Synthesis and biological evaluation of novel cytotoxic phospholipids for prostate cancer

Article abstract of DOI:10.1016/j.bmcl.2004.07.026

A series of serine amide phosphates (SAPs) and serine amide alcohols (SAAs) were prepared and evaluated for their in vitro cytotoxicity against five human prostate cancer cell lines. The SAR and biological activity of the synthesized compounds is reported

Full text of DOI:10.1016/j.bmcl.2004.07.026

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4919–4923
Synthesis and biological evaluation of novel cytotoxic
phospholipids for prostate cancerI
Veeresa Gududuru,a Eunju Hurh,b Gangadhar G. Durgam,a Seoung Soo Hong,a
Vineet M. Sardar,a Huiping Xu,b James T. Daltonb and Duane D. Millera,
*
aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee, Memphis, TN 38163, USA
bDivision of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
Received 25 March 2004; revised 13 July 2004; accepted 14 July 2004
Available online 13 August 2004
Abstract—We describe herein synthesis, SAR, and biological evaluation of a novel series of cytotoxic serine amide phosphates
(SAPs) for prostate cancer. These compounds were tested for their cytotoxicity in five human prostate cancer cell lines (DU-145,
PC-3, LNCaP, PPC-1, and TSU), and in CHO and RH7777 cells (negative controls). Comparison of anticancer effects of these com-
pounds with a standard chemotherapeutic agent 5-fluorouracil shows that they are very effective in killing prostate cancer cells with
low micromolar cytotoxicity and provide us a new lead for the development of drugs for prostate cancer.
Ó 2004 Elsevier Ltd. All rights reserved.
Prostate cancer is the most common malignancy affect-
ing men and is the second-leading cause of cancer deaths
in the US.1 The risk of developing prostate cancer is
associated with age,2 ethnicity,2 family history,3,4 diet,5
and other factors. Treatment for prostate cancer de-
pends on the stage at which cancer is found and the
age and health status of the patient. Clinically, localized
disease is potentially curable6 with standard surgery
and/or radiation therapy. Standard treatment involves
either removal of the entire prostate gland (radical pro-
statectomy) or radiation therapy aimed at the pelvic
area. However, almost half of the men initially diag-
nosed with local disease are found to have tumors,
which have advanced to the periprostatic area or beyond
at the time of surgery. A variety of chemotherapeutic
agents7 are used alone or in combination with radiother-
apy to treat the advanced disease. None of the conven-
tional approaches to cancer therapy have proven to be
highly successful for prostate cancer.
One promising drug development strategy for prostate
cancer involves identifying and testing agents that inter-
fere with growth factors and other molecules involved in
the cancer cellꢀs signaling pathways. Lysophosphatidic
acid8,9 (1, LPA) is a natural glycerophospholipid that
possesses a range of biological actions and is the best
characterized member of the phospholipid growth fac-
tors (PLGFs) family. LPA elicits its effects via multiple
subtypes of membrane spanning G protein-coupled
receptors (GPCR) that include the EDG and PSP family
of receptors. The most prominent effects of LPA include
stimulation of cell proliferation10 and tumor cell inva-
sion.11 LPA stimulates phospholipase D activity and cell
proliferation in PC-3 cell lines.12 One of the LPA recep-
tors, LPA3, has been detected in prostate tissue.13 Ovar-
ian cancer cells produce and respond to LPA.14
Moreover, vascular endothelial growth factor released
from ovarian cancer cells in response to LPA has been
reported to induce angiogenesis in endothelial cells.15
We found that one of the compounds (2) prepared in
our laboratory inhibits LPA induced chloride currents
in frog oocytes.16 Thus, we thought that it would be
interesting to determine the actions of a small library
of these agents in several prostate cancer cell lines. Pre-
liminary studies17 have shown that compounds 2 and 3
are effective in killing prostate cancer cells (Fig. 1).
