Synthesis and bioactivity of sphingosine kinase inhibitors and their novel aspirinyl conjugated analogs

Article abstract of DOI:10.1016/j.ejmech.2010.06.005

Sphingosine kinase (SphK) is a lipid kinase with oncogenic activity, and SphK inhibitors (SKIs) are known for their anti-cancer activity. Here, we report highly efficient syntheses of SKIs and their aspirinyl (Asp) analogs. Both SKIs and their Asp analogs were highly cytotoxic towards multiple human cancer cell lines; in several cases the Asp analogs were up to three times more effective. Furthermore, they were equally potent inhibitors of SphK. The pharmacokinetic study indicated that SKI-I-Asp cleaved efficiently to form SKI-I and the half-life of SKI-I was increased from ～7 h in SKI-I to ～10 h in SKI-I-Asp injected mice, thereby prolonging its effect. In summary, the Asp-conjugated SKIs seem to be promising prodrugs of SKIs where delivery in vivo remains a problem.

Full text of DOI:10.1016/j.ejmech.2010.06.005

European Journal of Medicinal Chemistry 45 (2010) 4149e4156
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and bioactivity of sphingosine kinase inhibitors and their novel
aspirinyl conjugated analogs
,
b
,
c
,
a
a
a
a
Arun K. Sharma a
, Ugir Hossain Sk , Melissa A. Gimbor , Jeremy A. Hengst , Xujun Wang ,
*
Jong Yun a c, Shantu Amin a
,
,b,c
a Department of Pharmacology, Penn State Hershey College of Medicine, Penn State Hershey Cancer Institute, CH72, 500 University Drive, Hershey, PA 17033, USA
b Chemical Carcinogenesis and Chemoprevention Program of Penn State Cancer Institute, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA
c Experimental Therapeutics Program of Penn State Cancer Institute, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Sphingosine kinase (SphK) is a lipid kinase with oncogenic activity, and SphK inhibitors (SKIs) are known
for their anti-cancer activity. Here, we report highly efficient syntheses of SKIs and their aspirinyl (Asp)
analogs. Both SKIs and their Asp analogs were highly cytotoxic towards multiple human cancer cell lines;
in several cases the Asp analogs were up to three times more effective. Furthermore, they were equally
potent inhibitors of SphK. The pharmacokinetic study indicated that SKI-I-Asp cleaved efficiently to form
SKI-I and the half-life of SKI-I was increased from w7 h in SKI-I to w10 h in SKI-I-Asp injected mice,
thereby prolonging its effect. In summary, the Asp-conjugated SKIs seem to be promising prodrugs of
SKIs where delivery in vivo remains a problem.
Received 24 March 2010
Received in revised form
3 June 2010
Accepted 4 June 2010
Available online 12 June 2010
Keywords:
Sphingosine kinase
Sphingosine kinase inhibitors
Aspirin
Ó 2010 Elsevier Masson SAS. All rights reserved.
Prodrugs
1. Introduction
lines [7]. Among these, two compounds SKI-I and SKI-II (Fig. 1)
were the most effective and most studied agents.
Sphingosine kinase 1 (SphK1) is an oncogenic lipid kinase
closely linked to signaling pathways associated with cancer cell
proliferation/survival, metastasis and multi-drug resistance (MDR)
(for review [1e6]). SphK1 regulates the fate of cells by catalyzing
the formation of sphingosine-1-phosphate (S1P) at the expense of
ceramide. Over-expression of SphK1 is commonly observed in
many tumor tissues relative to patient matched normal mammary
tissues [7,8]. Importantly, studies showed that high SphK1
expression levels were a prognostic factor for decreased metas-
tasis-free survival and overall poor outcome of patients with
hormone-independent (i.e. ERꢀ) tumors [9,10]. Based on these and
many other studies [11e24], it has been proposed that targeting
SphK1 is a novel approach for the treatment of cancers including
metastatic and/or MDR. In this regard, we have previously identi-
fied several “drug-like” compounds that potently inhibited SphK1
activity, and showed that these novel SphK inhibitors (SKIs) are
potent inducers of cytotoxicity against various human cancer cell
SKI-II has been shown to be an excellent agent that inhibits
endogenous S1P formation in intact cells, SKI-II has a high degree of
specificity towards SphK1 with low cross-reactivity towards other
kinases, SKI-II treatment effectively inhibits tumor growth in
a syngenic mouse model of breast cancer [7,25]. In contrast,
although our ongoing studies show that SKI-I is more potent than
SKI-II, the poor solubility and minimal bioavailability of these
compounds have become the main problems in their efficacy as
anti-cancer agents in in vivo animal studies. To overcome this
problem, we designed and synthesized prodrug conjugates of SKI-I
and SKI-II. Since both these inhibitors have a phenolic hydroxyl
group, esterification was considered to be the method of choice for
developing their prodrug conjugates. In this regard, we made use of
2-(acetoxy)benzoate (aspirinyl) conjugation. The rational for
making the aspirinyl derivatives of SKI-I and SKI-II as prodrug
conjugates was that the 2-(acetoxy)benzoic acid (aspirin), released
upon hydrolysis by esterase (Fig. 1), is already a well established
non-steroidal anti-inflammatory drug. It has shown chemo-
preventive effects against the risk of developing various cancers
[26e30] and heart diseases [31]. US Preventive Services Task Force
(USPSTF, March, 2009) has recommended the use of aspirin for the
primary prevention of coronary heart disease. Regular low dose
(w81 mg), amount in a baby aspirin is actually recommended for
* Corresponding author. Department of Pharmacology, Penn State Hershey
College of Medicine, Penn State Hershey Cancer Institute, CH72, 500 University
Drive, Hershey, PA 17033, USA. Tel.: þ1717 531 0003x285016; fax: þ1717 531 0244.
