochemistry and Molecular Biology, The University of Ferrara, Ferrara, Italy
c Department of Medicine and Therapeutics, Li Ka Shing Medical Sciences Building, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China
d Department of Surgery, Li Ka Shing Medical Sciences Building, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2,6-dimethoxylpyridinyl phosphine oxides have been synthesized and examined for their anti-
tumor activity. 2,6-Dimethoxy-3-phenyl-4-diphenylphosphinoylpyridine 2 has been employed as the
lead compound for this study. We found out that the presence of phosphine oxide on the 2,6-dimethoxyl-
pyridine ring is important for the antitumor activity; the presence of bromine on this core leads to a fur-
ther enhancement of its antitumor activity. This is the first reported work on the antitumor activity of the
2,6-dimethoxy-3,5-dibromopyridinyl phosphine oxide 5b towards MDAMB-231 breast cancer and
SKHep-1 hepatoma cell lines.
Received 3 November 2008
Revised 17 February 2009
Accepted 24 February 2009
Available online 27 February 2009
Keywords:
Pyridinyl phosphine oxides
Antitumor
Ó 2009 Elsevier Ltd. All rights reserved.
MDAMB-231 breast cancer
SKHep-1 hepatoma cell lines
Only a few previous reports have been published on the prepa-
ration of phosphine oxides and on the characterization of their
antitumor activity. Examples of studies reporting the antitumor
activities of phosphine oxides are those reported by Babbitt et al.
and Spasokova et al. Babbitt and co-workers reported the prepara-
tion of tris(2,2-dimethyl-1-aziridinyl) phosphine oxide (TEPA-132)
for treatment of leukemia cells in 1967.1 The Spasovska’s group re-
ported the synthesis of platinum(II), platinum(IV), and palla-
dium(II) complexes of amino substituted phosphine oxides
(dimethyl aminomethylphosphine oxide and methyl bis(amino-
methyl)phosphine oxide in 1990.2 The pyridinyl compounds have
been reported to show biological activity. On the other hand, Kle-
eman and his researchers reported that the 2,6-disubstituted pyr-
idines can function as herbicides.3 The reported studies aroused
our interest to explore the antitumor activities using simple pyrid-
inyl phosphine oxides. To begin with our assumption, compound 1
was produced and test as reference. In consideration of the biolog-
ical activity exhibited by phosphine oxide and 2,6-disubstituted
pyridines, we develop herein pyridinyl phosphine oxides based
on the lead compound 2,6-dimethoxy-3-phenyl-4-diphenylphos-
phinoylpyridine 2 (Fig. 1), and report the antitumor activities of
the synthesized compounds towards MDAMB-231 breast cancer
and SKHep-1 hepatoma cell lines, whereas the biological activities
of this type of phosphine oxides had not been reported previously.
In order to further probe with our assumptions, we employed our
previously reported synthetic scheme4 for making compounds. As
a strategic consideration, we firstly started the present study with
the easily available 2,6-dimethoxypyridine (3), which followed our
patented route of making pyridinyl phosphine ligands. The pres-
ence of substitution (i.e. methoxyl group) at positions 2 and 6
can avoid the interference of the pyridinyl nitrogen in the subse-
quent compounds synthesis in the reported scheme.
* Corresponding authors. Tel.: +852 3400 8705; fax: +852 2364 9932 (K.H.L.).
E-mail addresses: bcjoelam@inet.polyu.edu.hk (K.H. Lam), bcachan@polyu.
edu.hk (A.S.C. Chan), bccotang@inet.polyu.edu.hk (J.C.O. Tang).
