Synthesis, X-ray crystal structure study, and cytostatic and antiviral evaluation of the novel cycloalkyl-N-aryl-hydroxamic acids

Article abstract of DOI:10.1021/jm040878r

In vitro evaluation of the novel cycloalkyl-N-(4-chlorophenyl)-hydroxamic acids (2a-g) demonstrated that 2b,d,e exhibited rather marked inhibitory activity (IC₅₀ = 7-10 μM) against pancreatic carcinoma, 2b-d against colon carcinoma, 2d against

Full text of DOI:10.1021/jm040878r

8
84
J. Med. Chem. 2005, 48, 884-887
Brief Articles
Synthesis, X-ray Crystal Structure Study, and Cytostatic and Antiviral
Evaluation of the Novel Cycloalkyl-N-aryl-hydroxamic Acids
,
†
†
†
†
‡
§
Monika Barbari c´ ,* Stanko Ur sˇ i c´ , Viktor Pilepi c´ , Branka Zorc, Antonija Hergold-Brundi c´ , Ante Nagl,
|
|
⊥
⊥
⊥
⊥
Mira Grdi sˇ a, Kre sˇ imir Paveli c´ , Robert Snoeck, Graciela Andrei, Jan Balzarini, Erik De Clercq, and
Mladen Mintas*,
#
Faculty of Pharmacy and Biochemistry, University of Zagreb, 10000 Zagreb, Croatia, Faculty of Science, University of Zagreb,
1
0000 Zagreb, Croatia, Faculty of Textile Technology, University of Zagreb, 10000 Zagreb, Croatia, Rudjer Bo sˇ kovi c´ Institute,
1
0000 Zagreb, Croatia, Rega Institute for Medical Research, Katholieke Universiteit Leuven, 3000 Leuven, Belgium,
and Faculty of Chemical Engineering and Technology, University of Zagreb, 10000 Zagreb, Croatia
Received September 13, 2004
In vitro evaluation of the novel cycloalkyl-N-(4-chlorophenyl)-hydroxamic acids (2a-g)
demonstrated that 2b,d,e exhibited rather marked inhibitory activity (IC50 ) 7-10 µM) against
pancreatic carcinoma, 2b-d against colon carcinoma, 2d against laryngeal carcinoma, and
2
b,d against breast carcinoma. 2e showed the most pronounced anti-cytomegalovirus activity
-
1
(
EC50 ) 1.5 and 0.8 µg mL ) only at g5-fold lower than the cytotoxic concentration. 2d and 2f
-1
showed modest, albeit selective, activity against cytomegalovirus (2d, EC50 ) 7.3-8.9 µg mL ,
selectivity index 7-10; 2f, EC50 ) 7-13 µg mL , selectivity index 10).
-
1
Introduction
Scheme 1. Synthesis of Cycloalkyl-N-aryl-hydroxamic
Acids 2a-ga
Hydroxamic acids are important iron chelators and
microbial siderophores.1,2 They are associated with
diverse biological activities including antibacterial, anti-
3
fungal, and antitumor profiles. Succinyl, malonyl, and
glutaryl hydroxamates are known to inhibit matrix
metalloproteinases, a class of enzymes implicated in4
inflammatory, malignant, and degenerative diseases.
Phenoxyphenyl sulfone N-formylhydroxylamines (retro-
hydroxamates) have been recognized as potent, selec-
tive, and orally bioavailable matrix metalloproteinase
5
inhibitors. Antiinflammatory activity of hydroxamic
acids is also connected with inhibition of 5-lipoxygenase,
an enzyme involved in the biosynthesis of leukotrienes,
mediators of inflammatory and allergic disorders.6,7
Furthermore, hydroxamate-based compounds are effec-
8
9
tive urease, ribonucleotide reductase, or angiotensin
1
0
converting enzyme (ACE) inhibitors. Finally, some
hydroxamic acids are currently accepted therapeutic
agents, e.g. desferrioxamine B (treatment of iron over-
load), hydroxycarbamide (antineoplastic), ibuproxam,
oxametacin and bufexamac (antiinflammatory and an-
algesic drugs), and adrafinil (R-adrenergic agonist and
antidepressant).1 Taking the pharmacological potential
of this class of compounds into account, we have
synthesized the new type of N-aryl hydroxamic acids
containing various cycloalkyl groups connected to the
a
Reagents and conditions: (i) nitrosobenzene, MeCN, catalytic
amount of HCl, rt, 3-22 h; (ii) N-phenylhydroxylamine, N-
methylmorpholine, anhydrous toluene, rt, 3 h.
carbonyl group with the aim to evaluate their cytostatic
and antiviral activities.
1
Chemistry
Synthesis. A specific synthetic approach using acyl
chlorides and nitrosobenzene as the starting reagents
was applied for the preparation of hydroxamic acids
2a-f (Scheme 1). This method enabled a direct intro-
duction of the chloro-substituent in the para position
of the benzene ring.12 The nonhalogenated compound
*
To whom correspondence should be addressed. Phone: 385-1-48-
5
6-202. Fax: 385-1-48-56-201. E-mail: mlovrek@yahoo.com (M.B.);
mladen.mintas@pierre.fkit.hr (M.M.).
†
Faculty of Pharmacy and Biochemistry, University of Zagreb.
‡
Faculty of Science, University of Zagreb.
§
Faculty of Textile Technology, University of Zagreb.
|
Rudjer Bo sˇ kovi c´ Institute.
2
g was synthesized from cyclohexaneacetyl chloride and
⊥
Katholieke Universiteit Leuven.
#
N-phenylhydroxylamine by the usual method for hy-
Faculty of Chemical Engineering and Technology, University of
droxamic acid synthesis.1
3,14
Zagreb.
1
0.1021/jm040878r CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/14/2005

