Synthesis and characterization of novel bioactive 1,2,4-oxadiazole natural product analogs bearing the N-phenylmaleimide and N-phenylsuccinimide moieties

Article abstract of DOI:10.3762/bjoc.9.259

Taking into consideration the biological activity of the only natural products containing a 1,2,4-oxadiazole ring in their structure (quisqualic acid and phidianidines A and B), the natural product analogs 1-(4-(3-tert-butyl-1,2, 4-oxadiazol-5-yl)phenyl)pyrrolidine-2,5-dione (4) and 1-(4-(3-tert-butyl-1,2,4- oxadiazol-5-yl)phenyl)-1H-pyrrole-2,5-dione (7) were synthesized starting from 4-(3-tertbutyl-1,2,4-oxadiazol-5-yl)aniline (1) in two steps by isolating the intermediates 4-(4-(3-tert-butyl-1,2,4-oxadiazol-5-yl)phenylamino)-4-oxobutanoic acid (3) and (Z)-4-(4-(3-tert-butyl-1,2,4-oxadiazol-5-yl)phenylamino)-4-oxobut- 2-enoic acid (6). The two natural product analogs 4 and 7 were then tested for antitumor activity toward a panel of 11 cell lines in vitro by using a monolayer cell-survival and proliferation assay. Compound 7 was the most potent and exhibited a mean IC₅₀ value of approximately 9.4 μM. Aniline 1 was synthesized by two routes in one-pot reactions starting from tert-butylamidoxime and 4-aminobenzoic acid or 4-nitrobenzonitrile. The structures of compounds 1, 2, 4, 5 and 6 were confirmed by X-ray crystallography.

Full text of DOI:10.3762/bjoc.9.259

Synthesis and characterization of novel bioactive
1,2,4-oxadiazole natural product analogs
bearing the N-phenylmaleimide and
N-phenylsuccinimide moieties
Catalin V. Maftei1, Elena Fodor1, Peter G. Jones1, M. Heiko Franz2,
Gerhard Kelter3, Heiner Fiebig3 and Ion Neda*4,§
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 2202–2215.
1Institut für Anorganische und Analytische Chemie, Technische
Universität Carola Wilhelmina, Hagenring 30, D-38106 Braunschweig,
Germany, 2InnoChemTech GmbH, Hagenring 30, D-38106
Braunschweig, Germany, 3Oncotest GmbH, Am Flughafen 12–14,
79108 Freiburg, Germany and 4Institutul National de Cercetare
Dezvoltare pentru Electrochimie si Materie Condensata,
Str. Dr. A. Paunescu Podeanu Nr. 144, Ro-300569 Timisoara,
Romania
doi:10.3762/bjoc.9.259
Received: 01 August 2013
Accepted: 26 September 2013
Published: 25 October 2013
This article is part of the Thematic Series "Natural products in synthesis
and biosynthesis".
Email:
Guest Editor: J. S. Dickschat
Ion Neda* - neda@innochemtech.de
© 2013 Maftei et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
§ Fax: (+) 49 531 391 5387
Keywords:
antitumor activity; bioisosteres; maleimide; natural product analogs;
1,2,4-oxadiazoles
Abstract
Taking into consideration the biological activity of the only natural products containing a 1,2,4-oxadiazole ring in their structure
(quisqualic acid and phidianidines A and B), the natural product analogs 1-(4-(3-tert-butyl-1,2,4-oxadiazol-5-yl)phenyl)pyrrolidine-
2,5-dione (4) and 1-(4-(3-tert-butyl-1,2,4-oxadiazol-5-yl)phenyl)-1H-pyrrole-2,5-dione (7) were synthesized starting from 4-(3-tert-
butyl-1,2,4-oxadiazol-5-yl)aniline (1) in two steps by isolating the intermediates 4-(4-(3-tert-butyl-1,2,4-oxadiazol-5-yl)phenyl-
amino)-4-oxobutanoic acid (3) and (Z)-4-(4-(3-tert-butyl-1,2,4-oxadiazol-5-yl)phenylamino)-4-oxobut-2-enoic acid (6). The two
natural product analogs 4 and 7 were then tested for antitumor activity toward a panel of 11 cell lines in vitro by using a monolayer
cell-survival and proliferation assay. Compound 7 was the most potent and exhibited a mean IC50 value of approximately 9.4 µM.
Aniline 1 was synthesized by two routes in one-pot reactions starting from tert-butylamidoxime and 4-aminobenzoic acid or
4-nitrobenzonitrile. The structures of compounds 1, 2, 4, 5 and 6 were confirmed by X-ray crystallography.
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Introduction
The five-membered heterocyclic 1,2,4-oxadiazole motif is of
synthetic and pharmacological interest. It also forms an impor-
tant constituent of biologically active compounds including
natural products [1]. Sawyer et al. have described such com-
pounds as bioisosteres for amides and esters [2], with the 1,2,4-
oxadiazoles showing higher hydrolytic and metabolic stability.