Figure 1. Chemical structures of triphenylphosphine oxides and 2,6-dimethoxy-3-
phenyl-4-diphenylphosphinoylpyridine.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.091

K. H. Lam et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2266–2269
2267
Table 1
Using the commercially available compound 2,6-dimethoxy-
pyridine (3) as a starting material, 3-bromo-2,6-dimethoxypyri-
dine (4a) were synthesized by adding bromine in carbon
Growth inhibitory concentration (IC50) of compound 5b on the MDAMB-231 and
SKHep-1 human cancer cells as well as two non-malignant human hematological
disorder bone marrow cells [BM(1) and BM(2)]
tetrachloride slowly to
3
at temperature between À30 and
MDAMB-231
47.50 2.11
115.53 9.30
SKHep-1
BM(1)
BM(2)
À40 °C to give the product with 76% yield, while 3,5-dibromo-
2,6-dimethoxypyridine (4b) was the side product in this reaction.
To a solution of 4a or 4b in THF, lithium diisopropylamide (LDA)
in THF at À78 °C was added, followed by the addition of chlorodi-
phenylphosphine to produce 3-bromo-2,6-dimethoxy-4-diphenyl-
phosphino-pyridine or 3,5-dibromo-2,6-dimethoxy-4-diphenyl-
phosphino-pyridine of up to 95% yield. Dropwise addition of
hydrogen peroxide to a acetone-dichloromethane (1:1) solution
of the above compounds at 0 °C gave 3-bromo-2,6-dimethoxy-
4-diphenylphosphinoylpyridine (5a) or 3,5-dibromo-2,6-dime-
thoxy-4-diphenylphosphinoylpyridine (5b) with up to 96%
Compound 5b
IC50: ( M)
l
38.53 3.13
87.33 5.01
54.17 5.51
144.53 8.96
58.13 4.85
132.30 5.24
Cisplatinum
IC50: ( M)
l
Relative activities: 5b > 5a > 6a > 6b (or 2)
Cisplatinum was used as a positive reference antitumor agent. Results are based on
the one step ATP lite assay which documented the intracellular adenosine tri-
phosphate level. Reported results represent the mean standard deviation from
three mean values of triplicate experiments in which each experiment had tripli-
cate tests and a mean was calculated. The three independent experiments gave
similar results. Please refer to the text for details.
yield.
2,6-Dimethoxy-3-phenyl-4-diphenylphosphinoylpyridine
(6a) was synthesized via a Suzuki coupling of 6a with phenyl boro-
nic acid with a 89% yield, while 6b is the side product produced in
this reaction. All products were characterized by 1H, 31P, 13C NMR
and high-resolution mass spectrometry. All of the synthetic inter-
mediates are air-stable, and the overall synthetic steps are easy to
handle. Scheme 1 shows the synthetic scheme for compounds 4–
7.5 Details of the synthetic methods for the intermediates can be
referenced to our previous work in 2001.4
Human breast cancer MDAMB-231 and hepatoma SKHep1 cell
lines and consented non-malignant hematological disorder bone
marrow cells during presentation (female, 45 years old and male,
56 years old) were used for preliminary anti-cancer screening
and toxicology analysis purpose. A concentration of 1 Â 105/mL
of human cells seeded in the 96 wells microtitre plates for 24 h
were used. The tested compounds were prepared as a stock con-
centration of 100 mM in DMSO. All the tested compounds were
Due to the significant antitumor activity of aziridinyl phosphine
oxides and transitional metal complexes of the amino phosphine
oxides reported, we have tried to employ the simple and easily
available triphenylphosphine oxides (1) as the leads. However, this
compound was found to exhibit low activity. Surprisingly, the
replacement of one phenyl group of triphenylphosphine oxide with
2,6-dimethoxypyridine (2) was demonstrated to significant im-
prove the antitumor activity towards MDAMB-231 breast cancer
and SKHep-1 hepatoma cell lines. It is interesting to recognize that
both phosphine oxide and 2,6-dimethoxypyridine moieties are
important to account for the activity with reference to compounds
1 and 3. The addition of a bromo group to the core structure 2 can
improve the antitumor activity (compound 5a: mean growth
inhibitory percentage ꢀ30% on both MDAMB-231 and SKHep-1
at 100 lM when compared with control). Compound 5b, which
bears two bromo groups, exhibits a significant enhanced apoptotic
activity than that of 5a (bears 1 bromo group only) (Fig. 2). In con-
trast to compound 5, pyridinyl phosphine compound does not ex-
hibit any antiproliferative activity on MDAMB-231 and SKHep-1
human cancer cells.
added at a starting concentration of 100 lM, followed by a serial
of twofold dilutions and incubated for a further period of 48 h.