Encouraged by these results and due to important bio-
logical effects of LPA and its implications in the patho-
physiology of a variety of cancers we decided to
Keywords: Prostate cancer; LPA; New agents; Cytotoxicity.
q Part of this work was presented at 225th ACS National Meeting,
New Orleans, LA, March 23–27, 2003 and at 94th Annual AACR
Meeting, Washington, DC, July 11–14, 2003.
*
Corresponding author. Tel.: +1-901-448-6026; fax: +1-901-448-
3446; e-mail: dmiller@utmem.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.026

4920
V. Gududuru et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4919–4923
optimize the activity of SAPs for in vitro anticancer
activity against prostate tumor cell lines. We report in
this paper the synthesis, SAR, and in vitro cytotoxic
activity of SAP derivatives against five human prostate
cancer cell lines and two nontumor cell lines to deter-
mine their selectivity (Fig. 1).
alcohols 17 and 18 (Scheme 4). Compound 20, which
has an ethanolamine amide backbone rather than the
serine amide backbone was synthesized according to
the reported procedure.19
All compounds were characterized by 1H and 13C
NMR, mass spectroscopy and, in certain cases, elemen-
tal analysis.20
The general synthesis of SAPs and serine amide alcohols
(SAAs) is shown in Scheme 1. Commercially available
N-Boc-serine (R or S form) was allowed to react with
an appropriate amine in the presence of EDC/HOBt to
form amide 5. Amide 5 was treated with TFA to give
serine amide alcohol (6). Phosphorylation of amide 5
and concurrent removal of protecting groups under hy-
We examined the cytotoxicity of synthesized compounds
in five human prostate cancer cell lines (DU-145, PC-3,
LNCaP, PPC-1, and TSU) and in two negative control
cell lines (CHO and RH7777) using the sulforhodamine
B (SRB) assay.21 Cells were exposed to a wide range of
concentrations (0–100lM) of the particular compound
for 96h in 96-well plates. Cells were fixed with 10% tri-
chloroacetic acid, washed five times with water. The
plates were air dried overnight and fixed cells were
stained with SRB solution. The cellular protein-bound
SRB was measured at 540nm using a plate reader. Cell
numbers at the end of the treatment were measured.
IC50 (i.e., concentration that inhibited cell growth by
50% of untreated control) values were obtained by non-
linear regression analysis using WinNonlin. For com-
parative purposes and to understand the degree of
drogenolysis conditions using Pd/C in ethanol gave 2.
18
During the progress of this work a report
appeared
for the synthesis of 2b. However, according to our
knowledge these type of compounds (SAPs) have never
been examined for prostate cancer therapy. We synthe-
sized unsaturated analogues 9 and 11 by similar proce-
dures as shown in Scheme 2. Serine diamide
phosphates (SDAPs) and other amine derivatives were
synthesized starting from O-benzyl N-Boc-serine follow-
ing the earlier procedures (Scheme 3). LAH mediated
reduction of compound 5e gave long chain N-alkyl amino
O
O
O
O
P
OH
O
P
OH
O
P
HO
O
NH(CH2)13CH3
HO
O
O
(CH2)nCH3
HO
O
NH(CH2)13CH3
+
OH
O-
NH3
NHAc
3
2
1
Figure 1. Structures of LPA and SAPs.
O
O
O
ii
i
HO
NH(CH2)nCH3
HO
NH(CH2)nCH3
NHBoc
HO
OH
NHBoc
NH3+CF3COO-
6a-6g
5
4
O
O
O
P
O
P
OBn
iv
iii
HO
O
NH(CH2)nCH3
BnO
O
NH(CH2)nCH3
NHBoc
5
+
O-
NH3
2a-2f
7
Scheme 1. Reagents and conditions: (i) CH3(CH2)nNH2, EDC, HOBt, CH2Cl2, rt, 5h; (ii) TFA, CH2Cl2, rt, 0.5h; (iii) tetrazole, dibenzyl
diisopropylphosphoramidite, CH2Cl2, rt, 0.5h, H2O2, rt, 0.5h; (iv) H2, 10% Pd/C, EtOH, rt, 3h.