To the best of our knowledge, there are only a few examples of
natural products with a 1,2,4-oxadiazole core or a structure
based on it. The 3-substituted indole alkaloids, phidianidines A
and B (Figure 1), have been isolated by Carbone et al. from the
aeolid opisthobranch Phidiana militaris [3]. They are selective
inhibitors of the dopamine transporter DAT and partial agonists
of the μ opioid receptor [4]. Moreover, these selective mole-
cules are attractive as CNS targets because neither phidianidine
A nor B is cytotoxic. Another example of a natural product with
a oxadiazole core is quisqualic acid (Figure 1). This metabolite
was obtained from the seeds of Quisqualis indica and Q. fructus
[5,6] and is a strong agonist for AMPA (α-amino-3-hydroxy-5-
methyl-4-isoxazolepropionic acid) receptors and group I
metabotropic glutamate receptors [7].
Furthermore, 1,2,4-oxadiazoles are widely used in synthetic
chemistry, e.g., in the search for antitumor agents. Cancer
consists of more than one hundred different diseases, all of
which are characterized by the uncontrolled growth and spread
Figure 1: Natural products having a 1,2,4-oxadiazole core.
of abnormal cells. In this context, the identification of drugs acid (RGD) sequence. Antagonists of this receptor are able to
acting as apoptosis inducers represents an attractive approach inhibit angiogenesis. 1,2,4-Oxadiazolebutanoic acids such as C
for the discovery of new anticancer agents. 1,2,4-oxadiazole A were tested as non-peptidic analogs of αvβ3 antagonists [10].
(Figure 2) was found to act as an apoptosis agent by a high- Furthermore, substituted 1,2,4-oxadiazoles have been described
throughput screening (HTS) assay [8]. A series of 1,2,4-oxadia- as antirhinovirals [11], benzodiazepine receptor partial agonists
zole-5-carboxamides B have been synthesized and tested as [12], anti-inflammatory [13], muscarinic agonists [14], sero-
inhibitors of the glycogen synthase kinase 3 (GSK-3), a key toninergic (5-HT3) antagonists [15], and growth hormone secre-
regulator of both differentiation and cellular proliferation [9]. tagogues [16].
An alternative antitumor strategy involves the inhibition of The maleimide motif is also a useful five-membered hetero-
processes involved in tumor growth, e.g., angiogenesis. Inte- cycle in pharmacological chemistry. Kratz et al. synthesized
grin αvβ3 is a receptor that has been found on the surface of maleimide derivatives of doxorubicin and camptothecin. After
many tumor cells and recognizes the arginine–glycine–aspartic intravenous administration these designed anticancer drugs bind
Figure 2: Examples of 1,2,4-oxadiazole antitumorals.
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rapidly to circulating albumin [17-19]. Endogenous albumin (a) The condensation of amidoximes with carboxylic acid
could be seen as a drug carrier, as it accumulates in solid tumors derivatives.
according to the pathophysiology of tumor tissue [20,21].
Therefore, designed prodrugs have a higher antitumor efficacy (b) The dipolar cycloaddition of nitrile oxides to nitriles.
in vivo than drugs. Furthermore, maleimides possess strong
antifungal activities against important human opportunistic The general approach for the synthesis of 1,2,4-oxadiazoles is
pathogenic fungi. These antifungal drugs appear to be excellent illustrated in Scheme 1.
candidates for further development [22-27]. Barrett et al. point
out that the possibility of performing chemical modifications is Using route (a), the amidoxime route, the carboxylic acid has to
a requirement for developing novel drugs, a strong activity is be employed in an activated form. The activated carboxylic acid
just the starting point [28].
can be prepared beforehand or in situ by several methods [34],
e.g., as an acyl chloride or by the use of N,N′-carbonyldiimida-
Another moiety worth investigation is succinimide, because zole (CDI). In the first step the amidoxime is O-acylated with
N-phenylsuccinimides are regarded as some of the most effica- the activated derivative in a condensation reaction. The
cious agricultural fungicides [29,30]. They have also been O-acylated amidoxime can be isolated or it can immediately
shown to be selective nephrotoxic compounds [31,32].
undergo the cyclisation to the heterocyclic oxadiazole ring. This
cyclodehydration reaction takes place by heating to tempera-
Considering natural products with the 1,2,4-oxadiazol moiety, tures above 100 °C [35,36]. Microwave techniques have also
such as phidianidines A and B (selective inhibitors of DAT), we been employed in the synthesis of such heterocycles. The
decided to synthesize, isolate and characterize novel natural advantage of CDI is that it activates the carboxylic acid in situ
product analogs of 1,2,4-oxadiazole derivatives bearing and can be used for step 1 and step 2 in DMF.
N-phenylmaleimide or N-phenylsuccinimide functionalities in
order to improve their biological activity. The new derivatives Scheme 2 presents the one-pot synthesis of 4-(3-tert-butyl-
have been tested for in vitro antitumor activity toward a panel 1,2,4-oxadiazol-5-yl)aniline (1) starting from tert-butylamidox-
of 11 cell lines.
ime and 4-aminobenzoic acid. Activation of the 4-aminoben-
zoic acid with CDI and further acylation of the tert-butylami-
doxime with DMF as a solvent furnished the O-acylamidoxime,
Results and Discussion
Clapp reviewed the synthesis of 1,2,4-oxadiazoles [33]. He which was not isolated. After heating to 120 °C for four hours,
pointed out that two general methods dominate the practical it underwent a cyclodehydration reaction, delivering aniline 1
preparation (≈95%):
with 59% yield after purification.
Scheme 1: Common synthetic strategies toward 1,2,4-oxadiazoles; (a) amidoxime route; (b) 1,3 dipolar cycloaddition route.