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 885
Table 1. Inhibitory Activities of Cycloalkyl-N-aryl-hydroxamic Acids 2a-g on the Growth of Malignant Tumor Cell Lines and
Normal Fibroblasts (WI38)
a
50% inhibitory concentration or compound concentration required to inhibit cell proliferation by 50%.
Table 2. Activity of Hydroxamic Acids 2a-g Against
Cytomegalovirus in Human Embryonic Lung (HEL) Cell
Cultures
antiviral activity
cytotoxicity
a
-1
-1
EC50 (µg mL
)
(µg mL )
Compd.
a-g
AD-169
strain
Davis
strain
cell morphology cell growth
b
(CC50)c
2
(MCC)
2
2
2
2
2
2
2
a
b
c
d
e
f
>20
10
>20
8
100
100
g20
50
37
20
7.3
7.9
1.5
13
8.9
6.2
0.8
7
28
129
4.7
>50
>200
Figure 1. The molecular structure and labeling of 2d.
Displacement ellipsoids are drawn at the 20% probability level.
g4
100
100
The structures of the novel compounds were deduced
on the basis of analysis of IR spectra and chemical shifts
g
8
8
ganciclovir 0.37
cidofovir
0.37
0.29
g400
g 400
ndd
24
1
13
and coupling constants in their H and C NMR
spectra. The structure of 2d was confirmed by its X-ray
crystal structure analysis (Figure 1) (see Supporting
Information).
0.18
a
Effective concentration required to reduce virus plaque forma-
tion by 50%. Virus input was 100 plaque forming units (PFU).
b
Minimum cytotoxic concentration that causes a microscopically
c
detectable alteration of cell morphology. Cytotoxic concentration
required to reduce cell growth by 50%. Not determined.
d
Biological Results and Discussion
Cytostatic Activities. Hydroxamic acids 2a-g were
evaluated for their cytostatic activity against malignant
tumor cell lines: murine leukemia (L1210), human
T-lymphocyte cells (Molt4/C8 and CEM), colon carci-
noma (SW620), laryngeal carcinoma (Hep2), pancreatic
carcinoma (MiaPaCa2), breast carcinoma (MCF7), cer-
vical carcinoma (HeLa), and human normal fibroblasts
herpes simplex virus-2 (HSV-2; G), vaccinia virus,
vesicular stomatitis virus, herpes simplex virus-1
-
-
(TK KOS ACV), herpes simplex virus-1 (TK VMW
1837), parainfluenza-3 virus, reovirus-1, Sindbis virus,
Coxsackie virus B4, Punta Toro virus, and respiratory
syncytial virus. 2a-e and 2f were tested against HIV-1
and HIV-2, whereas 2d, 2e, and 2g were evaluated
+
(WI38) (Table 1). 2b,d,e exhibited rather marked in-
against thymidine kinase positive (TK ) and negative
-
hibitory activity (IC50 ) 7-10 µM) against pancreatic
carcinoma, 2b-d against colon carcinoma, 2d against
laryngeal carcinoma, and 2b,d against breast carci-
noma, but also against human normal fibroblasts. The
best selectivity against tested tumor cell lines vs normal
fibroblasts was exhibited by compounds 2a and 2c.
Antiviral Activities. Compounds 2b and 2d-g were
evaluated against herpes simplex virus-1 (HSV-1; KOS),
(TK ) strains of varicella-zoster virus (VZV) and cy-
tomegalovirus (CMV, Table 2). Of all compounds exam-
ined, 2e showed the most pronounced anti-CMV activity
-
1
(EC50 ) 1.5 and 0.8 µg mL ). On the other hand,
compounds 2d and 2f showed selectivity, albeit modest,
as antiviral agents against cytomegalovirus (2d, EC
5
0
-
1
) 7.3 and 8.9 µg mL , selectivity index (ratio CC50/
IC50) 7-10; 2f, EC50 ) 7-13 µg mL , selectivity index
-
1