E-mail address: aks14@psu.edu (A.K. Sharma).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.06.005

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A.K. Sharma et al. / European Journal of Medicinal Chemistry 45 (2010) 4149e4156
O
H
N
H
N
S
N
N
OCOCH3
O
N
H
O
N
O
O
Cl
OCOCH3
SKI-II-ASP
SKI-I-ASP
OH
S
H
H
N
N
N
N
N
H
N
HO
O
Cl
SKI-II
SKI-I
+
+
O
O
OH
OCOCH3
OH
OCOCH3
Aspirin
Aspirin
Fig. 1. Formation of SKI-I and SKI-II from corresponding aspirinyl derivatives.
people who are at a high risk of having heart diseases. At high doses
aspirin may cause undesirable side effects e.g. gastrointestinal
ulcers. However, in a cancer therapy setting, as is the case for SKIs,
the release of aspirin outweighs the risks. The release of aspirin is
therefore not expected to cause side effects and could potentially
add its anti-inflammatory properties.
In this study, we describe simple and novel synthetic method for
SKI-I and SKI-II and their aspirinyl substituted prodrugs SKI-I-Asp
(10) and SKI-II-Asp (14). The cytotoxicity and activity of aspirinyl
derivatives were compared with the corresponding parent
compounds and the suitability of the aspirinyl analogs was deter-
mined by evaluating their degradation pattern in in vitro culture
conditions and in a mouse model.
The solubility/bioavailability of SKI-I is the main hurdle in its
delivery to animals. The compound tends to precipitate in the body
and is thus not readily bioavailable. To address this problem, we
synthesized an aspirinyl conjugate of SKI-I. First approach was to
treat SKI-I with 2-acetoxybenzoyl chloride in the presence of
a base. However, this reaction led to multiple products resulting in
lower yields. Several attempts to enhance the yield of this step
failed. We then changed the strategy and treated 2-hydroxy-1-
naphthaldehyde (9) with 2-acetoxybenzoyl chloride in the pres-
ence of potassium carbonate to first synthesize intermediate 9
(Scheme 1). The use of triethylamine as a base instead of potassium
carbonate in this alkylation resulted in multiple products leading to
lower yield of 9. Condensation of 9 with hydrazide 3 finally gave the
desired SKI-I-Asp (10) in excellent yield (Scheme 1). The aspirinyl
linkage provided facile solubility in organic solvents and easy
purification of this compound.
2. Results
To generalize the feasibility of aspirinyl prodrugs, we also
synthesized 2-(acetoxy)benzoate derivative of SKI-II (Scheme 2).
SKI-II (13) was prepared by condensing 2-bromoketone (11) with
thiourea 12 following a literature method [36]. In contrast to SKI-I,
SKI-II reacted efficiently with 2-acetoxybenzoyl chloride in the
2.1. Chemistry
The synthesis of SKI-I was accomplished in just three steps as
outlined in Scheme 1. The synthesis of ester 2 was carried out using
a literature method [32], by reacting commercially available 20-
acenaphthone (1) and dimethyl oxalate in the presence of sodium
hydride with excellent yield. Treatment of 2 with anhydrous
hydrazine [33] led to the formation of pyrazole hydrazide 3 which
on condensation with 2-hydroxynaphthaldehyde under standard
conditions [34] furnished the desired SKI-I (4) in good yield.
Alternatively, the synthesis of SKI-I can also be achieved by first
hydrolyzing the ester 2 using 1 N HCl following a literature
procedure [35] to yield the corresponding acid 5 followed by
treatment with hydrazine [32] to the form the pyrazole derivative 6
as a white solid. Acid chloride derivative 7 was obtained by reaction
of 6 with thionyl chloride. Refluxing of 7 with anhydrous hydrazine
[33] in ethanol yielded hydrazide 3, which was then converted to
SKI-I (4) as described above.
pr
esence of triethylamine to yield the desired product SKI-II-Asp
(14). All the intermediates and the final products were purified by
silica gel column chromatography and characterized on the basis of
NMR and high-resolution mass spectra.
2.2. SKIs and their aspirinyl analogs induce human cancer cell
cytotoxicity
We have previously demonstrated that both SKI-I and SKI-II
compounds are potent inducers of cancer cell cytotoxicity [7]. To
determine whether aspirinyl linkage to SKI-I or SKI-II would alter
the cytotoxic potency, we performed the Sulforhodamine B (SRB)
cytotoxicity assays on human cancer cell lines which include brain
(U78MG), ovarian (HeLa), lung (H460, H226, H441, and A549),

A.K. Sharma et al. / European Journal of Medicinal Chemistry 45 (2010) 4149e4156
4151
N
O
O
OH
CO H
HN
O
OH
CO Me
CO H
(iii)
(i)
(ii)
5
6
2
1
(vii)
(iv)
N
HN
N
HN
H
N
H
CONHNH
(v)
COCl
(vi)
N
N
N
HO
O
3
7
SKI-I
4
20% yield
H
CHO
CHO
O
OH
(vii)
(viii)
H
N
N
OCOCH
O
N
N
O
O
O
90% yield
9
8
OCOCH
SKI-I-Asp
10
Scheme 1. Synthesis of SKI-I (4) and SKI-I-Asp (10). Reagents and conditions: (i) NaH, dimethyl oxalate, C6H6 and MeOH, RT, overnight (ii) 1 N HCl, 1,4-dioxane, reflux, 2 h (iii)
hydrazine hydrate, EtOH, reflux, 30 min (iv) SOCl2, C6H6, reflux, 5 h (v) hydrazine hydrate, EtOH, reflux, overnight (vi) 2-hydroxynaphthaldehyde, AcOH (cat.), DMSO (vii) K2CO3,
THF, 16 h, RT, (viii) AcOH (cat.), DMSO, RT, 3 h.
breast (MDA-MB-231), and pancreatic (Mia-PaCa2), and immor-
talized normal human mammary epithelial cell line, MCF-10A, and
mouse embryonic fibroblast (MEF) cell line (cancer cell lines).
Cancer cells (5 ꢁ 103) were cultured in a 96 well plate for 24 h, and
then treated with various concentrations of SKIs and their aspirinyl
conjugates or control DMSO for 72 h. As shown in Table 1, the
cytotoxic potency (indicated by IC50 values) of aspirinyl-linked SKI
analogs was similar or better than their parental SKI compounds.
