The influence of the substitution pattern on the molecular conformation of ureido-1,2,5-oxadiazoles, related to STAT3 inhibitors: Chemical behavior and structural investigation

Article abstract of DOI:10.1002/cbdv.201100339

Signal transducer and activator of transcription 3 (STAT3) is a protein constitutively activated by aberrant upstream tyrosine kinase activities in a broad spectrum of human solid and blood tumors. Therefore, the availability of drugs affecting STAT3 may have important therapeutic potential for the treatment of cancer. Pursuing our efforts in exploring the influence of the substitution pattern of the ureido 1,2,5-oxadiazole moiety on the molecular conformation, new compounds substituted at positions 3 and 4 on the furazane ring were synthesized. The inhibition properties vs. STAT3 of the novel compounds were evaluated in a dual-luciferase assay, using HCT-116 cells, and the results evidenced a moderate activity only for the compounds endowed with a planar arrangement. Crystallographic studies of the new derivatives were performed in order to evidence the peculiar chemical behavior and to evaluate how structural modulations affected the biological properties. Copyright

Full text of DOI:10.1002/cbdv.201100339

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
The Influence of the Substitution Pattern on the Molecular Conformation of
Ureido-1,2,5-oxadiazoles, Related to STAT3 Inhibitors: Chemical Behavior
and Structural Investigation
by Stefania Villa, Daniela Masciocchi, Arianna Gelain, and Fiorella Meneghetti*
Department of Pharmaceutical Sciences ꢀPietro Pratesiꢁ, University of Milan, via L. Mangiagalli 25,
IT-20133 Milano
(phone: þ39-02-503-19306; fax: þ39-02-503-19359; e-mail: fiorella.meneghetti@unimi.it)
Signal transducer and activator of transcription 3 (STAT3) is a protein constitutively activated by
aberrant upstream tyrosine kinase activities in a broad spectrum of human solid and blood tumors.
Therefore, the availability of drugs affecting STAT3 may have important therapeutic potential for the
treatment of cancer. Pursuing our efforts in exploring the influence of the substitution pattern of the
ureido 1,2,5-oxadiazole moiety on the molecular conformation, new compounds substituted at positions 3
and 4 on the furazane ring were synthesized. The inhibition properties vs. STAT3 of the novel compounds
were evaluated in a dual-luciferase assay, using HCT-116 cells, and the results evidenced a moderate
activity only for the compounds endowed with a planar arrangement. Crystallographic studies of the new
derivatives were performed in order to evidence the peculiar chemical behavior and to evaluate how
structural modulations affected the biological properties.
1. Introduction. – Signal transducer and activator for transcription 3 (STAT3) is a
latent cytosolic protein that directly relates extracellular signals (growth factors,
polypeptide, cytokines, etc.) from the membrane to the nucleus. STAT3 Proteins
constitute an important point of convergence for many signaling pathways that are
commonly activated in cancer cells. Binding of growth factors or cytokines to their
receptors results in the activation of intrinsic receptor tyrosine-kinase activity or
receptor-associated kinases, such as the Janus kinase (JAK) or Src tyrosine kinases.
These enzymes subsequently phosphorylate the cytoplasmic tails of the receptor to
provide the docking sites for the recruitment of monomeric STAT3, then STAT3s
themselves become substrates for tyrosine phosphorylation. Non-receptor tyrosine
kinases, such as the oncoproteins Src and Bcr-Abl (a fusion of the breakpoint-cluster
region (Bcr) and Abelson leukaemia (Abl) proteins), can phosphorylate STAT3
independently of receptor engagement. Phosphorylated STAT3 dimerizes and trans-
locates to the nucleus, where the dimers directly regulate gene expression. Whereas
STAT3 activation is tightly regulated in normal cells, the persistent activation of
tyrosine kinases in cancer causes constitutive activation of STAT3 [1–9]. This leads to
permanent changes in the expression of genes controlling fundamental cellular
processes, which are subverted in cancer cells. STAT3 is constitutively activated by
aberrant upstream tyrosine kinase activities in a broad spectrum of human solid tumors,
including breast, brain, colon, prostate, lung, pancreatic, pituitary, gastrointestinal,
ovarian, cervical tumors, and melanoma, as well as hematological malignancies such as
ꢂ 2012 Verlag Helvetica Chimica Acta AG, Zꢃrich

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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lymphomas and leukemias [10][11]. Since STAT3 inhibition leads to apoptosis in tumor
cells, but not in normal cells [12][13], it represents a promising target for therapeutic
intervention. At present, there is no report of an approved drug targeting STAT3, even
though a number of small molecules were discovered as potential STAT3 inhibitors
[14].