Afterwards, the evaluation of possible antiproliferative and/or
cytotoxicity of our novel synthesized compounds was assayed by
the one step ATP lite assay purchased from PerkinElmer (Nether-
lands) according to the technical manual provided (Table 1). Mor-
phological changes of cancer cells include cell rounding, bubbling
of membrane, lost of attachment property and shrinkage with
the apoptotic features were recorded under inverted microscopy
study (Fig. 2).6–8
To understand the possible underlying mechanism of how com-
pound 5b could induce cancer cell death, both MDAMB-231 and
SKHep-1 human cancer cells were pre-incubated with 10
z-VAD-fmk, a pan-capsase inhibitor, before the addition of 70
of compound 5b. After 48 h of incubation, cell viability was deter-
lM of
l
M
Scheme 1. Synthetic pathway for pyridinyl phosphine oxide.

2268
K. H. Lam et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2266–2269
Figure 2. Morphological changes of (B) MDAMB-231 and (D) SKHep-1 human cancer cells after a 48 h incubation with compound 5b (60 lM) when compared with the
untreated (A) MDAMB-231 and (C) SKHep-1 human carcinoma cells.
cultured non-malignant hematological disorder bone marrow cells
was observed, suggesting the benefits for the development of this
class of compounds as novel anti-tumor agents.
In conclusion, we have discovered the new usage (i.e. antitumor
studies in this work) of our reported pyridinyl type phosphorine
oxide moieties. Further work using this type of compounds and
their derivatives are in-progress.
Acknowledgments
We acknowledge a donation fund from Mr. KC Lo to Dr JCO Tang
for the establishment of Lo Ka Chung Centre for Natural Anti-can-
cer Drug Development (Laboratory B) and a Niche area grant of-
fered by the Hong Kong Polytechnic University to Dr KH Lam and
Dr. JCO Tang (BB8T, HK$100,000). Professor R Gambari is spon-
sored by AIRC (Italian Association for Cancer Research). Dr. CH Chui
is supported by the Research Assistant Professorship offered by
Professor GYM Cheng (Haematology Research Funding) and Chi-
nese University of Hong Kong. Special thanks should be given to
the reviewers for their constructive comments and suggestions
for this Letter.
Figure 3. Analysis of the possible reversal effects of
a pan caspase inhibitor
zVADfmk (10 M) on the cytotoxicity of compound 5b at a concentration of 70 lM
l
on MDAMB-231 and SKHep1 human carcinoma cells after 48 h of incubation.
Reported results represent the mean standard deviation from triplicate tests. The
Figure shows a representative experiment taken from three independent experi-
ments giving similar results. Asterisk indicates that there is a statistical significant
(p value <0.05, student’s t-test) between the compound 5b loaded cells and the
compound 5b plus zVADfmk loaded cells.
Supplementary data
mined again by the one step ATP lite assay system. As shown in
Figure 3, even preincubation of the pan caspase inhibitor could sig-
nificantly reverse the growth inhibitory effect of compound 5b on
both cancer cell lines and the mean percentage of survival cells was
found to increase near a double for MDAMB-231 while there was
near a triple for that of SKHep-1 human carcinoma cells, the treat-
ment itself could not recover the cell viability to the control level.
As caspase pathway is only one of the possible ways to mediate the
signal that is initiated by phosphine oxide treatment, we believe
that there would be any other possibilities being included as well.
Collectively, our experimental results show that caspase depen-
dent apoptotic pathway is recruited in our compound 5b induced
cancer cell death on both MDAMB-231 breast carcinoma cells
and SKHep-1 hepatoma cells in vitro. Interestingly, a differential
selective effects between the human cancer cell lines and primary
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.02.091.