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Scheme 2: One-pot synthesis of 4-(3-tert-butyl-1,2,4-oxadiazol-5-yl)aniline (1) by using the amidoxime route. i. 1.1 equiv CDI in DMF, 30 minutes; ii.
1.1 equiv CDI in DMF, 120 °C, 4 h.
The structure of compound 1 was confirmed by X-ray structure Following the second route, the 1,3-dipolar cycloaddition, with
determination (Figure 3 and Figure 4). It crystallizes with two the purpose of increasing the yield of compound 1, we used
molecules in the asymmetric unit, which differ in the relative p-toluenesulfonic acid (PTSA)–ZnCl2 as a catalyst for the syn-
orientation of the rings (interplanar angles 22° and 9°). Three of thesis of aniline 1 from amidoximes and organic nitriles [37].
the four NH hydrogens are involved in hydrogen bonds, leading tert-Butylamidoxime and 4-aminobenzonitrile were mixed in
to ribbons of H-bonded rings parallel to the a axis.
DMF with catalytic amounts of PTSA–ZnCl2. First, tert-butyl-
Figure 3: Molecular structure of 4-(3-tert-butyl-1,2,4-oxadiazol-5-yl)aniline (1). Atoms are drawn as 50% thermal ellipsoids. One hydrogen at N1’ is
eclipsed.
Figure 4: Packing diagram of compound 1. Hydrogen bonds are indicated as dashed lines.
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amidoxime is activated by PTSA–ZnCl2; resulting in the forma- to isolate the intermediate. The overall yield in this case was
tion of a Lewis acid–ammonia complex as a leaving group, 64%. The intermediate 2 was isolated to be fully charazterized.
giving rise to the formation of the nitrile oxide. The 1,2,4- After a series of tests by using various acids as catalyst (p-tolu-
oxadiazole moiety is established by the 1,3-dipolar cycloaddi- ensulfonic acid (PTSA), 2-mesitylenesulfonic acid (MSA) and
tion of nitrile oxide to the 4-aminobenzonitrile. However, the methanesulfonic acid (MeSA) in combination with ZnCl2 and
Lewis acid might also be involved in the formation of the hete- ZnBr2), MSA–ZnBr2 in acetonitrile proved to be the best
rocycle via a Lewis acid catalyzed [3 + 2] cycloaddition reac- combination for the preparation of compound 2 from tert-butyl-
tion. Unfortunately, the yield for this reaction was very low amidoxime and 4-nitrobenzonitrile under mild conditions. The
(<20%).
results are summarized in Table 1. The optimized yield was
93% (Scheme 3) which makes this route more practical than the
In order to obtain compound 1 by the 1,3 dipolar cycloaddition amidoxime route presented in Scheme 2 with a yield of only
route we changed the protocol. We synthesized the nitro deriva- 59%.
tive 2 (3-tert-butyl-5-(4-nitrophenyl)-1,2,4-oxadiazole) in situ
by using the same catalyst pair as in the first synthesis route. The structure of compound 2 was confirmed by X-ray structure
Then, we attempted to hydrogenate nitro compound 2 to the determination (Figure 5 and Figure 6). The interplanar
corresponding amine 1 in the same reaction pot without having angle in compound 2 is only 3° and the molecules are
Table 1: Optimal conditions for the 1,3-dipolar cycloaddition route.a
Entry
Catalyst 1
Catalyst 2
Solvent
Time (h)
Yield (%)
1
PTSA
MSA
ZnCl2
ZnCl2
ZnCl2
ZnBr2
ZnBr2
ZnBr2
ZnCl2
ZnCl2
ZnCl2
ZnBr2
ZnBr2
ZnBr2
DMF
5
64
76
56
70
79
58
82
92
63
86
93
65
2
DMF
3
3
MeSA
PTSA
MSA
DMF
12
5
4
DMF
5
DMF
3
6
MeSA
PTSA
MSA
DMF
12
2
7
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
8
2
9
MeSA
PTSA
MSA
12
2
10
11
12
2
MeSA
12
aGeneral conditions: tert-butylamidoxime (1 equiv), 4-nitrobenzonitrile (1 equiv), catalyst 1 (0.3 equiv), catalyst 2 (0.3 equiv), 80 °C.
Scheme 3: One-pot synthesis of 4-(3-tert-butyl-1,2,4-oxadiazol-5-yl)aniline (1) by using the 1,3-dipolar cycloaddition route.
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linked to ribbons parallel to the b axis by two C–H···O interac- The synthesis of amide 3 was performed under inert conditions
tions.
by mixing an equimolar amount of aniline 1 and succinic anhy-
dride in a minimum volume of dichloromethane (Scheme 4).
Compound 3 was obtained with a short reaction time and a high
yield (91%). The ester derivative methyl 4-(4-(3-tert-butyl-
1,2,4-oxadiazol-5-yl)phenylamino)-4-oxobutanoate (5) was
prepared by the addition of a diethyl ether solution of
diazomethane to a suspension of amide 3. The ester 5 was
afforded in a quantitative amount.
For the synthesis of imide 4 the starting material, amide 3, was
mixed with an equimolar amount of sodium acetate in acetic
anhydride and the mixture was heated for 4 h at 80–85 °C;
resulting in the corresponding N-arylsuccinimide 4 (87%).
Figure 5: Molecular structure of 3-tert-butyl-5-(4-nitrophenyl)-1,2,4-
oxadiazole (2). Atoms are drawn as 50% thermal ellipsoids.