An attempt to find inhibitors of this pathway, by the known dual-luciferase assay
system with cancer cell line harboring abnormally activated STAT3, the ureido-
oxadiazole derivative AVS-0288 (I) was disclosed as potential anticancer agent, due to
its ability to significantly decrease STAT3 activity [15]. To build up a validated
pharmacophoric model and to understand the bases for its STAT3 inhibitory activity,
we investigated the effects of a series of modifications structurally related to I. In
particular, we studied the effects of the chlorophenyl replacement to C(4) of the
heterocycle and simultaneously the insertion of a CH2 group between the ureido and
the N-phenyl moieties. The previous biological data revealed that, of the ureido series,
several derivatives showed interesting results [16]. The structureꢀactivity relationships
study (SAR) on these compounds highlighted that a polar substituent at C(4) had
detrimental effect on the activity, while the insertion of the benzylic side chain linked to
the ureido moiety was tolerated (compound 1a, R¼CH2OH). Starting from this point,
we investigated the molecular modifications of the substitution at C(4), synthesizing
two derivatives, 2a (R¼CHO) and 3a (R¼Me), related to 1a. They bear a substituent
selected to fine-tune the influence on the activity of lower-polarity groups, to establish
whether the presence of an aromatic moiety at C(4) is essential to confer the STAT3
inhibitory activity. The syntheses of 2a and 3a revealed significant differences in their
chemical behaviors compared to the previously reported oxadiazole derivatives [16].
The earlier studies indicated that an extended molecular conformation was critical for
the activity; we now suggest that the loss of the inhibitory potency of a number of
ureido derivatives could be mainly due to conformational changes that interfered at the
STAT3 binding site. Therefore, we synthesized the isomers (E)- and (Z)-4a (R¼
PhCH¼CH), which bear a benzyl (Bn) chain linked to the ureido moiety. The choice of
the styryl group was considered to clarify the molecular requirements at C(4), by
selecting a group able to confer a planar molecular conformation. Then, we prepared
the styryl derivatives (E)- and (Z)-4b with a Ph ring directly linked to the ureido
moiety, as in I, to evaluate if the conjugation effect along the entire molecule is required
for the extended conformation of the compounds. Taking into account the similarity in
the chemical structure among the active compound I and the considered derivatives,
little changes in functional groups were analyzed, in order to understand the SAR
associated to the flexibility of the ureido moiety, as it seems to play an important role
for the observed biological activity.
In this article, we describe the syntheses of 1a and 1b, 2a and 2b, 3a, (E)-4a and (E)-
4b, and (Z)-4a and (Z)-4b, highlighting the most interesting aspects of the reaction
paths, together with the investigation of their molecular conformation by means of X-
ray analysis, and then their biological data will be compared with the those of reference
compound.
2. Results and Discussion. – 2.1. Chemistry. The syntheses of compounds 1a and 1b
and 2a and 2b were performed starting from 6, which was prepared according to the

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
procedure described in [16]. The general synthetic approach, used to introduce the
planned functional groups in the oxadiazole ring, involved a condensation reaction
between the NH2 group of the key intermediates 3-amino-1,2,5-oxadiazoles, differently
substituted at C(4), and benzyl or phenyl isocyanate (BnNCO and PhNCO, resp.)
under microwave (MW) irradiation. For the synthesis of 1a and 1b, before the
condensation, the alcohol function was protected by ClCOOMe. The MW-assisted
reactions were necessary to obtain the designed compounds, as the conventional
conditions were not efficient. The toluene solutions of 7 and the corresponding
isocyanate in equimolar amounts were irradiated to obtain the expected ureido
derivatives 8a and 8b, selecting time and temperatures in order to optimize the yields
(Scheme 1). The OH group was then deprotected and oxidized with DessꢀMartin
periodinane in dry CH2Cl2 to give the corresponding aldehydes 2a and 2b. In solution,
compounds 2a and 2b underwent spontaneous cyclization to form 5a and 5b,
respectively obtained through the intramolecular reaction between the ureido N-atom
and the aldehyde function. This is in agreement with the intramolecular ring closures
Scheme 1. Synthesis of Compounds 1a and 1b and 2a and 2b

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1243
when aldehydes bind to N-atoms able to give hemi-aminals inside the stable medium-
sized rings [17–23]. It was not possible to follow the cyclization reaction by NMR, as
the equilibrium was completely shifted towards the cyclic form. Compounds 5a and 5b
were the only products isolated as established by 1H-NMR spectroscopy. The following
single-crystal X-ray analysis on 5a supported unambiguously the proposed structure.