Specifically, the IC50 values of SKI-I and its prodrug SKI-I-Asp
remained essentially the same towards brain and ovarian cancer
cell lines, whereas SKI-I-Asp was approximately w3 times more
potent than the parent SKI-I compound towards breast, pancreatic
and all the four lung cancer cell lines. Additionally, consistent with
our previous findings [7], the SKI-I and its aspirin derivative SKI-I-
Asp showed lower IC50 values compared to the SKI-II or its aspirin
derivative SKI-II-Asp. Similar results were observed in normal
human mammary epithelial cell line, MCF-10A. Together, these
results indicate that aspirinyl linkage of SKIs may improve the
cytotoxic potency of parental SKI compounds [7].
2.3. Metabolism of aspirinyl prodrugs of SKIs in cell culture media
As indicated above in cytotoxicity studies, the parent compound
SKI-II and its aspirinyl derivative 14, were equally cytotoxic in
various cancer cell lines (Table 1). On the other hand, aspirinyl
derivative 10 of SKI-I was more potent than the parent compound
SKI-I. This could be due to fact that aspirinyl-linked SKIs are more
stable and resistant to spontaneous rapid cleavage of the ester bond
in 10% FBS containing culture media. To test this potential possi-
bility of degradation, we subjected SKIs and their aspirinyl-linked
analogs to same culture condition (10% FBS). Specifically, SKI-I and
SKI-II were incubated in DMEM culture medium at 37 ꢂC for 2, 16,
and 72 h time points. Subsequently, samples were extracted with
ethyl acetate and analyzed for decomposition using HPLC. We
determined the ratio of asprinyl derivatives 10 and 14 to their
corresponding parent compounds 4 and 13, respectively (Fig. 2A).
In case of SKI-I-Asp (10), w40% of the aspirinyl derivative was
decomposed to SKI-I in just 2 h. After 16 h, w80% of the compound
was converted. After 72 h, the formation of SKI-I remained constant
at w82%. The SKI-II-Asp (14) derivative degraded to a nearly similar
extent in 72 h (Fig. 2B) but a little more slowly and steadily. About
15% SKI-II was formed in 2 h, w50% in 16 h and w75% at the end of
72 h i.e. about 25% SKI-II-Asp (14) was still remaining after 72 h. To
OH
O
S
OH
S
Br
(i)
N
H
N
+
H N
N
H
Cl
Cl
Table 1
SKI-II
13
IC50 (m
M) of SKIs and their aspirinyl analogs using SRB assaya (72 h treatment).
11
12
Cancer/normal Tissue
cell lines
SKI-I
SKI-I-Asp SKI-II
SKI-II-Asp
(ii)
U87MG
HeLa
H460
H226
H441
Brain
Ovary
Lung
Lung
Lung
1.0 ꢃ 0.2 1.5 ꢃ 0.5
1.0 ꢃ 0.3 1.0 ꢃ 0.1
3.2 ꢃ 1.0 1.2 ꢃ 0.5
5.7 ꢃ 2.1 1.7 ꢃ 0.3
2.0 ꢃ 1.2 0.6 ꢃ 0.2
3.8 ꢃ 1.6 0.9 ꢃ 0.1
4.4 ꢃ 1.7 1.3 ꢃ 0.9
4.8 ꢃ 2.6 1.6 ꢃ 0.5
4.1 ꢃ 1.5 2.6 ꢃ 1.2
10.7 ꢃ 1.1
8.1 ꢃ 1.6
9.1 ꢃ 1.5 11.2 ꢃ 2.9
O
9.2 ꢃ 2.3
8.6 ꢃ 1.7
8.0 ꢃ 0.9
7.8 ꢃ 0.8
8.2 ꢃ 1.0
7.7 ꢃ 1.4
9.1 ꢃ 0.8
6.7 ꢃ 1.6
5.7 ꢃ 1.9
5.9 ꢃ 2.1
8.4 ꢃ 1.4
8.7 ꢃ 1.4
S
OCOCH
O
N
H
N
A549
Lung
Cl
MDA-MB-231
Mia-PaCa2
MCF-10A
MEF
Breast
Pancreas
Breast
Fibroblast 5.0 ꢃ 1.3 5.5 ꢃ 0.7
SKI-II-ASP
14
14.5 ꢃ 2.2 11.3 ꢃ 2.0
8.9 ꢃ 1.2 9.4 ꢃ 1.8
Scheme 2. Synthesis of SKI-II (13) and SKI-II-Asp (14). Reagents and conditions: (i)
EtOH, reflux, 1.5 h, NH4OH (ii) Et3N, THF, overnight, RT.
a
All values represent the average from three independent experiments.

4152
A.K. Sharma et al. / European Journal of Medicinal Chemistry 45 (2010) 4149e4156
Asp (MW 591) structure (Fig. 3), suggesting that the dose of SKI-I-
A
100
50
Asp could be increased by nearly 40% without any additional
systemic toxicity. Furthermore, the concentration of SKI-I in SKI-I-
Asp injected mice decreased more slowly over time as compared to
SKI-I (Fig. 3). Therefore, the half-life of SKI-I in SKI-I injected mice
was w7 h which was increased in SKI-I-Asp injected mice to w10 h.
SKI-I
SKI-I-Asp
2.5. Sphingosine kinase activity assay
The data from the cytotoxicity assays, presented above, indicates
that the SKI lead compounds and their aspirinyl derivatives have
essentially equal cytotoxicity across a panel of cancer cell lines. This
finding suggests that the aspirinyl derivatives also have roughly
equal inhibitory activity towards SphK1. To confirm this, we treated
HeLa cells with increasing concentrations of the SKIs and their
aspirinyl derivatives for 24 h prior to performing in vitro SphK1
activity assays. As shown in Fig. 4, at lower doses (i.e., <1.25 mM),
the SKI-I-Asp and SKI-II-Asp derivative were equally effective at
inhibiting SphK1 as the parent SKI compounds. At higher doses
0
0
2
16
72
Time (hr)
B
100
SKI-II
SKI-II-Asp
(ꢄ5
mM), the aspirinyl derivatives were less effective than the
parent SKI compounds.
