Synthesis and antiproliferative activity of imidazole and imidazoline analogs for melanoma

Article abstract of DOI:10.1016/j.bmcl.2008.04.073

We have previously reported substituted 2-aryl-thiazolidine-4-carboxylic acid amides as potent and selective antiproliferative agents for melanoma. To understand the importance of the thiazolidine ring and to reduce potential complications associated with the two chiral centers, we designed and synthesized sets of new analogs by modifying this ring. These new analogs were tested in two melanoma cell lines and fibroblast cells (negative controls). Compared with the older analogs containing the thiazolidine ring, these new analogs have lower potency in general, but some of these analogs still have very good selectivity. These structure-activity studies indicated that the thiazolidine ring is very critical for the activity for these series of compounds.

Full text of DOI:10.1016/j.bmcl.2008.04.073

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3183–3187
Synthesis and antiproliferative activity of imidazole
and imidazoline analogs for melanoma
Jianjun Chen,a, Zhao Wang,a, Yan Lu,a James T. Dalton,b
a,
Duane D. Millera and Wei Li
*
aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center,
Memphis, TN 38163, USA
bDivision of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
Received 16 January 2008; revised 24 April 2008; accepted 28 April 2008
Available online 1 May 2008
Abstract—We have previously reported substituted 2-aryl-thiazolidine-4-carboxylic acid amides as potent and selective antiprolif-
erative agents for melanoma. To understand the importance of the thiazolidine ring and to reduce potential complications associ-
ated with the two chiral centers, we designed and synthesized sets of new analogs by modifying this ring. These new analogs were
tested in two melanoma cell lines and fibroblast cells (negative controls). Compared with the older analogs containing the thiazol-
idine ring, these new analogs have lower potency in general, but some of these analogs still have very good selectivity. These struc-
ture–activity studies indicated that the thiazolidine ring is very critical for the activity for these series of compounds.
Ó 2008 Elsevier Ltd. All rights reserved.
While most early stage melanoma can be cured surgi-
cally, melanoma cells that have migrated to other organs
are notoriously resistant to all existing treatments. The
5-year survival rate for patients with metastatic mela-
noma is less than 15%.1–4
We recently reported that substituted 2-aryl-thiazoli-
dine-4-carboxylic acid amides are promising potent
and selective antiproliferative agents for melanoma.14,15
Compared with Sorafenib that has been used in clinical
trials for melanoma, this class of compounds is more po-
tent and more selective based on in vitro cell assays. We
demonstrated that the activity of these compounds
strongly depends on the length of the side chain and
the nature of the substitutions on the aromatic ring. In
addition, we found that the chirality at position 4 in thi-
azolidine analogs is not critical for their activity.14 How-
ever, the importance of chirality at position 2 is
unknown, because all our previous compounds were
tested as diastereomers. Separation of these stereoiso-
mers by chromatographic methods turned out to be ex-
tremely difficult. We asked for assistance from scientists
from Chiralcel Technologies who tried both normal-
phase and reverse-phase conditions with their nine chiral
columns, but none of the combinations could separate
these stereoisomers satisfactorily (unpublished data).
In the arena of chemotherapeutic agents for advanced
melanoma, Dacarbazine (DTIC) is the only FDA-ap-
proved drug in the past 30 years. However, it provides
only less than 5% of complete remission in patients.3,5
Significant research and effort have resulted in many
promising drug candidates,6 either small molecules or
biological agents based on initial small clinical trials;
unfortunately, to date none has demonstrated a clear
advantage over DTIC in subsequent large, randomized
clinical trials.1,7–11 With the incidence of melanoma rap-
idly rising in the United States and other developed
countries, there is an urgent need to develop more effec-
tive drugs.6,12,13
Converting the thiazolidine ring to an imidazoline or
imidazole ring represents a very productive approach.
First, the nitrogen-containing ring would be very stable.
Imidazoline and imidazole rings are important biologi-
cal building blocks and are present in many existing
drugs.16 Second, the imidazoline ring contains only
one chiral center, and the imidazole ring contains no
Keywords: Melanoma; Thiazolidine; Imidazole analogs; Imidazoline
analogs; Structure–activity relationships.
*
Corresponding author. Tel.: +1 9014487532; fax: +1 9014486828;
e-mail: wli@utmem.edu
These authors equally contributed to the paper.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.073