By the same methodology [16] for preparing compound 3a, we detected an anomalous
reactivity of the precursor 9 during the reaction with BnNCO in toluene under MW
irradiations (Scheme 2). After cooling a solid fraction precipitated from the reaction
mixture, TLC analysis revealed the presence of one product, 3a, in the solution, while in
the solid fraction other two compounds, 10 and 11, were identified. Suitable crystals for
X-ray analysis of the two different portions were obtained, and the crystallographic
results confirmed that the toluene-soluble product was the expected 3a, while the solid
material was constituted by two not separable symmetrically disubstituted ureas 10 and
11 (see Scheme 2 and Crystallographic Studies). The formation of the N,N’-dibenzylur-
ea (11) is an expected side-product formed in presence of trace of H2O by nucleophilic
addition to the isocyanate C-atom [24]. On the contrary, the other compound, 10, was
an unusual product (to the best of our knowledge, only one patent in the literature
[25]), and its formation can be rationalized as outlined in Scheme 3. The starting amine
9 reacted with BnNCO to give the desired ureido derivative 3a. A thermal conversion
of 3a led to the formation of the corresponding isocyanate, which instantly reacted with
the initial amine 9 in excess to form the symmetrically substituted urea 10. As
continuation of our work, we investigated, in this series of compounds, the influence of
Scheme 2. Synthesis of Compound 3a
Scheme 3. Proposed Mechanistic Pathway for the Formation of Compound 10

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
an aromatic group at C(4) on both the activity and the conformation. Since the urea
linkage plays an active role in the solid-state conformation, and it governs the overall
shape of the molecules, the introduction at C(4), of a vinyl spacer, bearing a Ph group,
allowed us to verify if the resonance effect between the oxadiazole and the substituent
at C(4) is determinant for the trans/trans-conformation of the ureido moiety. We
synthesized compounds with the styryl moiety at C(4) by exploiting the aldehydic
reactivity of the cyclic hemiaminals 5a and 5b [26]. The products (E)-4a and (Z)-4a and
(E)-4b and (Z)-4b were obtained by a Wittig reaction between 5a and 5b, respectively,
and Bn(Ph3)PCl under basic condition in THF at reflux, as ca. 3 :1 (E)/(Z) mixtures
(Scheme 4). The (E)-and (Z)-isomers were easily separated by silica-gel chromatog-
raphy. The assignment of the (E)/(Z) geometry about the styryl p-bond was achieved
by H,H- coupling constants for the olefinic H-atom signals and confirmed by X-ray
analysis.
Scheme 4. Synthesis of compounds (E)-4a and (E)-4b and (Z)-4a and (Z)-4b
2.2. Biology. The biological data showed that compounds 3 and 5 were inactive,
while the two styryl isomers (E)-4a and (Z)-4a exhibited inhibitory activity of 15.71
and 21.20%, respectively (Table 1). In particular, (E)- and (Z)-4a exhibited inhibitory
Table 1. STAT3 Inhibitory Activity of Compounds 3–5 by Dual-Luciferase Assay
Compounds
n
R
Inhibitiona) at 2 mm [%]
3a
1
1
1
0
0
–
–
–
Me
<1
(E)-4a
(Z)-4a
(E)-4b
(Z)-4b
5a
PhCH¼CH
PhCH¼CH
PhCH¼CH
PhCH¼CH
PhCH2
Ph
15.71
21.20
<1
<1
<1
5b
<1
45.00
AVS-0288 (I)
–
a) The values are means of three experiments and the maximum deviation was less than 10%.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1245
activities comparable to several 4-phenyl-substituted derivatives [16], to support the
hypothesis concerning the important presence of an aromatic group at C(4). Isomers
(E)- and (Z)-4a retained a modest activity probably due to the electronic conjugation
of the styryl chain with the oxadiazole, which favored the establishment of stacking
interactions with the biological target. The drop in activity of these derivatives with
respect to I could be due to the presence of the flexible Bn chain linked to the ureido
moiety that disrupted the flat conformation of the molecule. To check if the above
hypothesis might have a correspondence in the solid state, isomers (E)- and (Z)-4b
with a Ph group linked to the ureido moiety were prepared, and the data obtained for
(E)- and (Z)-4b showed the absence of inhibitory activity; therefore, the styryl system
is not determinant to guarantee a planar arrangement and at the same time an extended
conformation.
2.3. Crystallographic Studies. The X-ray molecular structures of the compounds 1a,
5a, 3a, 10–11, (E)-4a, and (E)-4b are presented, as ORTEP views [27], in Fig. 1.