References and notes
1. Munson, A. E.; Babbitt, H. L. Cancer Chemother. Rep. 1967, 51, 253.
2. Dodov, N.; Varbanov, S.; Borisov, G.; Spasovska, N. J. Inorg. Biochem. 1990, 39,
201.
3. Kleeman, A.; Munro, D.; Patel, B. Eur. Pat. 1993, EP 572093.
4. Pai, C. C.; Lin, C. W.; Lin, C. C.; Chen, C. C.; Chan, A. S. C.; Wong, W. T. J. Am. Chem.
Soc. 2000, 122, 11513.
5. NMR spectral data. 3-Bromo-2,6-dimethoxypyridine (4a): 1H NMR (500 MHz,
CDCl3): d 3.90 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 6.23 (d, J = 8.5 Hz, PyH), 7.64 (d,
J = 8.5 Hz, PyH); 13C NMR (125 MHz, CDCl3): d 53.9, 54.4, 95.5, 102.9, 143.8,
158.6, 162.3; mass spectrum (ESI): 218.09 [M(Br79)+H]+ and 220.09
[M(Br81)+H]+. 3,5-Dibromo-2,6-dimethoxypyridine (4b): 1H NMR (500 MHz,
CDCl3): d 3.99 (s, 6H, OCH3), 7.86 (s, PyH); 13C NMR (125 MHz, CDCl3): d 55.3,
96.2, 146.2, 158.3; mass spectrum (ESI): 295.89 [M(Br79)(Br79)+H]+, 297.89

K. H. Lam et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2266–2269
2269
[M(Br79)(Br81)+H]+ and 299.89 [M(Br81)(Br81)+H]+. 3-Bromo-2,6-dimethoxy-4-
diphenylphosphinoylpyridine (5a): 1H NMR (500 MHz, CDCl3): d 3.85 (s, 3H,
OCH3), 3.93 (s, 3H, OCH3), 6.34 (d, J = 15 Hz, 1H, PyH), 7.35–7.32 (m, 4H, PhH),
7.45–7.42 (m, 2H, PhH), 7.56–7.52 (m, 4H, PhH); 13C NMR (125 MHz, CDCl3): d
54.3, 55.1, 98.9, 108.4, 128.9, 130.4, 131.3, 132.1, 132.2, 146.0, 146.8, 160.0,
161.9; 31P NMR (125 MHz, CDCl3): d 31.6; mass spectrum (ESI): 441.8 [M+Na]+.
cells were re-suspended in complete cell culture medium at a concentration of
approximately 1 Â 105/mL and counted manually using a haematocytometer
under an inverted microscope. Cancer cells seeded in the 96 wells microtitre
plates for 24 h were prepared for the phosphine oxides screening. The
compounds were dissolved in molecular biology grade dimethylsulfoxide
(DMSO). Cisplatinum was used as the positive reference and added at
starting concentration of 167 M. Compounds were added at starting
concentration of 100 M and followed by a serial of twofold dilutions and
a
3,5-Dibromo-2,6-dimethoxy-4-diphenylphosphinoylpyridine
(5b):
1H
NMR
l
a
(500 MHz, CDCl3): d 3.99 (s, 6H, OCH3), 7.44–7.47 (m, 4H, PhH), 7.54–7.56 (m,
2H, PhH), 7.70–7.74 (m, 4H, PhH); 13C NMR (125 MHz, CDCl3): d 55.1, 101.2,
128.5, 128.9, 132.1, 132.3, 132.5, 132.6, 133.3, 144.1, 144.8, 158.9, 159.0; 31P
NMR (125 MHz, CDCl3): d 32.9; mass spectrum (ESI): 495.93 [M(Br79)(Br79)+H]+,
497.93 [M(Br79)(Br81)+H]+ and 499.93 [M(Br81)(Br81)+H]+. 2,6-Dimethoxy-3-
phenyl-4-diphenylphosphinoylpyridine (6a): 1H NMR (500 MHz, CDCl3): d 3.85
(s, 3H, OCH3), 3.93 (s, 3H, OCH3), 6.34 (d, J = 15 Hz, 1H, PyH), 7.08–6.95 (m, 6H,
PhH) 7.35–7.32 (m, 4H, PhH), 7.45–7.42 (m, 2H, PhH), 7.56–7.52 (m, 4H, PhH);
13C NMR (125 MHz, CDCl3): d 54.0, 54.3, 105.5, 105.6, 105.7, 119.6, 119.7, 127.3,
128.4, 128.5, 128.6, 131.7, 131.8, 132.5, 133.8, 133.9, 145.7, 146.4, 161.4, 161.5,