The structures of compounds 4 and 5 were also confirmed by
X-ray structure analysis (Figure 7, Figure 8 and Figure 9). In
compound 4 the oxazoline and phenyl rings are approximately
coplanar (6°), but the pyrrolidine ring is rotated by 52° with
respect to the phenyl ring. The main packing interaction is an
Figure 6: Packing diagram of compound (2) showing C–H···O interac-
tions.
Imide derivatives have been found to possess a broad spectrum
of biological activities. A variety of methods have been reported
for the preparation of this class of compounds. The synthesis
follows a two-step protocol. First, it is necessary to synthesize
the amide derivative.
Figure 7: Molecular structure of 1-(4-(3-tert-butyl-1,2,4-oxadiazol-5-
yl)phenyl)pyrrolidine-2,5-dione (4). Atoms are drawn as 50% thermal
ellipsoids.
Scheme 4: Synthesis of 1-(4-(3-tert-butyl-1,2,4-oxadiazol-5-yl)phenyl)pyrrolidine-2,5-dione (4).
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good yield (84%). For the synthesis of imide 7 the amide 6 was
mixed with an equimolar amount of sodium acetate in acetic
anhydride and the mixture was heated for 4 h at 80–85 °C
resulting in the corresponding N-arylmaleimide 7 (75%).
The structure of compound 6 was also confirmed by X-ray
structure analysis (Figure 10 and Figure 11). In compound 6 the
Figure 8: Molecular structure of 4-(4-(3-tert-butyl-1,2,4-oxadiazol-5-
yl)phenylamino)-4 oxobutanoate (5). Atoms are drawn as 50% thermal
ellipsoids.
offset stacking parallel to the c axis (not shown). Compound 5
has an interplanar angle of 14°. The molecules are associated
into ribbons parallel to [110] by one long N–H···N and two
shorter C–H···O interactions.
The synthesis of amide 6 was performed by mixing an
equimolar amount of aniline 1 and maleic anhydride in
dichloromethane (Scheme 5). The amide 6 was obtained with a
Figure 10: Molecular structure of (Z)-4-(4-(3-tert-butyl-1,2,4-oxadiazol-
5-yl)phenylamino)-4-oxobut-2-enoic acid (6). Atoms are drawn as 50%
thermal ellipsoids.
Figure 9: Packing diagram of compound (5). Dashed lines indicate hydrogen bonds.
Scheme 5: Synthesis of 1-(4-(3-tert-butyl-1,2,4-oxadiazol-5-yl)phenyl)-1H-pyrrole-2,5-dione (7).
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Figure 11: Packing diagram of compound 6. Dashed lines indicate hydrogen bonds.
interplanar angle is 11°; the intramolecular hydrogen bond is
almost symmetrical (O4–H04 1.03(3), H04···O2 1.47(3)) Å. The
molecules are linked to form layers perpendicular to the hexag-
onal c axis by one C–H···O and one three-centre (N–H,
C–H)···O interaction. The double bond has a Z-configuration.
Antitumor activity
The in vitro antitumor activity of six synthesized compounds
toward a panel of 11 cell lines was assessed by using a mono-
layer cell survival and proliferation assay. By exhibiting a mean
IC50 value of 9.4 µM 7 was the most potent compound. Com-
pounds 1, 5, 4, 6 and 3 have only marginal antitumor activity
toward the 11 tested cell lines (Figure 12).
Figure 12: In vitro antitumor activity of compounds 1, 3–7 toward 11
human tumor cell lines.
An investigation of the activity of compounds in a cell line
panel representing various tumor histo-types, as performed in
this study, allows the analysis of potency and tumor selectivity marked in green) and the range of activity among the cell lines
and the identification of active compounds which qualify for (i.e., the IC50 value of the most resistant cell line divided by the
further preclinical development. Tumor selectivity of the com- IC50 value of the most sensitive cell line). Overall, a good anti-
pounds is illustrated in Figure 13 by a heat-map presentation of tumor potency (i.e., a mean IC50 < 30 µM) combined with a
the individual IC50 values. Good tumor selectivity is reflected good selectivity (i.e., a range of activity > 8 or at least 2 cell
by cell lines exhibiting above-average sensitivity (i.e., an indi- lines exhibiting above-average sensitivity) was obvious for
vidual IC50 value smaller than 0.5 of the mean IC50 value; compound 7.
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Beilstein J. Org. Chem. 2013, 9, 2202–2215.
Figure 13: Individual IC50 values [µM] of compounds 1, 3–7 in a panel of 11 human tumor cell lines.