Compound 1a presents a cis/trans arrangement of the two amide bonds due to the
formation of the intramolecular H-bond N4ꢀH4· · ·N2; distance, 2.21(1) ꢄ; angle,
130(1)8. The two rings form a dihedral angle of 57(1)8. A three-dimensional network,
stabilized by NꢀH· · ·O H-bonds, CpꢀH· · ·O intermolecular contacts, and aromatic
stacking involving the oxadiazole rings, characterizes the molecular assembly. The
bicyclic derivative 5a has a folded conformation, defined by the dihedral angle of 86(1)8
between the oxadiazole and Ph moieties. The six-membered ring C1/C2/C3/N3/C11/N4
adopts a half-boat conformation with puckering parameters QT ¼0.157(3) ꢄ and q2 ¼
106.8(9)8 [28]. The intermolecular H-bonds involve O31ꢀH· · ·O2I (distance 1.63(1) ꢄ,
angle 157(1)8 (I at 1ꢀx, ꢀy, 1ꢀz)) and N4ꢀH· · ·O3II (distance, 1.99(1) ꢄ, angle,
176(1)8 (II at xþ1, y, z)), with the formation of cyclic dimers further extended into a
two-dimensional network. The molecular packing is consolidated by pꢀp stacking of
the oxadiazole rings (centroidꢀcentroid distance of 3.34(1) ꢄ) and by weak CpꢀH· · ·N
contacts: C8ꢀH· · ·N2III (distance, 2.91(1) ꢄ; angle, 131(1)8 (III at xꢀ1, y, zþ1)). In the
crystal structure of 3a, as in 1a, the formation of the intramolecular H-bond N4ꢀH4· · ·
N2, (distance, 2.01(1) ꢄ; angle, 136(1)8) favors the cis/trans conformation of the ureido
moiety. The dihedral angle of 88(1)8 between the oxadiazole and the Ph moieties
evidences the curled-up shape of the molecule, determined by the different orientations
of the two rings. Its crystal packing is characterized by dimers stabilized by N3ꢀH· · ·
O2I H-bonds (distance, 1.91(1) ꢄ; angle, 176(1)8 (I at 2ꢀx, ꢀy, 1ꢀz)). In the
C7H8N6O3 ·C15H16N2O1 (10–11) co-crystals, the two molecules are located on a
crystallographic twofold axis coincident with the carboxylic bond. Compound 10 is
essentially planar, while 11 is characterized by a ꢀZꢁ shape, with the two symmetry-
related benzene moieties almost perpendicular to the ureido plane (dihedral angle,
77(1)8), with the dihedral angle of 15(1)8 between them. The two different units are
connected by two bifurcated H-bonds, the strong N3bꢀH· · ·O2cI (distance, 1.87(1) ꢄ;
I
II
1
1
angle, 155(1)8 ( at ꢅ2 ꢀy, ꢀ, ꢅ2 ꢀz)) and the looser one N3cꢀH· · ·O2b (distance,
2.19(1) ꢄ; angle, 151(1)8 (II at 1ꢀx, 1ꢀy, z)). The three-dimensional arrangement of
the molecules gives rise to the formation of columnar structures along the 42 axis of the
tetragonal cell. The formation of reciprocal H-bonds between the ureido H-atoms and
the C¼O O-atoms of 10 and 11 acted as stabilizing factors, giving reason to the
separation of the crystalline adduct 10–11 from the reaction mixture. Bifurcated H-

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 1. ORTEP [28] Views of the molecular structures with the relative atom-numbering scheme. In the
adduct 10–11, labeled atoms are related to unlabeled by binary symmetry (thermal ellipsoids at 30%
probability).
bonds are a common motif in diaryl-urea and their molecular complexes (Fig. 2)
[29][30].
The crystal structures of (E)-4a and (E)-4b confirmed the (E) configuration of the
styryl C¼C bond. In (E)-4a, the p-conjugated system is essentially planar with a
dihedral angle of 3.3(1)8 between the oxadiazole and Ph rings, while the two Ph rings
are almost orthogonal with a dihedral angle of 82(1)8. In (E)-4b, the Ph ring of the
styryl moiety is rotated by 53.4(1)8 with respect to the heterocyclic ring; that twist
disfavors the transmission of the resonance effect along the CPhꢀC¼CꢀC(het) moiety.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1247
Fig. 2. Partial packing diagram with the H-bond pattern involving compounds 10 and 11, leading to the
formation of the chain along the b axis (H-bond distances are reported)
This is revealed by the shorter values of the C12ꢀC13 and C1ꢀC3 bond lengths in (E)-
4a (1.456(4) and 1.44(4) ꢄ vs. 1.477(7) and 1.501(7) ꢄ for (E)-4a and (E)-4b, resp.),
characterized by a higher coplanarity between the two rings. In (E)-4b, the Ph group
linked to the ureido moiety is almost coplanar with the oxadiazole, having a dihedral
angle of 14(1)8, while the two Ph rings are rotated by 53.0(1)8. In both compounds, the
intramolecular H-bond N4ꢀH· · ·N3 is present, so the ureido group adopts a cis/trans-
conformation at the two amide bonds. In (E)-4a, the N4ꢀH· · ·N3 H-bond is
characterized by a distance of 1.93(1) ꢄ and an angle of 143(1)8, in (E)-4b, the same
atoms are engaged in a stronger interaction, as shown by the value of the distance of
1.54(6) ꢄ and of an angle of 160(1)8. The cis/trans ureido conformation in (E)-4b is
further stabilized by the intramolecolar contact C6ꢀH· · ·O2 of 2.13(1) ꢄ and an angle
of 140(1)8. The existence of this intramolecular interaction between the aromatic H-
atoms of the Ph ring with the ureido O-atom is a key feature which imparts additional
stability to the molecular conformation in the solid state. In the crystal, molecules of
(E)-4a and (E)-4b are arranged in a similar manner. The crystal packing is built up by
centrosymmetric dimers joined by N3ꢀH· · ·O2I H-bonds. Weak contacts link adjacent
dimers, type CpꢀH· · ·O and CpꢀH· · ·N in (E)-4a and (E)-4b, respectively, leading to
the formation of infinite chains. In the crystal of (E)-4a, p–p interactions are also
present between nearly parallel oxadiazole and styryl moieties of neighboring
molecules stacked along the c axis in a head-to-tail manner (centroid-to-centroid
distance of 4.1(1) ꢄ).