50
0
3. Discussion
The aspirinyl analogs of SKIs were designed to take advantage of
the fact that apart from an easy release of the parent compound by
the hydrolysis of the ester linkage by esterases on administration in
vivo, aspirin will be released as a side product. Aspirin is a well
established anti-inflammatory drug and does not require any
additional study on determining its toxicity or ADME profile.
Therefore, its release in the process is not expected to cause any
additional systemic toxicity and in fact could potentially add its
anti-inflammatory effect in addition to the anti-cancer effect of
SKIs. The synthesis of SKI-I was accomplished in just three steps in
a good overall yield. The aspirinyl analog of SKI-I could not be
synthesized from SKI-I treatment with 2-acetoxybenzoyl chloride,
however pretreatment of 2-acetoxybenzoyl chloride with
2-hydroxynapthaldehyde followed by condensation led to a very
good yield of SKI-I-Asp (10) (Scheme 1). The synthesis of SKI-II-Asp
(14) on the other hand was efficiently obtained from SKI-II,
synthesized in just one step (Scheme 2), by treatment with 2-
acetoxybenzoyl chloride.
0
2
16
72
Time (hr)
Fig. 2. Serum metabolism of (A) SKI-I-Asp and (B) SKI-II-Asp in vitro: 1 mg/ml of each
compound in DMSO was dissolved in 5 mL 10% FBS supplemented DMEM or serum
free DMEM media in 15 mL conical tubes. The tubes were placed in a shaking incubator
at 37 ꢂC. At 0, 2, 16 and 72 h samples were taken out, filtered, and analyzed using HPLC.
The percentage of SKIs and SKI-Asp peaks was calculated for each time point and
plotted.
determine if the degradation is due to an enzymatic reaction or
solely due to the pH dependent hydrolysis in the media, we incu-
bated SKI-I-Asp and SKI-II-Asp with serum free media. Interest-
ingly, both SKI-I-Asp and SKI-II-Asp remained unchanged even
after 72 h of incubation at 37 ꢂC. This suggested that the degrada-
tion is due to some enzymatic hydrolysis.
Taken together with the cytotoxicity data, this suggests that the
effect seen for aspirinyl derivatives in the in vitro studies may not
solely be due to the decomposition to the parent compounds, but
that the aspirin derivatives themselves may also have some
contribution towards the overall observed cytotoxicity. This argu-
ment is strengthened by the fact that the cells absorbed most of the
compound in the initial 1e2 h when 60e85% of aspirinyl deriva-
tives of both SKI-I and SKI-II were still present within the cell.
The comparison of the cytotoxicity of SKIs and their corre-
sponding aspirinyl analogs showed that SKI-I-Asp was w3 times
more effective than SKI-I in killing breast, pancreas and all the lung
cancer cells tested (Table 1), while being equally cytotoxic to brain
2.4. Metabolism of aspirinyl prodrug of SKI-I in mice
To determine the decomposition patterns of SKI-Asp analogs in
vivo, mice were injected with SKI-I and its prodrug SKI-I-Asp
(60 mg/kg body weight) and the blood was collected at 1, 2, 4, 8,
and 16 h time points. Each sample was subjected to HPLC analysis
and compared with SKI-I and SKI-I-Asp standards. One hour after
injection, the HPLC profile of blood from SKI-I-Asp treated mice
revealed the presence of only the SKI-I peak and did not show any
SKI-I-Asp signal. The maximum serum concentration (Cmax) of SKI-I
was observed 2 h after injection of both SKI-I and its corresponding
aspirinyl analog. Interestingly, the Cmax of SKI-I from SKI-I-Asp mice
was w50% of SKI-I injected mice, instead of being w70% as
expected from the molar ratio of SKI-I (MW 407) fragment in SKI-I-
Fig. 3. Pharmacokinetics in mice (C3H-HEJ strain): Mice were injected with con-genic
mouse AML cell line called p210-GFP. GFP is fluores-tag. Both the SKI-I and SKI-I-Asp
were injected 60 mg/kg (ip). Serum was collected at 1, 2, 4, 8, and 16 h time points and
analyzed by HPLC.

A.K. Sharma et al. / European Journal of Medicinal Chemistry 45 (2010) 4149e4156
4153
blood, and a slow decline in SKI-I concentration in SKI-I-Asp
injected mice suggests that a part of SKI-I-Asp may be absorbed in
tissues and release into blood stream overtime, keeping blood
levels of the SKI-I from decreasing rapidly and thereby prolonging
the effect of the drug.
The systemic toxicity and potency of a drug is generally decided
by its concentration in blood at a given time. The lower concen-
tration of SKI-I at a given time in SKI-I-Asp treated mice also
suggests that SKI-1-Asp dose may be increased to match compa-
rable Cmax of SKI-I in the blood after 2 h, as observed for SKI-I
treated mice, without causing additional systemic toxicity, thus
lowering the dosing frequency.
A
90
80
70
60
50
40
30
20
10
0
SKI-I
SKI-I-Asp
DMSO 0.25 uM 1.25 uM 5 uM 25 uM
At lower doses (i.e., <1.25
were equally effective at inhibiting SphK1 as the parent SKI
compounds, while at higher doses (ꢄ5 M), the aspirinyl deriva-
mM), aspirinyl conjugates 10 and 14
B
m
80
70
60
50
40
30
20
10
0
SKI-II
SKI-II-Asp
tives were less effective than the parent SKI compounds (Fig. 4).
Whether this is due to the fact that either aspirinyl-linked SKI
results in less affinity for SphK1 or less availability of the active SKI
(as indicated in Fig. 2) due to slow conversion of aspirinyl deriva-
tives to active SKI by cleavage of the ester bond, is not known at this
time. However, the fact that the SKI-I-Asp (10) readily cleaves to
SKI-I in vivo, a little lower SphK1 activity shown by aspirinyl analogs
at higher concentrations would not be of much importance. In
either case, it is clear that aspirinyl-linked SKI compounds are
potent inhibitors of SphK1 demonstrating the feasibility of parent
SKI compounds modifications to improve their cytotoxic potency
without the loss of specificity towards SphK1.