Conclusion
at 0.00 ppm. In the case of multiplets, the signals are reported as
Two novel 1,2,4-oxadiazol natural product-inspired derivatives intervals. Signals were abbreviated as s, singlet; d, doublet; t,
bearing N-phenylmaleimide or N-phenylsuccinimide moieties triplet; q, quadruplet; m, multiplet. IR spectra were recorded
were synthesized in two steps starting from 1,2,4-oxadiazole with a Bruker Vertex 70 ATR. Mass spectra (electronic ionisa-
amine 1. All intermediate derivatives were isolated, character- tion, 70 eV) were recorded on a Finnigan MAT 8400-MSS and
ized and tested in vitro for antitumor activity toward a panel of Finnigan MAT 4515. High resolution mass spectra were
11 cell lines by using a monolayer cell survival and prolifera- recorded on a Finnigan MAT 95 XP. Melting points were
tion assay. With an IC50 value of 9.4 µM, compound 7 was the measured with a Schropp Gerätetechnik MPM-HV2. The
most active, probably because maleimide is able to rapidly bind melting points are uncorrected. Column chromatography was
to the cysteine-34 position of circulating albumin, transported carried out by using Merck silica gel 60 (70–200 mesh).
as an albumin conjugate (macromolecular drug), and then
released in the target tissue by acid related cleavage or enzy- Procedures
matic cleavage. Compounds 1, 3, 4, 5 and 6 have no or only Synthesis of 4-(3-tert-butyl-1,2,4-oxadiazol-5-
marginal activity toward the 11 cell lines tested. Considering yl)aniline (1)
the bioactive natural products (quisqualic acid and phidiani- a. 1.0 g (7.2 mmol) of 4-aminobenzoic acid was dissolved under
dines A and B), it is of great pharmacologic interest to synthe- an inert atmosphere in 20 mL of DMF, and 1.28 g (7.9 mmol)
size new derivates of 1,2,4-oxadiazole with an efficient bio- of CDI solved in 30 mL of DMF was added. After 30 minutes
logical transport in cells by using natural transporters such as of stirring at room temperature 0.91 g (7.9 mmol) of tert-butyl-
amino acids, peptides or sugars. Moreover, it is desirable to amidoxime was added. A second portion of CDI (1.28 g,
reduce, or even remove completely, the secondary effects to 7.9 mmol) was added, and the reaction mixture was heated
minimize the toxicity and increase the selectivity. All deriva- under reflux until the reaction was complete. The mixture was
tives were obtained with a high purity (at least 97% based on cooled to room temperature and poured into 500 mL of
1H NMR) and good to high yields (75–96%). The structural water–ice mixture. The formed solid was filtered off, washed
assignments were corroborated by X-ray structure analysis.
with water, dried and flash chromatographed (silica, ethyl
acetate/hexane 2:1), yielding after purification 0.92 g
(4.2 mmol, 59%) of 1 as a yellow solid.
Experimental
General
All reagents were purchased from commercial sources (Sigma- b. To a mixture of 0.116 g (1 mmol) of tert-butylamidoxime in
Aldrich or Acros) and used without further purification. 5 mL of solvent (acetonitrile or DMF) under an inert atmos-
Solvents were of analytical grade. 1H and 13C NMR spectra phere was added 0.15 g (1 mmol) of 4-nitrobenzonitrile. To this
were recorded at room temperature on a Bruker Avance 200 reaction mixture was added 0.3 mmol of acid catalyst 1
operating at 200 MHz for 1H and 50 MHz for 13C. Chemical (57.0 mg PTSA, 70.8 mg MSA or 28.8 mg MeSA) and
shifts (δ) are reported relative to the tetramethylsilane peak set 0.3 mmol of catalyst 2 (40.9 mg ZnCl2 or 67.5 mg ZnBr2), and
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Beilstein J. Org. Chem. 2013, 9, 2202–2215.
the mixture was heated to 80 °C until no further change was Synthesis of 4-(4-(3-tert-butyl-1,2,4-oxadiazol-5-
observed (2–12 hours). Then the reaction mixture was cooled to yl)phenylamino)-4-oxobutanoic acid (3)
room temperature, 10 mg of Pt/C 10% catalyst was added, and 0.3 g (1.38 mmol) of 4-(3-tert-butyl-1,2,4-oxadiazol-5-
hydrogen was bubbled through the solution until intermediate 2 yl)aniline (1) was mixed under inert conditions with an
was no longer detected. The reaction mixture was filtered equimolar amount (0.138 g, 1.38 mmol) of succinic anhydride
through a layer of Celite, and then the solvent was removed in 20 mL of DCM. This mixture was vigorously stirred at room