8
86 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
0). These compounds therefore represent anti-CMV
Brief Articles
1
N-Hydroxy-N-phenyl-cyclohexylacetamide (2g). N-Phe-
nylhydroxylamine (0.33 g, 3 mmol) dissolved in toluene (30
mL) and N-methylmorpholine (0.3049 g, 3 mmol) in toluene
lead compounds for further synthetic optimization. 2b
showed some activity against HSV-2 (G) (IC50 ) 9.6 µg
mL ) while 2e showed some anti-HIV-1 activity in
CEM cell cultures (IC50 ) 10 µM) (see Supporting
Information).
(
15 mL) were added simultaneously to a stirred solution of
-
1
cyclohexaneacetyl chloride (0.482 g, 3 mmol) in toluene (55
mL), previously cooled to 0 °C. The reaction mixture was
allowed to warm gradually to room temperature, stirred for 3
h, and then extracted with water, saturated sodium hydrogen
carbonate solution, 1% HCl solution, and water. The organic
layer was dried over anhydrous sodium sulfate, filtered, and
evaporated under reduced pressure. Triturating of the oily
product with cyclohexane and cooling to 0 °C gave the
crystalline product 2g. Yield: 0.335 g (48%). Mp: 116-119
Conclusions
The main purpose of this study was to evaluate new
types of hydroxamic acid derivatives 2a-g for their
cytostatic and antiviral activities. Among the evaluated
compounds, 2a and 2c showed the best selectivity
against malignant tumor cell lines. 2e exhibited the
most pronounced anti-CMV activity (EC50 ) 1.5 and 0.8
µg mL ) only at g5-fold lower than the cytotoxic
concentration. 2d and 2f showed modest, albeit selec-
tive, activity against cytomegalovirus. These compounds
are therefore anti-CMV leads for further synthetic
optimization.
°
C.
X-ray Determination. Details of the crystallographic
determination are given in the Supporting Information. Crys-
tallographic data excluding structure factors for the struc-
ture reported in this paper have been deposited with the
Cambridge Crystallographic Data Center as supplementary
publication number CCDC-234603. Copies of data can be
obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, U.K. [fax, +44-(0)1223-336033;
e-mail, deposit@ccdc.cam.ac.uk].
-
1
Experimental Section
Acknowledgment. Support for this study was
provided by the Ministry of Science of the Republic
of Croatia (Projects 0006543, 006431, 0125003, and
Synthetic Procedure. (a) To a stirred solution of acyl
chloride 1a, 1d, or 1e (8 mmol), concentrated HCl (0.003 mL),
and sodium dithionite (5 mg) in acetonitrile (40 mL) was added
a solution of nitrosobenzene (7.2 mmol) in acetonitrile (40 mL)-
dropwise during 1-3 h. The reaction mixture was stirred
additionally for 3-22 h, and the solidified product was filtered
off. Evaporation of the mother liquor under reduced pressure
gave the oily residue, which was triturated with light petro-
leum (bp 40-80 °C). Separation of the organic phase and
standing at room temperature afforded 2a, 2d, or 2e as
crystalline products. (b) To a solution of nitrosobenzene (3
mmol) in acetonitrile (25 mL) were added acyl chlorides 1b,
0
0981499). This study was performed in the framework
of the COST program, action D13/0006/99. We thank
Lizette van Berckelaer for excellent technical assistance
in performing the antitumor cell activity assays, as well
as Ann Absillis, Anita Van Lierde, Frieda De Meyer,
Anita Camps, and Lies Vandenheurck for excellent
technical assistance in performing the antiviral activity
assays.
1
c, or 1f and concentrated HCl (0.25 mL) dropwise. The
reaction mixture was stirred at room temperature for 5-22
h. Products 2b and 2c were isolated in the following way: The
reaction mixture was neutralized with sodium hydrogen
carbonate and filtered and the mother liquor evaporated under
reduced pressure. The residual oil was triturated three times
with light petroleum (bp 40-70 °C). Products 2b and 2c
crystallized slowly from the separated petroleum extract.
Compound 2e was recrystallized from an acetonitrile/acetone
Supporting Information Available: Experimental de-
1
13
tails; H/ C NMR, IR, elemental analysis, and crystallographic
6
data; cytotoxicity and antiviral activity data of 2a-g in E SM
cell cultures; protocols for the biological testing. This material
is available free of charge via the Internet at http://
pubs.acs.org.
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JM040878R