O
O
O
ii
i
HO
NHC8H16
NH3+Cl-
HO
OH
C8H17
HO
NHC8H16
C8H17
NHBoc
NHBoc
8
9
4
O
O
O
O
P
O
iv
iii
8
HO
P
O
NHC8H16 C8H17
O
O
NHC8H16
C8H17
O-
NHBoc
10
+
NH3
11
Scheme 2. Reagents and conditions: (i) C8H17(CH:CH)C8H16NH2, EDC, HOBt, CH2Cl2, rt, 5h; (ii) 2M HCl/Et2O, rt, overnight; (iii) tetrazole,
di-tert-butyl diisopropylphosphoramidite, CH2Cl2, rt, 0.5h, H2O2, rt, 0.5h; (iv) TFA, CH2Cl2, rt, 0.5h.

V. Gududuru et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4919–4923
4921
O
O
O
ii, iii
i
BnO
NHR2
BnO
NHR2
NHBoc
BnO
OH
NHBoc
NHR3
14
13
12
O
O
O
v, vi
iv
HO
P
O
NHR2
HO
NHR2
O-
NHR3
NHR3
16
15
Scheme 3. Reagents and conditions: (i) R2NH2, EDC, HOBt, CH2Cl2, rt, 5h; (ii) TFA, CH2Cl2, rt, 0.5h; (iii) TEA, R3SO2Cl or R3NCO or R3COCl;
(iv) H2, 10% Pd/C, EtOH, rt, 3h; (v) tetrazole, dibenzyl diisopropylphosphoramidite, CH2Cl2, rt, 0.5h, H2O2, rt, 0.5h; (vi) H2, 10% Pd/C, EtOH, rt,
3h.
O
prostate cell line. The inhibitory activity of sulfonamide
derivatives 15b and 16b and urea derivative 15c in all
five prostate cancer cell lines showed a general decreas-
ing trend suggesting that derivatization of C2 amine
group is not tolerable for their ability to kill prostate
cancer cells.
i, ii
HO
NH(CH2)17CH3
HO
NH(CH2)17CH3
NH3+Cl-
NHBoc
17
5e
ii
HO
NH(CH2)17CH3
N(Me)H2+Cl-
18
To further investigate the extent of structural tolerance
permitted in the serine amide backbone region, we re-
placed the serine amide group with simple ethanolamine
amide by synthesizing compounds 19 and 20. However,
these ethanolamine amide analogues were less potent
and particularly compound 19 did not show any activity
against DU-145, PC-3, and LNCaP prostate cancer cell
lines.
Scheme 4. Reagents and conditions: (i) TFA, CH2Cl2, rt, 0.5h (ii) (a)
LAH, Et2O, reflux, 7h; (b) HCl.
cytotoxicity we tested 5-fluorouracil against all five pros-
tate cancer cell lines. The results are summarized in
Table 1.
When the amide group in serine amide alcohols was re-
duced to produce long chain N-alkyl amino alcohols 17
and 18, these analogues retained cytotoxicity and were
very effective in killing prostate cancer cell lines with
low micromolar cytotoxicity. To determine the selectiv-
ity, several of synthesized compounds were also exam-
ined for their cytotoxicity in CHO and RH7777 cells
as negative controls. Many of the potent compounds
showed similar cytotoxicity and were nonselective in
their action against prostate cancer cell lines and non-
tumor negative control cells.
From the cytotoxicity data it is clear that most of the
compounds tested showed good anticancer activity
against all five prostate cancer cell lines. It is notewor-
thy, to mention that serine amide alcohols (6b,e,f) with-
out a phosphate group are as effective as SAPs. A direct
relationship was observed between length of the alkyl
chain and cytotoxicity of the tested compounds.
Accordingly, all of these compounds showed an alkyl
chain length dependent cytotoxicity. Compounds with
shorter alkyl chains (2a, 6b, 15d, 16d) are less cytotoxic
than analogues with longer alkyl chains (see Table 1).
Compound 2f emerged as one of the most potent SAPs
tested so far with an IC50 of 1.8lM against PPC-1 cell
line. However, serine amide alcohols are more potent
than corresponding SAPs when the alkyl chain length
is below 18C, but no significant difference in the cytotox-
icity is observed between serine amide alcohols and
SAPs with alkyl chain more than 18C.