under reduced pressure. The solid that remained was dried and temperature for 5 hours. The remaining residue was freed from
flash chromatographed (silica, ethyl acetate/hexane 2:1), solvent, suspended in 50 mL of water, and the pH value
yielding after purification 1 as a yellow solid. Yield: PTSA/ adjusted to 2–3 with a 1 N solution of HCl. The resulting
ZnCl2/DMF 139.0 mg, 0.64 mmol, 64%; MSA/ZnCl2/DMF suspension was filtered, and the product was dried in vacuum. It
165.0 mg, 0.76 mmol, 76%; MeSA/ZnCl2/DMF 121.6 mg, was purified by flash chromatography (silica gel, ethyl acetate/
0.56 mmol, 56%; PTSA/ZnBr2/DMF 152.0 mg, 0.7 mmol, hexane 1:1), yielding 0.398 g (1.25 mmol, 91%) of 3 as a white
70%; MSA/ZnBr2/DMF 171.5 mg, 0.79 mmol, 79%; MeSA/ solid after purification. MS m/z: 317 (M+, 15), 299 (55), 217
ZnBr2/DMF 125.9 mg, 0.58 mmol, 58%; PTSA/ZnCl2/CH3CN (50), 202 (100), 120 (55); HRMS: calcd. for C16H19N3O4+,
178.1 mg, 0.82 mmol, 82%; MSA/ZnCl2/CH3CN 199.8 mg, 317.13701; found, 317.13717; 1H NMR (200 MHz, DMSO-d6)
0.92 mmol, 92%; MeSA/ZnCl2/CH3CN 136.8 mg, 0.63 mmol, δ 12.24 (bs, 1H), 10.42 (s, 1H, -NH), 8.07–8.02 (m, 2H),
63%; PTSA/ZnBr2/CH3CN 186.7 mg, 0.86 mmol, 86%; MSA/ 7.96–7.92 (m, 2H), 2.67–2.59 (m, 4H), 1.38 (s, 9H); 13C NMR
ZnBr2/CH3CN 201.9 mg, 0.93 mmol, 93%; MeSA/ZnBr2/ (50 MHz, DMSO-d6) δ 28.1 (-CH3), 28.5, 31.1, 31.9, 117.6,
CH3CN 141.1 mg, 0.65 mmol, 65%. MS m/z: 217 (M+, 60), 202 118.8, 128.6, 143.3, 170.7, 173.6, 174.3, 177.5; mp 201–203
(5), 120 (100); HRMS: calcd. for C12H15N3O+, 217.12096; °C; IR (ATR): 1/λ 3385 (w), 2964 (w), 1696 (s), 1608 (m),
found, 217.12159; 1H NMR (200 MHz, CDCl3) δ 7.87–7.78 1594 (w), 1506 (s), 1492 (m), 1464 (w), 1410 (m), 1394 (w),
(m, 2H), 6.68–6.56 (m, 2H), 4.08 (bs, 2H), 1.32 (s, 9H); 13C 1351 (m), 1337 (m), 1313 (m), 1281 (w), 1249 (w), 1200 (m),
NMR (50 MHz, CDCl3) δ 27.8 (-CH3), 31.7, 113.5, 113.7, 1177 (m), 1159 (s), 994 (w), 859 (m), 830 (w), 802 (w), 773
129.1, 149.7, 174.7, 177.3; mp 120–122 °C; IR (ATR): 1/λ (m), 681 (m), 612 (m) cm−1.
3437 (w), 3326 (w), 3218 (w), 2966 (w), 2360 (w), 2162 (w),
1699 (w), 1652 (w), 1603 (s), 1581 (m), 1522 (w), 1510 (m), Synthesis of 1-(4-(3-tert-butyl-1,2,4-oxadiazol-5-
1491 (m), 1463 (m), 1442 (m), 1415 (w) 1394 (w), 1351 (s), yl)phenyl)pyrrolidine-2,5-dione (4)
1329 (w), 1291 (w), 1197 (m), 1186 (s), 1172 (s), 1096 (w), 0.3 g (0.94 mmol) of 4-(4-(3-tert-butyl-1,2,4-oxadiazol-5-
1028 (w), 962 (w), 845 (m), 835 (m), 777 (s), 700 (w), 645 (s) yl)phenylamino)-4-oxobutanoic acid (3) was mixed with an
cm−1.
equimolar amount of sodium acetate (0.077 g, 0.94 mmol) in
15 mL of acetic anhydride, and the mixture was then heated for
Representative procedure for the synthesis of 3-tert- 4 h at 80–85 °C. After cooling, the product was washed with
butyl-5-(4-nitrophenyl)-1,2,4-oxadiazole (2) 100 mL H2O at pH ≈ 2–3. The solid was dried in vacuum and
To a mixture of 0.116 g (1 mmol) of tert-butylamidoxime in flash chromatographed (silica, ethyl acetate/hexane 2:1),
acetonitrile under an inert atmosphere was added 0.150 g yielding after purification 0.244 g (0.81 mmol, 87%) of 4 as a
(1 mmol) of 4-nitrobenzonitrile in molar ratio 1:1. To this reac- white solid. MS m/z: 299 (M+, 55), 284 (15), 202 (100); HRMS:
tion mixture was added 0.3 mmol (0.07 g) of 2-mesitylenesul- calcd. for C16H17N3O3+, 299.12644; found, 299.12695;
fonic acid and 0.3 mmol (0.067 g) of ZnBr2, and the mixture 1H NMR (200 MHz, CDCl3) δ 8.18–8.09 (m, 2H), 7.48–7.39
was heated to 80 °C for 2 hours. After the reaction was finished (m, 2H), 2.80 (s, 4H), 1.34 (s, 9H); 13C NMR (50 MHz, CDCl3)
the heating was removed, and the mixture was cooled to room δ 28.3 (-CH3), 32.3, 124.2, 126.6, 128.6, 135.3, 174.0, 175.5,
temperature. The solvent was concentrated under reduced pres- 178.3; mp 136–138 °C; IR (ATR): 1/λ 2970 (w), 2875 (w),
sure, and ethyl acetate was added. The mixture was washed 1776 (w), 1709 (s), 1613 (w), 1520 (w), 1500 (w), 1465 (w),
with sodium hydrogen carbonate solution, water and brine. The 1419 (w), 1382 (m), 1351 (w), 1275 (w), 1197 (m), 1171 (s),
organic phase was dried over anhydrous sodium sulfate, 923 (w), 904 (w), 841 (m), 810 (w), 771 (w), 737 (w), 697 (m),
filtered, and the solvent was removed. The resulting residue was 659 (w) cm−1.