As demonstrated by the crystallographic results (Fig. 3), all derivatives, 1a, 3a, (E)-
4a, and (E)-4b, are characterized by a constrained conformation, resulting from the
rotation of the ureido moiety, due to the formation of a NhetH· · ·Nurea intramolecular
H-bond, differently to I.
In the crystal structure of I [16], we found that the two ureido H-atoms are not
involved in intramolecular contacts, thus they could participate in H-bonds at the

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 3. Superimposition of the crystal structures of I (violet), 1a (blue), 3a (green), and (E)-4a (light
blue) and (E)-4b (orange) achieved by overlaying the oxadiazole rings. H-Atoms are omitted for clarity.
macromolecular binding site. In addition, its flat conformation could favor the p-
interactions with the aromatic residue present in the binding pocket. Beside that, the
pendant halogen atoms play a very important role in anchoring I in the binding site in
extended conformation, so to prevent in this way the formation of the NhetH· · ·Nurea
interaction. On the basis of these findings, we can assume that the binding mode of the
ureido derivatives related to I requires that the oxadiazoleꢀureido group adopts an
extended orientation. This could be provided by positive hydrophobic interactions at
the active site, thus validating the Ph rings as anchor for hydrophobic arms capable to
interact with the target. This hypothesis is supported by the presence of a
intermolecular interaction network in the crystal structure of I involving the halogen
atoms, implicated in keeping a flat molecular conformation. Further, the lipophilicity of
these moieties can modulate in a suitable way the absorption properties of the
molecule, as they can find in the hydrophobic side chains, present in the binding site, a
favorable environment. In this respect, it is important to underline that CꢀF· · ·HꢀC
type interactions were reported to play an important role in crystal packing and in the
biological systems [31]. This suggests that different contacts of the hydrophobic part of
the molecule may guide the H-bonding interaction pattern of the ureido moiety, and
the overall molecular conformation in the bound and unbound forms.
3. Conclusions. – The present work highlights that the biological inactivity of 1a and
3a could be explained as they lack both of the key requirements previously indicated:
an aromatic moiety at C(4) and the extended conformation. Besides, the introduction
of a styryl moiety led to a moderate STAT3 inhibition in isomers (E)- and (Z)-4a.
Nevertheless, (E)- and (Z)-4b lost the inhibitory properties, indicating that the styryl
chain could not always place the Ph ring in a suitable orientation inside the binding
pocket, and the resonance effect was not determinant to guarantee an extended
conformation and a planar arrangement. The crystal structure of (E)-4b showed that
the substitution of the flexible Bn chain linked to the ureido moiety with a Ph ring did

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1249
not thoroughly influence the trans/trans-arrangement of the ureido bond. Indeed, in
this compound, the folded conformation could be also stabilized by an additional
CpꢀH· · ·O intramolecular interaction. From the SAR point of view, we propose the
important role played at the binding site of the halogen atoms, present in the reference
compound I, in keeping the extended conformation, implying that the substitution
pattern on both phenyl rings should be taken into account as a critical factor. In I, the
intermolecular contacts of the CF3 group could prevent the formation of the
intramolecular NhetH· · ·Nurea H-bond, so the two NꢀH donors and the C¼O acceptors
of the ureido moiety remained available for guest binding.
In summary, the detailed solid-state investigation of these derivatives provided
useful insight on the different factors contributing to the pre-organization of the
variable conformations of the ureido-oxadiazole group, supporting the hypothesis that
conformational changes have a strong effect on the STAT3 inhibitory properties, and
affording new information for further lead optimization of this class of compounds.
Experimental Part
1. General. Reagents were purchased from SigmaꢀAldrich and were used without any further
purification. Compounds 6 and 9 were synthesized according to literature methods [16][32]. The
reactions were performed under conventional conditions or by microwave (MW) irradiation (Biotage-
Initiator.2). Reactions were monitored by TLC on aluminium-backed Merck Silica Gel 60 plates
(0.2 mm). Intermediates and final compounds were purified by flash chromatography using Merck silica
gel 60 (70–230 mesh). The purity of final compounds was determined by HPLC analysis and were ꢁ
95%. M.p.: in open cap. tubes on a Bꢀchi Melting Point B-540 apparatus. 1H-NMR Spectra: at r.t., Varian
300 MHz Oxford instrument, chemical shifts (d) in ppm from TMS resonance in the indicated solvent
(TMS: 0.0 ppm). All new compounds gave satisfactory C, H, N elemental analyses (ꢂ0.4%).