In summary we have shown that SAPs and SAAs, deriv-
atives of LPA, represent a novel class of cytotoxic phos-
pholipids for prostate cancer. We have designed and
synthesized a number of SAP derivatives and evaluated
for their inhibitory activity toward the growth of human
prostate cancer cell lines. Several of these, such as 2f, 17,
and 18 are potent inhibitors of prostate tumor cell pro-
liferation at low micromolar cytotoxicity. Despite their
high cytotoxicity, the same compounds were non selec-
tive against nontumor CHO cells and receptor-negative
RH7777 cells. This initial report suggests that further
optimization is necessary to increase the selectivity.
Efforts are in progress in our laboratory to enhance
potency and selectivity of this class of compounds for
the treatment of prostate cancer.
IC50 values for enantiomers of serine amide alcohols
(6c,d) and SAPs (2b,c) are approximately equivalent,
which suggests that chirality is not important for the an-
tiproliferative activity of these compounds in prostate
cancer. Introduction of a double bond in the alkyl chain
lowered the potency of both serine amide alcohol 9 and
SAP 11.
To understand the importance of the amine functional-
ity we derivatized the amine group to the corresponding
Set B amide, sulfonamide and urea derivatives. Serine
diamide phosphate 16d with a shorter alkyl chains failed
to demonstrate cytotoxicity at concentration below
100lM in four prostate cancer cell lines except TSU
Acknowledgements
This research was supported by a Grant from the
Department of Defense (DAMD17-01-1-083).

Table 1. Anti-proliferative effects of synthesized compounds in prostate cancer and negative control cell lines
O
O
H
N
R2
R1O
NHR2
R1O
NHR2
R1O
NHR2
NH2 R3
Set C
R1O
+
NH3
+
NHR3
O
Set A
Set D
Set B
Set
Compound (chirality)
IC50 (lM)
RH7777a
R1
R2
R3
CHOa
DU-145b
PC-3b
LNCaPb
PPC-1b
TSUb
31.5
A
2a (2R)
2b (2R)
2c (2S)
2d (2R)
2e (2R)
2f (2S)
6a (2S)
6b (2R)
6c (2R)
6d (2S)
6e (2R)
6f (2R)
6g (2S)
9 (2S)
PO3H
PO3H
PO3H
PO3H
PO3H
PO3H
H
C10H21
C14H29
C14H29
C18H37
C19H39
C20H41
C8H17
––
––
––
––
––
––
––
––
––
––
––
––
––
––
––
ND
ND
ND
ND
3.7
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
50.2
20.6
32.0
11.7
5.7
36.0
>50
>50
19.1
15.3
>20
>100
35.0
10.2
10.3
5.2
44.7
10.1
19.7
7.2
22.1
>10
>10
5.6
>10
>10
4.8
5.0
5.8
3.6
1.8
1.8
7.8
10.8
11.1
>100
ND
ND
ND
2.5
>100
>100
31.0
8.1
>100
15.9
6.3
>100
H
C
C
10H21
14H29
52.2
8.2
6.9
5.4
5.1
7.0
6.9
16.0
26.0
7.5
9.2
4.4
3.9
6.6
5.5
12.4
H
H
C14H29
18H37
C19H39
20H41
10.0
3.8
6.2
2.2
H
C
2.6
3.2
H
2.4
4.1
5.3
6.6
5.3
3.9
1.8
2.6
H
C
ND
H
PO3H
C8H17–CH:CH–C8H16
C8H17–CH:CH–C8H16
5.2
11.9
6.8
28.6
5.9
39.2
6.6
12.2
5.1
21.1
11 (2S)
B
14a (2S)
14b (2S)
14c (2S)
14d (2S)
15b (2S)
15c (2S)
15d (2S)
16b (2S)