dried in vacuum and flash chromatographed (silica, ethyl
acetate/hexane 2:1), yielding after purification 0.232 g Synthesis of methyl 4-(4-(3-tert-butyl-1,2,4-oxadi-
(0.94 mmol, 94%) of 2 as a white solid. MS m/z: 247 (M+, 25), azol-5-yl)phenylamino)-4-oxobutanoate (5)
232 (45), 150 (100); 1H NMR (200 MHz, CDCl3) δ 8.43–8.28 A suspension of 0.3 g (0.94 mmol) of 4-(4-(3-tert-butyl-1,2,4-
(m, 4H), 1.45 (s, 9H); 13C NMR (50 MHz, CDCl3) δ 28.4, 32.6, oxadiazol-5-yl)phenylamino)-4-oxobutanoic acid (3) in 10 mL
124.2, 129.9, 144.6, 148.9, 174.2, 178.4.
of diethyl ether was cooled in an ice bath, and a solution of
2211

Beilstein J. Org. Chem. 2013, 9, 2202–2215.
diazomethane in diethyl ether 2.5 N was added dropwise until yielding after purification 0.176 g (0.59 mmol, 75%) of 7 as a
the white solid disappeared, and the gas evolution stopped. The white solid. MS m/z: 297 (M+, 60), 282 (15), 200 (100); HRMS:
solvent was removed under high vacuum to afford 0.298 g calcd. for C16H15N3O3+, 297.11079; found, 297.11127;
(0.9 mmol, 96%) of 5 as a white solid. No further purification 1H NMR (200 MHz, CDCl3) δ 8.26–8.14 (m, 2H), 7.62–7.54
was necessary. MS m/z: 331 (M+, 10), 299 (50), 284 (15), 202 (m, 2H), 6.88 (s, 2H), 1.44 (s, 9H); 13C NMR (50 MHz, CDCl3)
(100); HRMS: calcd. for C17H21N3O4+, 331.15266; found, δ 28.2 (-CH3), 32.3, 123.2, 125.5, 128.5, 134.2, 134.8, 168.6,
331.15275; 1H NMR (200 MHz, CDCl3) δ 8.34 (s, 1H-NH), 174.0, 178.3; mp 123–125 °C; IR (ATR): 1/λ 3474 (w), 3096
8.11–8.02 (m, 2H), 7.70–7.66 (m, 2H), 3.72 (s, 3H), 2.82–2.67 (w), 2975 (w), 2927 (w), 1713 (s), 1614 (w), 1567 (w), 1521
(m, 4H), 1.42 (s, 9H); 13C NMR (50 MHz, CDCl3) δ 27.8 (m), 1502 (m), 1466 (w), 1395 (m), 1385 (m), 1351 (w), 1306
(-CH3), 28.3, 31.3, 31.7, 51.4, 118.7, 119.3, 128.4, 141.0, (w), 1238 (w), 1195 (m), 1149 (s), 1064 (w), 1022 (w), 988 (w),
169.5, 173.0, 173.9, 177.6; mp 144–146 °C; IR (ATR): 1/λ 949 (w), 905 (w), 842 (m), 827 (s), 771 (m), 737 (w), 700 (s),
3391 (w), 2966 (w), 1738 (m), 1702 (m), 1607 (m), 1595 (w), 686 (m), 652 (w), 624 (w) cm−1.
1507 (s), 1492 (m), 1524 (w), 1392 (w), 1351 (m), 1327 (s),
1274 (w), 1251 (w), 1197 (m), 1172 (s), 1156 (s), 1097 (w), 991 X-ray structure determinations
(w), 988 (w), 863 (m), 836 (w), 797 (w), 773 (m), 699 (w), 690 Data are summarized in Table 2. Intensities were determined at
(w), 617 (s) cm−1.
100 K on Oxford Diffraction diffractometers by using mono-
chromated Mo Kα or mirror-focussed Cu Kα radiation. Struc-
tures were refined on F2 using the program SHELXL-97 [38].
Hydrogen atoms were refined either freely (NH), by using rigid
Synthesis of (Z)-4-(4-(3-tert-butyl-1,2,4-oxadiazol-5-
yl)phenylamino)-4-oxobut-2-enoic acid (6)
0.3 g (1.38 mmol) of 4-(3-tert-butyl-1,2,4-oxadiazol-5- methyl groups allowed to rotate but not tip or by using a riding
yl)aniline (1) was mixed under inert conditions with an model starting from calculated positions. Structure 5 was
equimolar amount of 0.135 g (1.38 mmol) of maleic anhydride treated as a non-merohedral twin.
in 20 mL of DCM. This mixture was vigorously stirred at room
temperature for 3 hours. The remaining residue was freed from CCDC–924911 (1), CCDC–951265 (2), CCDC–924912 (4),
solvent and suspended in 50 mL of water, and the pH value was CCDC–924913 (5), CCDC–924914 (6) contain the supplemen-
adjusted to 2–3 with a 1 N solution of HCl. The resulting tary crystallographic data for this paper. These data can be
suspension was filtered, the product dried in vacuum and flash obtained free of charge from the Cambridge Crystallographic
chromatographed (silica, ethyl acetate/hexane 2:1), yielding Data Centre via http://www.ccdc.cam.ac.uk/.
after purification 0.365 g (1.15 mmol, 84%) of 6 as a white
In vitro antitumor activity toward human
solid. MS m/z: 315 (M+, 15), 297 (50), 217 (55), 200 (100), 120
(75); HRMS: calcd. for C16H17N3O4+, 315.12136; found, tumor cell lines
315.12144; 1H NMR (200 MHz, DMSO-d6) δ 12.94 (bs, 1H), Antitumor activity of the compounds was tested in a monolayer
10.73 (s, 1H, -NH), 8.14–8.03 (m, 2H), 7.96–7.85 (m, 2H), cell survival and proliferation assay with human tumor cell
6.61–6.32 (m, 2H), 1.38 (s, 9H); 13C NMR (50 MHz, DMSO- lines. Studies carried out with panels of human tumor cell lines
d6) δ 28.0 (-CH3), 31.9, 118.3, 119.4, 128.7, 130.1, 131.4, of different origin and histotype allow for the analysis of
142.7, 163.6, 166.8, 174.2, 177.5; mp 214–216 °C; IR (ATR): potency and tumor selectivity of test compounds.