2.1. Synthesis of (4-Amino-1,2,5-oxadiazol-3-yl)methyl Methyl Carbonate (7). Compound 7 was
synthesized according to the procedure reported in [16].
2.2. General Procedure for the Synthesis of Methyl (4-Ureido-1,2,5-oxadiazol-3-yl)methyl Carbonate
Derivatives 8a and 8b. A mixture of 7 (50 mg, 0.29 mmol) and the appropriate isocyanate (0.40 mmol,
0.05 ml) in toluene (1 ml), in a sealed vial, was irradiated in a MW synthesizer at 300-Wat a temp. of 1808
for 40 min. After cooling, a white jelly-like solid began to precipitate. The solid was filtered and washed
with toluene.
[4-(3-Benzylureido)-1,2,5-oxadiazol-3-yl]methyl Methyl Carbonate (¼{4-[(Benzylcarbamoyl)-
amino]-1,2,5-oxadiazol-3-yl}methyl Methyl Carbonate; 8a). Yield: 31%. 1H-NMR (CDCl3): 3.80 (s,
MeO); 4.60 (d, J¼5.7, CH2); 5.35 (s, CH2O); 7.25–7.35 (m, 5 arom. H); 8.60 (s, NH, exchangeable with
D2O).
[4-(3-Phenylureido)-1,2,5-oxadiazol-3-yl]methyl Methyl Carbonate (¼ Methyl {4-[(Phenylcarba-
moyl)amino]-1,2,5-oxadiazol-3-yl}methyl Carbonate; 8b). Yield: 35%. 1H-NMR (CDCl3): 3.80 (s, MeO);
5.40 (s, CH2O); 7.20–7.60 (m, 5 arom. H); 8.60 (s, NH, exchangeable with D2O); 9.20 (s, NH,
exchangeable with D2O).
2.3. General Procedure for the Synthesis of 1-Aryl-3-[4-(Hydroxymethyl)-1,2,5-oxadiazol-3-yl]urea
Derivatives 1a and 1b. Compound 8a and 8b (50 mg, 0.16 mmol), resp., was dissolved in 1% soln. of
K2CO3 in MeOH (2 ml), and the resulting soln. was stirred at reflux for 2 h. After completion of the
reaction, MeOH was removed in vacuo. The residue was diluted with AcOEt (3 ml) and washed with
H2O (2ꢃ1 ml). The org. phase was dried (Na2SO4) and evaporated to obtain the white solid.
1-Benzyl-3-[4-(hydroxymethyl)-1,2,5-oxadiazol-3-yl]urea (1a). Quant. yield. 1H-NMR (CDCl3): 4.55
(d, J¼5.7, CH2); 4.80 (s, CH2); 7.25–7.40 (m, 5 arom. H); 8.10 (s, NH, exchangeable with D2O); 10.30 (s,
NH, exchangeable with D2O).
1-[4-(Hydroxymethyl)-1,2,5-oxadiazol-3-yl]-3-phenylurea (1b). Quant. yield. 1H-NMR (CD3OD):
4.85 (s, CH2); 7.10–7.50 (m, 5 arom. H).

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2.4. General Procedure for the Synthesis of 7-Hydroxy-6,7-dihydro[1,2,5]oxadiazolo[3,4-d]pyri-
midin-5(4H)-one Derivatives 5a and 5b. To an ice-cold soln. of the compound 1a and 1b (50 mg,
0.20 mmol), resp., in dry CH2Cl2 (1.5 ml), DessꢀMartin periodinane (131.5 mg, 0.31 mmol) was added in
one portion. The mixture was stirred at 08 for 30 min and then at r.t. for 24 h. The reaction was quenched
at 08 by stirring with a sat. aq. solns. of Na2S2O3 (1 ml) and of NaHCO3 (1 ml) for 10 min, to destroy any
unreacted DessꢀMartin reagent. The org. phase was then separated, dried (Na2SO4), and concentrated in
vacuo. The crude product was purified by flash chromatography (petroleum ether (PE)/AcOEt 6 :4) to
give the cyclic hemiaminals 5a and 5b as light yellow powders.
6-Benzyl-6,7-dihydro-7-hydroxy[1,2,5]oxadiazolo[3,4-d]pyrimidin-5(4H)-one (5a). Yield: 85%.
1
Light-yellow powder. M.p. 194–1968. H-NMR (DMSO): 4.40 (d, J¼15, CH2); 4.90 (d, J¼15, CH2);
6.10 (d, J¼9, CH, collapses to a s on D2O exchange); 7.20–7.30 (m, 5 arom. H); 7.40 (d, J¼9, OH,
exchangeable with D2O); 11.3 (s, NH, exchangeable with D2O). 13C-NMR (DMSO): 40.8 (CH2); 70.4
(OH); 120.8 (arom. CH); 120.9 (2 arom. CH); 120.9 (2 arom. CH); 130.8 (C); 140.4 (arom. C); 150.1 (C);
150.1 (C¼O).