3184
J. Chen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3183–3187
chiral center at all. These attributes may alleviate or
even eliminate the need for future chiral separations.
pounds 5–11 by using EDC/HOBt as coupling
reagents.19
In this letter, we report focused structure–activity rela-
tionship studies for the central thiazolidine ring, the ef-
fect of spacers, and an attempt to remove the chiral
centers. Specifically, we modified the structures by (a)
replacing sulfur with nitrogen or carbon to understand
the importance of the heteroatom in the ring, (b) intro-
ducing one or two double bonds in the ring (imidazo-
line and imidazole) to understand the importance of
the chiral centers, and (c) introducing various spacers
between the five-member ring and the aromatic ring
to understand the relative spatial arrangements for
the two rings.
The general synthesis of 2-aryl-imidazoline-4-carboxylic
acid amides is shown in Scheme 2.
Compounds 15–22 were synthesized under reported con-
ditions.20 The intermediate imidate was prepared from
corresponding acetonitrile or benzonitrile.21,22 Then imi-
date was cyclized with 1,2-diaminepropionic acid to give
the imidazoline monoacid.20 This monoacid coupled
with amine to yield the final compounds.
The general synthesis of 2-aryl-thiazolidine-4-carboxylic
acid amides with spacers is shown in Scheme 3. Com-
pounds 23–29 were synthesized by known procedures;14
The general synthesis of 2-aryl-imidazole-4-carboxylic
acid amides is shown in Scheme 1.
(p-acetylamino)-phenylactaldehyde
was
prepared
from (p-acetylamino)-phenylethanol by known proce-
dures;23 (p-acetylamino)-phenylethanol was synthesized
from (p-amino)-phenylethanol.24
Briefly, L-tartaric acid was converted to a dinitrate
that reacted with phenylaldehyde to give 2-phenyl
imidazole 4,5-dicarboxylic acid (compound 3) under
reported conditions.17 The diacid 3 was converted to
monoacid 4 through standard procedures,18 and this
monoacid was coupled with alkylamines to give com-
Compound 30, in which sulfur was replaced by a car-
bon, was obtained by coupling reaction with (2S, 5R)-
5-phenyl-pyrrolidine-1, 2-dicarboxylic acid 1-tert-butyl
ester, and hexadecylamine under EDC/HOBt condi-
R
OH
ONO2
COOH
CHO
H
N
COOH
a
b
HOOC
HOOC
COOH
+
N
OH
ONO2
COOH
R
1
3
2
R
R
H
H
c
d
N
R1R2NH
N
+
N
N
NR1R2
COOH
O
5-11
4
Scheme 1. Reagents and conditions: (a) HNO3, H2SO4; (b) NH4OH, H2O; (c) Ac2O, reflux; (d) EDC, HOBt, Et3N, DMF, 0 °C–rt.
R
R
NH
a
(CH2)n
(CH2)n
N
HCl
OEt
R=NH2
R=-NHC(O)CH3
d
b
O
NR1R2
COOH
N
R
R
N
c
(CH2)n
(CH2)n
N
H
N
H
15-22(for 15-21,n=0; for 22,n=1)
Scheme 2. Reagents and conditions: (a) HCl, EtOH, ether, 0 °C; (b) MeOH, Et3N, (S)-2,3-diaminopropanoic acid monohydrochloride, reflux; (c)
R1R2NH, EDC, HOBt, Et3N, DMF, 0 °C–rt; (d) Ac2O, MeOH, rt.