1/λ 3288 (w), 3104 (w), 2974 (w), 1707 (m), 1633 (w), 1616
(w), 1586 (m), 1566 (w), 1532 (s), 1520 (s), 1496 (s), 1467 (m), Ten out of the eleven tested cell lines were established at
1409 (w), 1396 (w), 1348 (w), 1325 (m), 1281 (w), 1267 (w), Oncotest from patient-derived human tumor xenografts
1188 (m), 1101 (w), 976 (m), 899 (w), 849 (s), 776 (m), 737 passaged subcutaneously in nude mice [39]. The origin of the
(w), 738 (w), 632 (w), 615 (m) cm−1.
donor xenografts was described [40,41]. The cell line HT-29
was kindly provided by the National Cancer Institute (Bethesda,
MA, USA). Cells were cultured in RPMI 1640 medium, supple-
mented with 10% fetal calf serum and 0.1 mg/mL gentamicin
Synthesis of 1-(4-(3-tert-butyl-1,2,4-oxadiazol-5-
yl)phenyl)-1H-pyrrole-2,5-dione (7)
0.25 g (0.79 mmol) of (Z)-4-(4-(3-tert-butyl-1,2,4-oxadiazol-5- under standard conditions (37 °C, 5% CO2). Authenticity of all
yl)phenylamino)-4-oxobut-2-enoic acid (6) was mixed with an cell lines was proven by STR analysis at the DSMZ (Braun-
equimolar amount of sodium acetate (0.065 g, 0.79 mmol) in schweig, Germany).
15 mL of acetic anhydride, and the mixture was then heated for
4 h at 80–85 °C. After cooling, the product was washed with A modified propidium iodide assay was used to assess the com-
100 mL of water at pH ≈ 2–3. The solid was dried in vacuum pounds’ activity toward human tumor cell lines [42]. Briefly,
and flash chromatographed (silica, ethyl acetate/hexane 2:1), cells were harvested from exponential phase cultures by
2212

Beilstein J. Org. Chem. 2013, 9, 2202–2215.
Table 2: Crystallographic data for compounds 1, 2, 4–6.
Compound
1
2
4
5
6
Formula
C12H15N3O
217.27
C12H137N3O3
247.25299.33
C16H17N3O3
299.33
C17H21N3O4
331.37
C17H21N3O4
331.37
Mr
Habit
colourless tablet
0.15 × 0.15 × 0.1
monoclinic
colourless tablet
0.24 × 0.12 × 0.07
orthorhombic
colourless needle
0.11 × 0.05 × 0.02
monoclinic
colourless plate
0.18 × 0.09 × 0.02
monoclinic
colourless prism
0.07 × 0.05 × 0.03
trigonal
Cryst. size (mm)
Crystal system
Space group
P21/c
Pbca
P21/c
P
R
Cell constants:
a (Å)
15.6514(10)
9.0961(6)
18.4999(12)
90
6.7175(4)
13.8116(6)
26.1018(9)
90
13.8569(4)
15.3562(4)
6.8926(2)
90
5.4494(5)
6.1403(6)
25.283(2)
86.906(8)
85.277(8)
72.926(8)
805.57
2
28.0192(6)
28.0192(6)
10.0740(8)
90
b (Å)
c (Å)
α (°)
β (°)
114.371(8)
90
90
94.918(2)
90
90
γ (°)
90
120
V (Å3)
2399.1
8
2421.7
8
1461.27
4
6849.3
18
Z
Dx (Mg m−3)
μ (mm−1)
F(000)
1.203
0.08
1.356
0.10
1.361
0.80
1.366
1.376
0.84
0.81
928
1040
−173
0.71073
56.6
632
352
2988
T (°C)
−173
−173
−173
−173
Wavelength (Å)
2θmax
0.71073
52.8
1.54184
152
1.54184
152
1.54184
152
Refl. measured
Refl. indep.
Rint
41755
4908
55696
2483
0.074
166
16680
3034
28530
3156
3747
0.064
311
0.027
202
0.067
219
Parameters
wR(F2, all refl.)
R(F, >4σ(F))
S
226
0.179
0.062
1.07
0.057
0.035
0.73
0.061
0.031
0.81
0.086
0.032
1.06
0.098
0.039
1.04
max. Δ/ρ (e Å−3)
0.17
0.18
0.21
0.38
0.17
trypsinization, counted and plated in 96-well flat-bottom PROJECT NR. 341-/05.10.2011 – Immunomodulatory Fluoro-
microtiter plates at a cell density dependent on the cell line glycopeptide Molecular Architectures, Ion Neda).
(4.000–20.000 cells/well). After 24 h recovery period to allow
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