-10
DMSO 0.25 uM 1.25 uM 5 uM 25 uM
Fig. 4. SphK1 activity assay: HeLa cells were treated for 24 h with either DMSO
(vehicle) or increasing concentrations of SKIs or their aspirinyl derivatives. In vitro
SphK1 activity assays were performed on 10
m
g of cytosol in triplicate (n ¼ 3).
4. Conclusion
and ovarian cancer cells. However, SKI-II and SKI-II-Asp (14) were
equally effective in killing various cancer cells e.g. brain, ovarian,
lung, breast, and pancreatic cancer cells. This suggested that the
observed activity of aspirinyl derivatives may actually be the
activity of parent SKIs that get cleaved during the cells treatment.
On the other hand equal cytotoxicity could as well be the result of
a coincidental similar activity of the SKI-Asp derivatives. To deter-
mine if the activity was due to the aspirinyl derivatives themselves
or due to the release of parent SKI inhibitors, metabolism studies of
the aspirinyl derivatives SKI-I-Asp (10) and SKI-II-Asp (14) were
conducted in culture conditions in the presence as well as in the
absence of serum. Incubation of 10 and 14 in cell culture media
(DMEM supplemented with 10% FBS, without cells) indicated that
both the aspirinyl analogs cleaved over time to release the corre-
sponding parent compounds but about 18e25% of the Asp analogs
still remained at the end of 72 h treatment (the time used for the
treatment of cells in SRB assay). This suggested that similar cyto-
toxicity observed for aspirinyl conjugates 10 and 14 and corre-
sponding parent compounds in SRB assay, may either be due to an
enzyme secreted from the cells to the culture medium that cata-
lyzed the cleavage further to release the parent SKIs or SKI-Asp
analogs themselves contribute to the overall cytotoxicity of these
compounds. It is important to note that no degradation of aspirinyl
analogs was observed in the serum free media (DMEM, without 10%
FBS or cells) indicating that degradation in media with serum is due
to some secreted cellular enzymatic activity.
In summary, practical syntheses of SKI-I and SKI-II and their
aspirinyl derivatives were accomplished in high overall yields. The
syntheses are not only short and efficient but also provide
a strategy to procure additional analogs for further structur-
eeactivity studies. Both the parent SKIs and their aspirinyl deriv-
atives showed similar cytotoxicity in SRB assays in most cases
except that in lung, breast and pancreatic cancer cell lines, SKI-I-
Asp was w3 times more effective as compared to SKI-I. The SphK1
activity of parent SKIs and their corresponding aspirinyl conjugates
was comparable at lower doses while at higher doses of 5 and
25
mM, the aspirin analogs showed lower activity. The degradation
pattern under the cell culture media conditions indicated that
w75e82% of the aspirinyl derivatives were cleaved to the corre-
sponding parent compounds by the end of the assays (72 h). In vivo
however, the SKI-I-Asp was completely metabolized to SKI-I within
1 h. Collectively, the results suggest that aspirinyl prodrug conju-
gates of the SKI-I and SKI-II may be effective surrogates for the
parent compounds. Further studies are, however, warranted to
determine the efficacy of these compounds.
5. Experimental section
5.1. Chemistry
Melting points were recorded on a Fischer-Johns melting point
apparatus and are uncorrected. 1H and 13C NMR spectra were
recorded using a Bruker Avance 500 MHz spectrometer. Chemical
The degradation of aspirinyl analogs was not complete under in
vitro culture conditions. An ideal prodrug, once administered, is
metabolized in vivo to an active metabolite. Therefore, to establish
the cleaving ability of aspirinyl group from the SKI-Asp prodrugs in
vivo, we evaluated this phenomenon in mice with SKI-I-Asp.
Interestingly, no trace of SKI-I-Asp was observed just after 1 h of
administration. The concentration of SKI-I in the serum in SKI-I-Asp
injected mice, peaked at the same time (2 h after injection) but was
lower as compared to SKI-I injected mice. The lower than expected
concentration of SKI-I, the lack of presence of SKI-I-Asp in the
shifts (d) were reported in parts per million (ppm) downfield from
the internal standard. The signals are quoted as s (singlet),
d (doublet), t (triplet), m (multiplet) and dt (doublet of triplet).
High-resolution MS (EI) were determined at the Chemistry
Instrumentation Center, State University of New York at Buffalo, NY.
Thin-layer chromatography (TLC) was developed on aluminum-
supported pre-coated silica gel plates (EM industries, Gibbstown,
NJ). Column chromatography was conducted on silica gel (60e200

4154
A.K. Sharma et al. / European Journal of Medicinal Chemistry 45 (2010) 4149e4156
mesh). The decomposition of aspirinyl derivatives was analyzed by
M) Bondclone
Method 2: To a solution of methyl ester 2 (0.45 g, 1.76 mmol) in
EtOH (20 mL) was added hydrazine hydrate (0.055 mL, 12.3 mmol)
and the reaction mixture was refluxed for 6 h under nitrogen. The
reaction mixture was cooled to room temperature and the
precipitated white solid was filtered and washed with mixture of
ethanol:hexanes (1:9) to yield 0.35 g (79%) of 3 as a white solid; mp
HPLC (Waters Breeze System) on a 4.6 ꢁ 250 mm (5
m
C18 reverse-phase column (Phenomonex) using solvent system 1
[solvent A (0.1% TFA in water) and solvent B (acetonitrile), using
a linear gradient program from A:B (70:30) to 50% B over 20 min, to
65% B next 5 min, to 70% B next 20 min, to 100% B next 5 min] or
solvent system 2 (A: Water, B: Methanol; 50% B to 75% B in
0e10 min, 75% B to 100% B in 10e45 min, 100% B in 45e50 min).
>270 ꢂC; 1H NMR (MeOH-d4):
d 9.31 (1H, s), 8.82e8.74 (4H, m),
8.32e8.38 (2H, m), 8.1 (1H, s); HRMS (ESI) calcd for C14H12N4O.Naþ,
275.0903; found, 275.0904.