161.8, 161.9; 31P NMR (125 MHz, CDCl3): d 27.6; mass spectrum (ESI): 416.17
[M+H]+; 438.15 [M+Na]+. 2,6-Dimethoxy-4-diphenylphosphinoylpyridine (6b): 1H
NMR (500 MHz, CDCl3): d 3.88 (s, 6H, OCH3), 6.52 (d, J = 12 Hz, 2H, PyH), 7.51–
7.53 (m, 4H, PhH) 7.61–7.63 (m, 2H, PhH), 7.64–7.66 (m, 4H, PhH); 13C NMR
(125 MHz, CDCl3): d 54.4, 104.4, 129.2, 132.5, 132.6, 132.9, 133.0, 163.9, 164.0;
31P NMR (125 MHz, CDCl3): d 28.9; mass spectrum (ESI): 340.11 [M+H]+.
6. (a) Cell lines and cell culture. Human breast cancer MDAMB-231 and hepatoma
SKHep-1 cell lines were used for preliminary anti-cancer screening of these
compounds. Both cancer cell lines were routinely maintained in RPMI cell
culture medium supplemented with 5% heat inactivated fetal bovine serum and
penicillin/streptomycin antibiotics (complete cell culture medium). The cells
were incubated in a humidified cell culture incubator at 37 °C with 5% CO2.; (b)
Antiproliferation of phosphosine oxides. Cancer cells were removed from 75 cm3
sterile cell culture flasks with onefold trypsin and neutralized with fetal bovine
serum. After washing with phosphate buffered saline and centrifugation, cancer
l
incubated for a further 48 h. The effective concentration of DMSO in each case
was approximately 0.1% by volume. Untreated control received either total
complete cell culture medium or 0.1% DMSO. Afterwards, the evaluation of
possible anti-proliferative potential of our synthesized compounds was
evaluated by the one step ATP lite assay cell proliferation kit purchased from
PerkinElmer (Netherlands). The one step ATP lite assay is a homogeneous
experiment of determining the number of viable cells in culture based on
quantitation of the ATP present, which is an indicator of metabolically active
cells. The homogeneous assay procedure involves adding the single reagent
directly to cells cultured in serum-supplemented medium. This results in cell
lysis and generation of a luminescent signal proportional to the amount of ATP
present. The amount of ATP is directly proportional to the number of cells
present in culture.; (c) Morphological investigation of cancer cells. MDAMB-231
and SKHep-1 human carcinoma cells were seeded at a concentration of 1 Â 105/
mL. After 24 h, phosphine oxides were added (compound 5b at 60 lM) and
incubated for 48 h. Any morphological changes were compared with the vehicle
control under the inverted microscope.
7. Kok, S. H. L.; Chui, C. H.; Lam, W. S.; Chen, J.; Lau, F. Y.; Wong, R. S. M.; Cheng, G.
Y. M.; Lai, P. B. S.; Leung, T. W. T.; Tang, J. C. O.; Chan, A. S. C. Bioorg. Med. Chem.
Lett. 2007, 17, 1155.
8. Kok, S. H. L.; Gambari, R.; Chui, C. H.; Yuen, M. C. W.; Lin, E.; Wong, R. S. M.; Lau,
F. Y.; Cheng, G. Y. M.; Lam, W. S.; Chan, S. H.; Lam, K. H.; Cheng, C. H.; Lai, P. B. S.;
Yu, M. W. Y.; Cheung, . F.; Tang, J. C. O.; Chan, A. S. C. Bioorg. Med. Chem. 2008, 16,
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