J. Chen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3183–3187
3185
tions, then deprotection of Boc with TFA yielded 30
(Scheme 4).
Replacing the imidazole ring with an imidazoline ring
slightly improved the activity in general (compounds
15–19), with substantial improvement when trimethoxyl
or amino acetyl substitutions were added (compounds
20–21). The structure activity for the side chain was sim-
ilar to that of the thiazolidine analogs, with a saturated
C14–C16 chain or an unsaturated C18 chain to be opti-
mal for activity against melanoma cells. Similarly, elec-
tronic donating substitutions on the paraposition of
the aromatic ring increased activity (compounds 20–
21). Replacing sulfur with carbon in the five-member
ring produced an inactive compound (30). Collectively,
these results indicate that the thiazolidine ring is critical
for the activity of this series of compounds.
We examined the antiproliferative activity of these imi-
dazoline and imidazole analogs in two melanoma cell
lines (human A375 cells and mouse B16 cells) and in a
fibroblast cell line. The activity on fibroblast cells served
as a control to determine the selectivity of these com-
pounds against melanoma. We used the standard sulfo-
rhodamine B (SRB) assay, which has been shown to
provide very reliable results based on the literature25
and our own work.14,15 Briefly, cells seeded in round-
bottom 96-well plates were exposed to a wide range of
concentrations for 48 h before they were fixed with
10% trichloroacetic acid. After they were washed five
times with water and air-dried overnight, cells were
stained with SRB solution, and total proteins were mea-
sured at 560 nm with a plate reader. IC50 (i.e., concen-
tration that inhibited cell growth by 50% of DMSO-
treated controls) values were obtained by nonlinear
regression analysis with GraphPad Prism (GraphPad
Software, San Diego, CA).
When a spacer was inserted between the imidazoline/thi-
azolidine ring and the phenyl ring (compounds 22–29),
there is no noticeable improvement in their activity
(compound 20 vs compound 22). Increasing the size of
the spacer by replacing one hydrogen in the methylene
group with a methyl group reduced the activity (com-
pound 23 vs 26, and compound 24 vs 27). Both of these
results seemed to indicate detrimental effects of spacers
for the activity. Finally, replacing the side chain with a
fluorene group resulted in complete loss of activity
(compound 23 vs 25). However, with proper parasubsti-
tution on the aromatic ring, the activity dramatically im-
proved (compound 25 vs compound 29) for this series of
compounds. On the other hand, with a C16 side chain,
the substitution on the phenyl ring had the opposite ef-
fect (compound 23 vs 26) in sharp contrast to the non-
chain analogs such as 29 and may indicate a different
mechanism of action when the long side chain is re-
placed by a conjugated ring system.
The ability of these new analogs to inhibit the growth of
melanoma cancer cell lines and fibroblast cells is sum-
marized in Table 1. Because of the lack of bioactivation
in vitro, DTIC is inactive as expected. We selected
Sorafenib (Bay43-9006) as the reference standard, be-
cause it has been used extensively in clinical trials for
melanoma.6,26 At this early stage, imidazoline analogs
were used as a diastereomeric mixture to select the most
promising compounds for further development.
In general, none of the new analogs is as active as the
lead compound. Although they do not contain any chi-
ral centers, analogs containing an imidazole ring (com-
pounds 5–11) did not have activity below 10 lM.
In conclusion, we synthesized new thiazolidine analogs,
based on our previous studies, by focusing on the struc-
Boc
H
N
R2
COOH
R2
R
R2
COOH
R
R
N
a
b
C
H
CH CHO
C
H
S
S
e,f
c
R=-NHC(O)CH3,R2=H
R
O
O
H
N
Boc
N
R2
R1
R2
R1
N
R
d
N
C
H
H
C
H
S
H
S
23-29
Scheme 3. Reagents and conditions: (a) L-cysteine, EtOH, H2O, rt; (b) (Boc)2O, NaOH, 1,4-dioxane, H2O; (c) R1NH2, EDC, HOBt, Et3N, CH2Cl2,
0 °C–rt; (d) CF3COOH, CH2Cl2; (e) 4-aminophenethyl alcohol, acetic anhydride, MeOH; (f) Dess-Martin reagent, CH2Cl2.
CONHC16H33
NH
CONHC16H33
NBoc
COOH
NBoc
a
b
30
Scheme 4. Reagents and conditions: (a) C16H33NH2, EDC, HOBt, Et3N, CH2Cl2; (b) CF3COOH, CH2Cl2 (44.7%, 2 steps).