5.1.1. Methyl-(2-hydroxy-4-naphthalen-2-yl-4-oxo-but-2-enoic)
acetate (2)
5.1.6. 5-Naphthalen-2-yl-1H-pyrazol-3-carboxylic acid (2-
To a suspension of sodium hydride (4.17 g, 116.2 mmol, 60% oil
dispersion) in benzene (150 mL) at 0 ꢂC was added methanol (5 mL)
dropwise. To this cold mixture was added a solution of 2-aceto-
naphthone (10 g, 58.7 mmol) and dimethyl oxalate (13.88 g,
117.5 mmol) in benzene (150 mL) dropwise. The mixture was
allowed to warm to room temperature, stirred overnight, quenched
with 1 N HCl solution, and filtered. The residue was purified
through silica gel column chromatography using methylene chlo-
ride:hexanes (7:3) as an eluent to yield 7.5 g (50%) of 4 as a yellow
hydroxynaphthalen-1-ylmethylene)-hydrazide (4)
To a solution of hydrazide 3 (1.0 g, 4.0 mmol) in DMSO (50 mL)
was added 2-hydroxy-1-naphthaldehyde (1.02 g, 5.9 mmol) in
DMSO (10 mL) followed by a catalytic amount of acetic acid. The
reaction mixture was stirred at RT overnight, poured in water
(100 mL), and the solid thus formed was filtered and washed with
methylene chloride:hexanes (7:3). The crude product was purified
by crystallization from THFehaxenes mixture to afford 1.5 g (94%) of
1 as a pale yellow solid; mp 182e183 ꢂC; 1H NMR (DMSO-d6):
d 14.1
solid; mp 104e106 ꢂC; 1H NMR (Acetone-d6):
d
8.83 (1H, s), 8.21
(1H, s), 12.9 (1H, s), 12.3 (1H, s), 9.65 (1H, s), 8.25 (1H, s), 8.07e7.9
(7H, m), 7.64e7.56 (3H, m), 7.45e7.41 (2H, m), 7.25(1H, d, J ¼ 7.5 Hz);
(1H, d, J ¼ 8.0 Hz), 8.15e8.06 (2H, m), 8.04 (1H, d, J ¼ 2.0 Hz), 7.73
(1H, t, J ¼ 6.4 Hz), 7.68 (1H, t, J ¼ 8.0 Hz), 7.35 (1H, s), 3.95 (3H, s);
HRMS (ESI) calcd for C15H12O4.Hþ, 257.0811; found, 257.0815.
13C NMR (DMSO-d6):
d 158.4, 147.5, 133.5, 133.2, 132.2, 129.5, 129.2,
128.6,128.3,128.2,127.3,127.1,124.6,124.1,124.0,121.4,119.4,109.1;
HRMS (ESI) calcd for C25H18N4O2.Hþ, 407.1503; found, 407.1510.
5.1.2. 2-Hydroxy-4-naphthalen-2-yl-4-oxo-but-2-enoic acid (5)
To a suspension of 2 (13.0 g, 50.7 mmol) in 1,4-dioxane (200 mL)
at room temperature was added 1 M HCl (200 mL) and the mixture
was refluxed for 4 h. The reaction mixture was cooled to room
temperature and the solvent was removed under reduced pressure.
The crude yellow solid thus precipitated was filtered, and washed
with water and diethyl ether. The crude solid was crystallized from
toluene and ethyl acetate to yield 12.0 g (97%) of acid 5 as a yellow
5.1.7. 1-Formyl-2-(2-acetoxybenzoyloxy)naphthalene (9)
To a solution of acetylsalicyloyl chloride (1.5 g, 8.7 mmol) in dry
THF (10 mL) was added dropwise to a mixture of 2-hydroxynaph-
thaldehyde (1.0 g, 5.81 mmol) and K2CO3 (1.2 g, 8.7 mmol) in THF
(40 mL) at 0 ꢂC under nitrogen atmosphere. The reaction mixture
was warmed to room temperature and stirred vigorously for 16 h.
The mixture was poured into water (40 mL) and extracted with
ethyl acetate (3 ꢁ 50 mL). The combined organic phases were
washed with brine (100 mL), dried (MgSO4), and evaporated in
vacuo. The crude product thus obtained was purified through
column chromatography over silica using methylene chloride:
hexanes (1:1) mixture to afford 1.3 g (72%) of 9 as a white solid; mp
solid; mp 170e171 ꢂC; 1H NMR (Acetone-d6):
d 8.85 (1H, s), 8.21
(1H, d, J ¼ 7.0 Hz), 8.16e8.14 (2H, m), 8.06 (1H, d, J ¼ 7.0 Hz), 7.73
(1H, t, J ¼ 6.5 Hz), 7.68 (1H, t, J ¼ 7.0 Hz), 7.35 (1H, s); MS (ESI) 243
(Mþ þ 1).
5.1.3. 5-Naphthalen-2-yl-1H-pyrazole-3-carboxylic acid (6)
117e119 ꢂC; 1H NMR (DMSO-d6):
d 10.66 (1H, s), 9.06 (1H, d,
A mixture of 5 (12.3 g, 49.6 mmol), anhydrous hydrazine (3.97 g,
124.0 mmol) and absolute EtOH (250 mL) was heated to reflux for
30 min. The precipitated hydrazide was filtered, washed with
diethyl ether and dried under vacuum to give 6 (8.7 g, 74%) as
J ¼ 8.5 Hz), 8.41 (1H, d, J ¼ 8.5 Hz), 8.33 (1H, d, J ¼ 7.5 Hz), 8.12 (1H,
d, J ¼ 8.5 Hz), 7.84 (1H, t, J ¼ 8.0 Hz), 7.8e7.74 (m, 1H), 7.69 (1H, t,
J ¼ 8.0 Hz), 7.57e7.54 (m, 2H), 7.39 (1H, d, J ¼ 8.0 Hz), 2.25 (3H, s);
MS (ESI) 357 (Mþ þ Na).
a white solid; mp 265e267 ꢂC; 1H NMR (DMSO-d6):
d 8.4 (1H, s),
8.03e7.93 (4H, m), 7.51e7.57 (2H, m), 7.35 (1H, s); HRMS (ESI) calcd
5.1.8. SKI-I-Asp (10)
for C14H10N2O2.Naþ, 261.0634; found, 261.0648.
