On the behaviour of the (Z)-phenylhydrazones of some 5-alkyl-3-benzoyl-1,2,4-oxadiazoles in solution and in the gas phase: kinetic and spectrometric evidence in favour of self-assembly

Article abstract of DOI:10.1016/j.tet.2007.11.014

Rate constants, k_A,R, for the rearrangement of the (Z)-phenylhydrazones (1a-e) of a series of 5-alkyl-3-benzoyl-1,2,4-oxadiazoles substituted at C(5) with linear alkyl chains of different length (from C₄ up to C₁₂) into th

Full text of DOI:10.1016/j.tet.2007.11.014

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 733e740
www.elsevier.com/locate/tet
On the behaviour of the (Z )-phenylhydrazones of some
5-alkyl-3-benzoyl-1,2,4-oxadiazoles in solution and in the gas
phase: kinetic and spectrometric evidence in favour of self-assembly
Antonella Fontana a, Susanna Guernelli b, Paolo Lo Meo c, Elisabetta Mezzina b,
b
b,
a
Stefano Morganti b, Renato Noto c, , Egon Rizzato , Domenico Spinelli , Romina Zappacosta
*
*
a Dipartimento di Scienze del Farmaco, Universita` ‘‘G. d’Annunzio’’, Via dei Vestini 31, 66013 Chieti, Italy
b Dipartimento di Chimica Organica ‘‘A. Mangini’’, Universita` di Bologna, Via San Giacomo 11, 40126 Bologna, Italy
c Dipartimento di Chimica Organica ‘‘E. Paterno`’’, Universita` di Palermo, Viale delle Scienze, Parco d’Orleans II, pad. 17, 90128 Palermo, Italy
Received 6 September 2007; received in revised form 16 October 2007; accepted 1 November 2007
Available online 6 November 2007
Abstract
Rate constants, kA,R, for the rearrangement of the (Z )-phenylhydrazones (1aee) of a series of 5-alkyl-3-benzoyl-1,2,4-oxadiazoles
substituted at C(5) with linear alkyl chains of different length (from C4 up to C12) into the relevant 4-acylamino-2,5-diphenyl-1,2,3-triazoles
(2aee) have been measured in dioxan/water in the base-catalyzed region (pSþ 10.5e12.6). For each substrate log kA,R are linearly related to
pSþ. The significant decrease of the slopes of these straight lines (from 0.96 down to 0.78) upon increasing the length of the linear alkyl chain
at C(5) and that of the reactivity (down to 14e26%) upon increasing the substrate concentration suggest a decrease of the polarity of the ‘actual’
reaction medium and provide indirect evidence of the tendency of the substrates (Z )-1aee to self-assemble. To confirm the above outcome direct
1
evidence of the formation of self-assemblies in solution were obtained from H NMR and spectrofluorimetry measurements while ESI-MS
experiments point out the presence of aggregated substrates also in the gas phase.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
such as a perfluoroalkyl,3 favoured new irreversible ring-
degenerate rearrangements3a as well as new ANRORC-like
(Addition of a Nucleophile followed by Ring Opening and
Ring Closure) processes eventually associated with ring-enlar-
gement.3bef
Our research group is involved by long time in the kinetic
study of the mononuclear rearrangement of heterocycles
(MRH) or BoultoneKatritzky reaction (BKR).1,2
Our interest in this and in general in ring interconversion of
azoles is based on both the intriguing mechanistic aspects of
the rearrangement (depending on both the proton concentration
and the structure of the investigated substrates) and the wide
synthetic applicability of ring interconversion of 1,2,4-oxadi-
azoles.1e3 E.g., a strong electron-withdrawing substituent,
In order to extend the study of the reactivity in MRH of
(Z)-phenylhydrazones of 3-benzoyl-1,2,4-oxadiazoles, we have
synthesized a new series of 5-alkyl-substituted substrates with
the aim of evaluating the effect of linear alkyl chains of differ-
ent lengths (from C4 up to C12) on the rearrangement rates of
(Z )-1aee into the relevant triazoles 2aee (Scheme 1).
The presence of long linear alkyl chains at C(5) of the
1,2,4-oxadiazole ring could favour self-assembly and conse-
quently could affect the course of the rearrangement. In this
paper we will therefore investigate the supramolecular behav-
iour of the title substrates by collecting evidence on their abil-
ity to self-assemble both in solution (by using kinetics,
* Corresponding authors. Tel.: þ39 091 596919 (R.N.); tel.: þ39 051
2095689; fax: þ39 051 2095688 (D.S.).
E-mail addresses: rnoto@unipa.it (R. Noto), domenico.spinelli@unibo.it
(D. Spinelli).
0040-4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.11.014