16d (2S)
OBn
OBn
OBn
OBn
H
C
18H37
C18H37
18H37
H
SO2Me
3.0
>50
18.5
9.2
9.9
11.2
6.2
47.3
>20
10.9
Not active
>20
2.9
16.7
>20
6.8
>50
>20
12.9
9.2
>20
ND
>50
20
>50
C
CO NH Ph (3,5-difluoro)
COC7H15
SO2Me
15.9
10.0
20.5
15.3
93.8
35.7
79.0
C8H17
C18H37
22.9
23.1
20
31.3
13.6
>20
35.0
16.0
4.0
10.2
>20
12.9
20
H
C
18H37
CO NH Ph (3,5-difluoro)
COC7H15
SO2Me
>20
>100
H
C8H17
C18H37
C8H17
>100
>50
>100
>100
43.2
>100
81.5
>50
>100
81.2
17.8
>100
PO3H
PO3H
50
>100
15.1
Not active
COC7H15
C
D
17 (2R)
18 (2R)
H
H
C
18H37
C18H37
H
Me
2.2
1.7
2.9
2.5
4.1
3.2
2.6
2.4
5.1
3.3
1.9
1.6
2.2
1.1
19
20
H
PO3H
C8H17–CH:CH–C8H16
C8H17–CH:CH–C8H16
––
––
>20
>50
>20
>50
Not active
>50
Not active
>50
Not active
Not active
>20
50
Not active
>50
5-FU
––
––
––
––
––
11.9
12.0
4.9
6.4
3.6
a Control cell lines.
b Prostate cancer cell lines.

V. Gududuru et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4919–4923
4923
Fischer, D. J.; Virag, T.; Nusser, N. U.S. Patent. 811,838,
2003.
References and notes
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20. All synthesized compounds were purified by column
chromatography and purity was confirmed by elemental
analysis and HPLC. Characteristic data for some com-
pounds is given below.
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17. Miller, D. D.; Tigyi, G.; Dalton, J. T.; Sardar, V. M.;
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Compound 2e: 1H NMR (300MHz, CF3CO2D) d 0.77 (br
s, 3H), 1.20 (br s, 32H), 1.53 (br s, 2H), 3.32–3.33 (m, 2H),
4.50 (br s, 1H), 4.67–4.72 (m, 2H); 13C NMR (300MHz,
CF3CO2D) d 11.92, 21.61, 25.78, 27.65, 28.13, 28.46,
28.49, 28.62, 28.67, 28.74, 31.0, 40.71, 54.27, 63.54, 164.99;
MS (ESI) m/z 449 [MÀH]. Anal. Calcd for C22H47N2O5PÆ
0.5EtOH: C, 58.64; H, 10.51; N, 6.22. Found: C, 58.33; H,
10.64; N, 5.91.
Compound 16b: 1H NMR (300MHz, CF3CO2D) d 0.79 (t,
J = 6.6Hz, 3H), 1.22 (br s, 30H), 1.56 (m, 2H), 3.20 (s,
3H), 3.35 (t, J = 6.9Hz, 2H), 4.50–4.70 (br m, 3H); 13C
NMR (300MHz, CF3CO2D) d 11.77, 21.50, 25.69, 27.63,
28.05, 28.35, 28.42, 28.51, 28.56, 28.63, 30.97, 39.70, 40.55,
56.04, 67.72, 66.73, 169.85; MS (ESI) m/z 513.2
[MÀH].
1
Compound 18: H NMR (300MHz, DMSO-d6) d 0.83 (t,
J = 6.9Hz, 3H), 1.23 (br s, 32H), 2.66 (br s, 3H), 2.92 (m,
2H), 3.2 (m, 2H), 3.52 (br s, 1H), 3.62–3.82 (m, 2H), 9.11
(br s, 1H), 9.35 (m, 2H); 13C NMR (300MHz, DMSO-d6)
d 13.85, 21.99, 25.26, 25.87, 28.44, 28.6, 28.73, 28.87, 28.9,
28.95, 30.0, 31.2, 44.39, 47.33, 47.45, 56.16, 57.05; MS
(ESI) m/z 357.5 [M+H].
Compound 20: The spectroscopic properties of this
compound were inconsistent with the assigned structure
reported in the literature.19.
21. Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simon,
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