6,7-Dihydro-7-hydroxy-6-phenyl[1,2,5]oxadiazolo[3,4-d]pyrimidin-5(4H)-one (5b). Yield: 80%.
1
Light-yellow powder. M.p. 182–1848. H-NMR (CDCl3): 6.35 (d, J¼8.7, CH, collapses to a s on D2O
exchange); 7.30–7.50 (m, 5 arom. H); 7.55 (d, J¼8.7, OH, exchangeable with D2O); 11.40 (s, NH,
exchangeable with D2O).
Synthesis of 1-Benzyl-3-(4-methyl-1,2,5-oxadiazol-3-yl)urea (3a), 1,3-Bis(4-methyl-1,2,5-oxadiazol-
3-yl)urea (10), and 1,3-Dibenzylurea (11). To a soln. of 4-methyl-1,2,5-oxadiazol-3-amine (9; 50 mg,
0.50 mmol) in toluene (1.5 ml), BnNCO (0.60 mmol, 0.08 ml) was added at r.t. The mixture was
irradiated in MW synthesizer at 300 W at 1708 for 20 min. After cooling, a white solid was collected by
filtration and washed with toluene. This solid was an adduct of 10 and 11 (yield 40%, molar ratio 1:1).
The remaining mixture was evaporated in vacuo and the crude residue was chromatographed on SiO2
(PE/AcOEt 8 :2) to give 3a (70 mg, 60%). White solid. M.p. 197–1998. 1H-NMR of 3a: (CDCl3) 2.35 (s,
Me); 4.55 (d, J¼5.7, CH2); 7.20–7.40 (m, 5 arom. H); 7.90 (br. s, NH, exchangeable with D2O); 9.90 (br. s,
NH, exchangeable with D2O). The adduct 10–11 was characterized by X-ray analysis.
2.5. General Procedure for the Synthesis of 3-(4-styryl-1,2,5-oxadiazol-3-yl)urea Derivatives (E)- and
(Z)-4a, and (E)- and (Z)-4b. A mixture 5a and 5b (50 mg, 0.20 mmol), resp., Bn(Ph3)PCl (90.4 mg,
0.23 mmol), K2CO3 (31.8 mg, 0.43 mmol), 18-crown-6 (cat. quantity), and THF (2 ml) as a solvent was
stirred at reflux for 1 h. The solvent was evaporated in vacuo, and the residue was treated with brine and
extracted with AcOEt (3ꢃ1 ml). The org. layer was evaporated in vacuo, and the crude product was
purified by flash chromatography (PE/AcOEt 7:3).
1-Benzyl-3-{4-[(E)- and (Z)-2-phenylethenyl]-1,2,5-oxadiazol-3-yl}urea ((E) and (Z)-4a). Yield:
50% of (E)-4a as a pale yellow solid (m.p. 192–1948) and 19% of (Z)-4a as a yellow solid (m.p. 170–
1728).
1
Data of (E)-4a. H-NMR (CDCl3): 4.59 (d, J¼5.7, CH2); 7.20 (d, J¼15.5, CH); 7.20–7.67 (m, 10
arom. H); 7.60 (d, J¼15.5, CH); 7.98 (t, J¼5.7, NH, exchangeable with D2O); 9.96 (br. s, NH,
exchangeable with D2O);
1
Data of (Z)-4a. H-NMR (CDCl3): 4.48 (d, J¼5.7, CH2); 6.44 (d, J¼12.2, CH); 7.11 (d, J¼12.2,
CH); 7.20–7.40 (m, 10 arom. H); 7.58 (t, J¼5.7, NH, exchangeable with D2O); 9.60 (s, NH, exchangeable
with D2O).
1-Phenyl-3-{4-[(E)- and (Z)-2-phenylethenyl]-1,2,5-oxadiazol-3-yl}urea ((E) and (Z)-4b). Yield:
55% of (E)-4b as a yellow solid (m.p. 201–2038) and 25% of (Z)-4b as a yellow solid (m.p. 173–1758).
Data of (E)-4b. 1H-NMR (CDCl3): 7.03 (d, J¼15.1, CH); 7.10–7.57 (m, 10 arom. H); 7.61 (d, J¼15.1,
CH); 8.70 (br. s, NH, exchangeable with D2O); 9.45 (br. s, NH, exchangeable with D2O).
Data of (Z)-4b. 1H-NMR (CDCl3): 6.51 (d, J¼12.1, CH); 7.03 (br. s, NH, exchangeable with D2O);
7.19 (d, J¼12.1, CH); 7.10–7.50 (m, 10 arom. H); 9.17 (br. s, NH, exchangeable with D2O).