3186
J. Chen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3183–3187
Table 1. Antiproliferative activity of imidazoline and imidazole analogs and their comparison with that of Sorafenib and the lead thiazolidine
compounds (ND: not detected)
Structure
Compound
R
R1
R2
IC50 SEM (lM)
A375
B16-F1
Fibroblast
5
H
H
H
H
H
H
H
n-C10H21
n-C12H25
H
H
H
H
H
H
H
>30
>30
>30
ND
ND
H
N
6
ꢀ30
7
n-C14H29
n-C16H33
18.7 1.0
16.5 0.6
20.7 0.8
16.1 0.5
>30
38.2 2.1
39.7 1.5
>30
42.7 2.2
27.9 1.0
47.4 1.3
23.9 1.2
ND
8
NR1R2
N
R
9
n-C18H37
(E)-Octadec-8-enyl
p-Bromophenyl
O
10
11
31.6 1.1
>30
15
16
17
18
19
20
21
H
n-C14H29
n-C16H33
n-C18H37
H
7.1 0.1
10.5 0.2
54.3 0.9
8.3 0.2
>20
7.2 0.2
5.7 0.3
50.5 1.3
12.0 0.2
>20
17.3 2.4
59.6 1.2
111.4 3.8
24.6 2.8
>20
O
H
H
H
H
NR1R2
N
R
R
H
(E)-Octadec-8-enyl
OCH3
n-C16H33
H
3,4,5-Trimethoxyl
3,4,5-Trimethoxyl
p-NHCOCH3
CH3
H
N
H
4.1 0.6
4.6 0.1
3.8 0.1
2.4 0.1
13.3 1.2
14.8 1.9
n-C16H33
H
O
NR1R2
N
H2
C
22
3,4,5-Trimethoxyl
n-C16H33
H
3.9 0.1
4.0 0.1
15.8 2.1
N
H
23
24
25
26
27
28
29
H
n-C16H33
H
8.1 1.0
10.3 0.5
174 11
13.1 0.5
21.2 0.9
191 14
6.1 0.1
6.5 0.1
11.6 0.2
118 8.0
12.2 0.3
16.5 1.0
171 21
4.0 0.1
15.3 0.9
15.5 1.9
>100
O
H
(E)-Octadec-8-enyl
2-Fluorene
n-C16H33
H
H
N
R2
R1
H
H
R
N
H
CH3
CH3
H
55.7 7.3
237 39
>100
C
H
H
S
H
(E)-Octadec-8-enyl
n-C16H33
2-Fluorene
p-NHCOCH3
p-NHCOCH3
H
15.3 0.5
O
n
-C16H33
NH
NH
30
13.7 0.4
11.5 0.3
46.6 1.5
DTIC
Sorafenib
>100
5.4 0.5
>100
4.9 0.3
ND
15.1 1.2
IC50 values expressed with standard error.
ture–activity relationship studies of the central five-mem-
ber ring. Although the current compounds displayed
lower potency when compared with our lead thiazolidine
analogs, they may have the distinct advantage of being
more stable in vivo with the reduced necessity of chiral
separations. Some of these new compounds have activity
similar to Sorafenib. Further modification of these com-
pounds to improve their potency is currently in progress.
Our ultimate goal is to identify several compounds that
are highly potent, are highly selective against melanoma
cells, and have very promising ADME characteristics for
subsequent in vivo studies.
University of Tennessee Health Science Center; NIH/
NCI 1R15CA125623-01A2 (W.L.); and the Van Vleet
Endowed Professorship (D.D.M.). Zhao Wang
acknowledges the support of the Alma and Hal Reagan
Fellowship. We thank Dr. David Armbruster for his edi-
torial assistance.
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