To a solution of 3 (0.396 g, 1.57 mmol) in DMSO (20 mL) was
added salicyloyl derivative 9 (0.5 g, 1.57 mmol) followed by a cata-
lytic amount of acetic acid. The mixture was stirred overnight at
room temperature, poured into water (30 mL) and extracted with
ethyl acetate. The organic layer was washed with brine (3 ꢁ 30 mL),
dried (MgSO4), and concentrated in vacuo. The residue was purified
by silica gel column chromatography using a mixture of methylene
chloride and hexanes (3:7) to afford 0.6 g (67%) of 10 as a white
5.1.4. 5-Naphthalen-2-yl-1H-pyrazole-3-carbonyl chloride (7)
To a suspension of 6 (8.0 g, 33.6 mmol) in dry benzene (150 mL)
under nitrogen was added thionyl chloride (4.9 mL, 67.2 mmol)
dropwise with stirring at room temperature. The reaction mixture
was refluxed for 5 h and the volatiles were removed under reduced
pressure. The crude solid thus obtained was triturated with diethyl
ether and filtered to yield 7 (7.8 g, 91%) as a yellow solid; mp 185 ꢂC
solid; mp 183e185 ꢂC; 1H NMR (DMSO-d6):
d 9.34 (1H, s), 9.11 (1H,
(dec.); 1H NMR (DMSO-d6):
d
8.42 (1H, s), 8.03e7.93 (4H, m),
s), 8.41 (2H, d, J ¼ 7.5 Hz), 8.26e7.96 (6H, m), 7.84 (1H, t, J ¼ 7.5 Hz),
7.59e7.51 (2H, m), 7.36 (1H, s); HRMS (EI) calcd for C14H9ClN2O,
256.0398; found, 256.0406.
7.74 (1H, t, J ¼ 7.0 Hz), 7.66 (1H, t, J ¼ 7.0 Hz), 7.60e7.57 (3H, m),
7.44e7.39 (3H, m), 2.25 (3H, s); 13C NMR (DMSO-d6):
d 169.8, 163.1,
158.7, 151.3, 149.1, 147.3, 144.3, 143.7, 135.7, 133.4, 133.2, 132.9, 132.3,
131.0, 129.2, 129.1, 128.6, 128.5, 128.2, 127.4, 127.2, 127.0, 126.8,
126.7,126.4,124.7,124.6,124.1,122.4,122.2,121.5,104.5, 21.2; HRMS
(ESI) calcd for C34H24N4O5.Naþ, 591.1639; found, 591.1624.
5.1.5. 5-Naphthalen-2-yl-1H-pyrazol-3-carboxylic acid hydrazide
(3)
Method 1: Anhydrous hydrazine (1.69 mL, 54.7 mmol) was
added to a suspension of 7 (7.0 g, 27.3 mmol) in EtOH (250 mL) and
the mixture was refluxed overnight. The precipitated white solid
was filtered, washed with EtOH and dried under vacuum to yield
6.3 g (91%) of 3 as a white solid.
5.1.9. 4-[4-(4-Chlorophenyl)thiazol-2-ylamino]phenol (13)
To a solution of haloketone (1.4 g, 5.99 mmol) in ethanol (20 mL)
was added 1-(4-hydroxyphenyl)-2-thiourea (1.0 g, 5.99 mmol) and

A.K. Sharma et al. / European Journal of Medicinal Chemistry 45 (2010) 4149e4156
4155
the reaction mixture was refluxed for 2 h. The hydrochloride
formed was filtered and washed with ether and crystallized from
aqueous ethanol. The hydrochloride was boiled with excess
ammonium hydroxide. The solid thus obtained was filtered,
washed with water and crystallized from 95% alcohol to yield 1.14 g
(60%) of 13 as a white crystalline solid; mp 179e180 ꢂC; 1H NMR
510 nm. Based on the absorbance results, IC50 for each SKI
compounds were determined using the Prism software (GraphPad
Prism 5.1).
5.3. In vitro SphK1 activity assay
(DMSO-d6):
d
9.94 (1H, s), 9.14 (1H, s), 7.91 (2H, d, J ¼ 9.0 Hz),
HeLa uterine cancer cells, were preincubated for 24 h with
increasing concentrations of SKIs or their derivatives with DMSO as
a vehicle control. Cells were flash frozen in liquid nitrogen and cell
lysates were centrifuged at 100,000g for 30 min at 4 ꢂC. Superna-
7.48e7.45 (4H, m), 7.29 (1H, s), 6.75 (2H, d, J ¼ 9.0 Hz); 13C NMR
(DMSO-d6):
d
164.9, 152.9, 149.3, 134.0, 133.7, 132.3, 129.1 (2ꢁ
CHarom), 127.8 (2ꢁ CHarom), 119.8 (2ꢁ CHarom), 116.0 (2ꢁ CHarom),
103.1; HRMS (ESI) calcd for C15H11ClN2OS.Hþ, 303.0353; found,
303.0355.
tants were removed and 10
a standard in vitro SphK1 activity assay as follows. Briefly, samples
were combined with 50 -erythro-sphingosine, 200 M ATP
and 2 Ci [ L final reaction volume of SKAAB
32P] ATP in a 100
buffer (20 mM Tris pH 7.4, 1 mM -mercaptoethanol, 1 mM EDTA,
mg of total protein was added to
mM
D
m
5.1.10. SKI-II-Asp (14)
m
g-
m
A solution of acetylsalicyloyl chloride (0.98 g, 4.5 mmol) in dry
THF (5 mL) was added dropwise to a mixture of 2 (1.0 g, 3.3 mmol)
and Et3N (0.45 g, 4.5 mmol) in THF (20 mL) at 0 ꢂC under nitrogen
atmosphere. The reaction mixture was warmed to room tempera-
ture and stirred vigorously overnight. The mixture was poured into
water (50 mL) and extracted with ethyl acetate (3 ꢁ 50 mL). The
combined organic phases were washed with brine (100 mL), dried
(MgSO4), and evaporated in vacuo. The crude product thus obtained
was purified through silica gel column chromatography using
methylene chloride:hexanes (3:1) mixture to afford 1.2 g (70.7%) of
b
0.1% Triton X-100, 1 mM Na3VO4, 15 mM NaF, 0.5 mM 4-deoxy-
pyridoxine) for 30 min at 37 ꢂC with shaking. Kinase reactions were
terminated by the addition of 10
were extracted by the addition of 400
100:200 v/v and 125 L Chloroform and 125
phase containing lipids was dried down under nitrogen stream.