3. Cell Cultures. The cancer cell lines were obtained from American Type Culture Collection. Human
breast cancer cell lines MDA-MB-468, and MDA-MB-231, and human colon cancer cell line SW620 were
maintained in RPMI 1640 (Gibco/BRL). Human colon cancer cell line HCT-116 was maintained in
McCoyꢁs 5A (Gibco/BRL). All culture media were supplemented with 10% heat-inactivated fetal bovine

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1251
serum (FBS; Gibco/BRL). Cell cultures were maintained at 378 under a humidified atmosphere of 5%
CO2 in an incubator.
4. Transient Transfection and Dual-Luciferase Assays. The STAT3 inhibitory activities of the new
synthesized compounds 3–5 were evaluated by a modified procedure of dual-luciferase assay in human
colorectal carcinoma cells HCT-116, characterized by uncontrolled expression of STAT3 [33].
Colorectal carcinoma HCT-116 cells were co-transfected with pSTAT-TA-Luc carrying firefly
luciferase gene (dependently expressed by STAT3 activity), and pRL-TK carrying renilla luciferase gene
(independently expressed by STAT3 activity). HCT-116 Cells were seeded at a density of 10ꢃ105 cells in
100 mm2 culture plate. Beetle luciferin and coelenterazine were used as substrates for the two enzymes,
resp. The cells were co-transfected with pSTAT3-TA-Luc (27 mg/plate) and internal control plasmid
pRL-TK (9 mg/plate) containing the Renilla luciferase gene. Cells were treated with the compounds at
concentration of 2 mm and incubated for 24 h. All plasmids used in this experiment were purchased from
Promega. The transfection was carried out using TransFectin (Bio-Rad) according to the manufacturerꢁs
protocol. After 5 h of transfection, the cells were trypsinized and seeded onto sterilized black bottom 96-
well plates at a density of 1ꢃ104 cells per well. On the following day, cells were treated with test
compounds and incubated for 24 h. Firefly and Renilla luciferase activities were measured using dual-
light reporter gene assay kit (Promega) on Wallac Victor2 (Perkin-Elmer, Inc., Wellesley, MA). Renilla
luciferase activity was determined to calibrate transfection efficiency and cytotoxicity of chemicals.
Relative STAT3 activity was calculated by dividing the firefly luciferase activity with Renilla luciferase
activity in each transfection experiment.
5. Cell Proliferation Assay. Cells were seeded at a density of 5,000 cells per well in 96-well plates in
RPMI 1640 or McCoyꢁs medium containing 10% FBS. They were replenished with fresh complete
medium containing either test compound or 0.1% DMSO. After incubation for 24 or 48 h, the cell
proliferation reagent WST-1 (Roche Applied Science) was added to each well. WST-1 Formazan was
quantitatively measured at 450 nm with an enzyme-linked immunosorbent assay reader (Bio-Rad).
6. X-Ray Crystallographic Analysis. The crystals of 1a and 5a were obtained by crystallization from
H2O/MeOH 1:2 as colorless prisms, while 3a, 10–11 and (E)-4a crystallized from MeOH/EtOH 1:1 soln.
as transparent needles. Crystals of (E)-4b were obtained from the slow evaporation of a MeOH soln.
They were mounted on an Enraf Nonius CAD-4 diffractometer using MoKa (a¼0.71073 ꢄ) radiation at
293(2) K. The lattice parameters were determined by least-squares refinements of 25 high-angle
reflections. The structures were solved by direct methods [34], and the refinements were carried out by
full-matrix least-squares. All non-H-atoms were refined anisotropically. The H-atoms of 1a, 3a, (E)-4a,
and (E)-4b were detected in a difference Fourier synthesis and refined with isotropic thermal factors,
while all H-atoms of 10–11 and 5a were introduced at calculated positions, in their described geometries,
and allowed to ride on the attached C-atom with fixed isotropic thermal parameters (1.2 Ueq of the parent
C-atom). Refinements were carried out with SHELX-97 [35]. Geometrical calculations were carried out
with the program PARST [36]. A summary of the crystal data, data collection, and structure refinement
is presented in Table 2.
CCDC-739436 (1a), CCDC-796642, (5a) CCDC-758866 (3a), CCDC-811038 (10–11), CCDC-
818273 ((E)-4a), and CCDC-846458 ((E)-4b) contain the supplementary crystallographic data for this
paper (excluding structure factors). These data can be obtained free of charge via www.ccdc.cam.ac.uk/
conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge
CB21EZ, UK; fax: (þ44)1223-336-033; or deposit@ccdc.cam.ac.uk).
Supplementary Information. Elemental analyses and 1H-NMR spectra of compounds 1–5, as well as
crystallographic data of 1a, 5a, 3a, 10–11, (E)-4a and (E)-4b are available from the corresponding
author.
The authors thank Prof. Byoung-Mog Kwon, Laboratory of Chemical Biology and Genomics, Korea
Research Institute of Bioscience and Biotechnology and Department of Biomolecular Science, Korea
University of Science and Technology, for the biological tests, and the University of Milano (PUR 2008)
for the financial support.

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Received October 19, 2011