Samples were then resuspended in 30 L chloroform and applied to
m
L 6 N HCl and the labeled lipids
L of Chloroform/MeOH
L 1 M KCl. The organic
m
m
m
m
a Silica Gel TLC plate (Whatman, Florham Park, NJ) and the lipids
were separated using a butanol:water:acetic acid 3:1:1 v:v:v
solvent system. The plates were analyzed by X-ray exposure and
the region of the TLC plate corresponding to the Rf value (0.32) of
sphingosine-1-phosphate (S1P) was scraped and the amount of S1P
formed was determined by liquid scintillation counting [38].
14 as a white solid; mp 150e152 ꢂC; 1H NMR (DMSO-d6):
d 8.25 (1H,
dd, J ¼ 8.0 and 2.0 Hz), 7.81 (2 H, d, J ¼ 9.0 Hz), 7.68 (1H, dt, J ¼ 8.0
and 2.0 Hz), 7.50 (1H, d, J ¼ 9.0 Hz), 7.44 (1H, d, J ¼ 7.5 Hz), 7.40 (1H,
d, J ¼ 8.5 Hz), 7.21 (3 H, d, J ¼ 8.5 Hz), 76.84 (1 H, s), 2.35 (3H, s); 13C
NMR (DMSO-d6):
d 169.7, 163.7, 163.5, 150.8, 149.3, 144.6, 139.6,
135.5, 133.8, 132.5, 132.2, 129.1 (2ꢁ CHarom), 127.9 (2ꢁ CHarom),
127.0, 124.6,122.9, 122.6 (2ꢁ CHarom), 118.2 (2ꢁ CHarom), 104.4, 21.2;
HRMS (ESI) calcd for C24H17ClN2O4S.Hþ, 465.0670; found,
465.0680.
5.4. Degradation of SKI-Asp analogs
1 mg/mL of each compound in DMSO was dissolved in 5 mL 10%
FBS supplemented DMEM or serum free DMEM media in 15 mL
conical tubes. The tubes were placed in a shaking incubator at 37 ꢂC.
At 0, 2, 16, and 72 h samples were taken out, filtered, and analyzed
5.2. Cell lines and culture conditions
using Waters HPLC on Bondclone 10 mm C18 column and solvent
The following cell lines were employed for cytotoxicity and in
vivo inhibitory studies: HeLa (ATCC: CCL2), U87MG (ATCC: HTB-14),
H460 (ATCC: HTB-177), H226 (ATCC: CRL-5826), H441 (ATCC: HTB-
174), A549 (ATCC: CCL-185), MDA-MB-231 (ATCC: HTB-26), and
MiaPaCa2 (ATCC: CRL-1420). These cell lines were cultured in
DMEM supplemented with 10% FBS at 37 ꢂC in a 5% CO2 atmo-
sphere. Additionally, immortalized normal human mammary
epithelial cell line MCF-10A (ATCC: CRL-10317), and mouse
embryonic fibroblast, MEF (Dr. H.G. Wang, Pennsylvania State
College of Medicine) were used as normal control cell lines.
system 1 or solvent system 2. The percentage of SKIs and their
aspirin conjugates peaks was calculated for each time point and
plotted.
5.5. Pharmacokinetic property studies
To determine pharmacokinetic properties of SKIs and their
aspirinyl-linked analogs, we intraperitoneally injected mice (12e15
weeks old) with SKI compounds at concentration of 30 mg/kg.
Subsequently, whole blood was collected from mice at various time
points (1, 2, 4, 8 and 16 h). Collected blood was coagulated and
serum was collected by centrifugation. Subsequently, serum was
subjected to HPLC using solvent system 1 or 2 to determine the
levels of SKIs or their aspirinyl-linked analogs. The amount of SKI
compounds in serum was calculated by area under the curve using
a standard curve and plotted against time.
5.2.1. Cytotoxicity assays
Sulforhodamine B (SRB) colorimetric assay was used to deter-
mine the cytotoxic effects of SKIs and their aspirinyl-linked SKI
compounds according to the methods described by Vichai et al.,
[37] with minor modifications. Briefly, all cancer cell lines (5 ꢁ 103)
were plated in a 96 well plate and 24 h later triplicate wells were
exposed to DMSO (vehicle) or increasing concentrations (0.001,
5.6. Statistical analysis
0.005, 0.025, 0.1, 0.5, 1.0, 2.5, 5.0, 25.0, and 50.0 mM) of SKIs and
their aspirinyl prodrugs for 72 h. After 72 h of treatments, cells were
fixed in 10% trichloroacetic acid (TCA) for 1 h, TCA was discarded
and plates were extensively washed with slow-running tap water.
After air-drying the plates overnight at room temperature, cells in
Statistical analysis was undertaken using the One-way ANOVA
followed by an appropriate post hoc test. Results were considered
significant at a P-value of <0.05.
each well were stained with 100
mL of 0.057% (wt/vol) of SRB
solution for 30 min and then the plates were rinsed four times with
1% (vol/vol) acetic acid to remove unbound dye. Plates were then
allowed to dry at room temperature, and subsequently cells stained
with SRB were incubated with 10 mM Tris base solution. After 1 h of
solubilizing SRB stain, the absorbance (O.D.) was measured at
Acknowledgement
This study was supported by the Penn State Hershey Cancer
Institute of the Penn State College of Medicine. The authors thank
Dr. Jyh-Ming Lin, Solution Phase NMR Facility at Core Research

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A.K. Sharma et al. / European Journal of Medicinal Chemistry 45 (2010) 4149e4156
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Facilities of the Penn State College of Medicine, for recording of
NMR spectra.
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