734
A. Fontana et al. / Tetrahedron 64 (2008) 733e740
The slopes of the rate profiles (Fig. 1) decrease from 0.96
C6H5
H
N
C6H5
C6H5
down to 0.78 on increasing the length of the linear 5-alkyl
chain (Table S1). The rather low slope4e6 values obtained
for (Z )-1d and (Z )-1e suggest a tendency of these substrates
to self-assemble into clusters of different size. The observed
rate constants are probably the outcome of different contribu-
tions from mononuclear rearrangement of each aggregated or
monomer form of the substrate.
R
B-
N
N
N
C6H5
-
δ
H B
N
N
Nα
C6H5
O
N
N
N
N
H
N
R
R
Oδ-
O
C6H5
2a-e
TS
(Z)-1a-e
a: R = C4H9
e: R = C12H25
d: R = C10H21
b: R= C6H13 c: R = C8H17
Scheme 1. Base-catalyzed rearrangement of the (Z )-phenylhydrazones 1aee
into the relevant triazoles 2aee.
In order to gain more information on the association pro-
cess we carried out a further kinetic investigation on the
MRH at different concentrations of (Z )-phenylhydrazone of
3-benzoyl-5-hexyl-1,2,4-oxadiazole (1b) (from 3.0ꢁ10ꢂ5 up
to 5.0ꢁ10ꢂ4 M), keeping constant the borate buffer concentra-
tion. The measured rate constants at different pSþ values are
reported in Table S2 of Supplementary data and the plots of
log kA,R versus [(Z )-1b] at three different pSþ in Figure 2.
The data in Figure 2 point to an ‘apparent’ significant de-
crease, observed for the first time, of the reactivity upon in-
creasing the concentration of (Z )-1b (14e26% in the pSþ
range 12.1e10.8) up to a critical (Z )-1b concentration. This
reactivity decrease, at each investigated pSþ, could be related
to a change in the state of aggregation of the substrate. Above
the critical breakpoint concentrations the reactivity reaches
a minimum plateau value suggesting that the MRH process
is brought about only by a largely prevailing and less reactive
substrate self-assembled into an aggregate with an optimum
aggregation number. Indeed at this high (Z )-1b concentration
the aggregated form of the substrate should prevail over the
monomer form. The breakpoint concentrations depend slightly
on the pSþ value, but anyway (Z )-1b appears to be completely
aggregated above 1ꢁ10ꢂ4 M.
1H NMR spectrometry, and spectrofluorimetry) and in the gas
phase (by using ESI-MS).
2. Results and discussion
2.1. Kinetic investigation
The rearrangement of (Z )-1aee into 2aee has been studied
in dioxan/water (D/W; 1:1, v/v) at different proton concentra-
tions. The apparent first-order rate constants, kA,R, and the cor-
responding rate profile (plot of log kA,R vs pSþ) are reported in
Table S1 of Supplementary data and Figure 1, respectively.
Figure 1 highlights that in the investigated pSþ range: (a)
the reactivity is largely affected by proton concentration as
has already been observed for other base-catalyzed
MRHs;2,4,5 (b) all the (Z )-phenylhydrazones (1aee) investi-
gated show similar reactivity, e.g., at pSþ ca. 11.5 the reactiv-
ity ratio between the investigated substrates is 1.00ꢀ0.15 in
line with the mechanism proposed (SNi)2aed and the strictly
similar electronic effects of the investigated 5-alkyl groups;2e
(c) only at the lowest and at the highest investigated pSþ does
the reactivity of the five substrates become significantly differ-
This behaviour of (Z )-1b well agrees with that observed for
the rearrangement of the same substrate in water/b-CD at sim-
ilar proton concentrations.2f In the latter case the much larger
decrease (ca. 31) of the rearrangement rate with respect to the
reaction in D/W was ascribed to the more hydrophobic envi-
ronment of the cavity of b-CD compared with D/W solutions.
In the present case the long linear chain at C-5 favours the
ent, spanning from (kA,R)1a/(k )
A,R 1e 0.74 up to 1.7 at pSþ 10.5
and 12.5, respectively.
-2.0
-2.7
-2.8
(Z)-1a
(Z)-1b
(Z)-1c
(Z)-1d
(Z)-1e
-2.5
-3.0
-3.5
-4.0
-4.5
-3.4
-3.6
-3.8
-4.0
10.5
11.0
11.5
pS+
12.0
12.5
0.0000
0.0002
[(Z)-1b] (M)
0.0004
Figure 1. Plot of log kA,R for the rearrangement of (Z )-1aee into 2aee in D/W
at 313 K versus pSþ.
Figure 2. Plot of log kA,R for the rearrangement of (Z )-1b into 2b in D/W
versus concentration of (Z )-1b at pSþ 10.80 ( ), 11.38 ( ), and 12.10 ( ).

A. Fontana et al. / Tetrahedron 64 (2008) 733e740
735
formation of aggregated substrate, which provides a more hy-
drophobic environment in which the rearrangement takes
place.
2.2. Spectrometric evidence for self-assembly
The self-association of (Z )-1aee and also of 2aee has
1
been studied by H NMR and spectrofluorimetry in solution
and by ESI-MS in the gas phase. Strong support for the occur-
rence of self-assembly has been obtained by all the three
techniques.
1
Figure 4. H NMR spectra of a 1.1ꢁ10ꢂ4 M solution of (Z )-1a recorded at T
298 K in solution of D2O/CD3OD (v/v): (a) 10:1, (b) 4:1, (c) 2.5:1, (d) 1.7:1,
(e) 0:1.
1
2.2.1. H NMR measurements
1
We examined the H NMR behaviour of the only (Z )-1a
(R¼C4H9) derivative, because of the very low solubility of
the other Z-phenylhydrazones (1bee) in water. Nevertheless
a small amount of CD3OD (3% v) was added to the D2O so-
lution in order to ensure the complete sample dissolution.
Spectra were recorded at different (Z )-1a concentrations
(from 1ꢁ10ꢂ4 up to 8.0ꢁ10ꢂ4 M) in a wide range of temper-
ature (298e333 K).
By increasing the temperature from 298 to 333 K an im-
provement of the resolution of all signals of the 1H NMR spec-
trum of 1.0ꢁ10ꢂ4 M (Z )-1a solution (Fig. 3) was observed, in
agreement with a disaggregation occurring at higher tempera-
ture. Figure 3 points out higher shifts for aliphatic protons with
respect to the aromatic ones, suggesting that only aliphatic
hydrophobic chains are involved in the aggregation process.
To examine whether the aggregation process occurs for
Figure 5. 1H NMR spectra of a 1.2ꢁ10ꢂ4 M solution of 2a recorded at T 298 K
in solution of D2O/CD3OD (v/v): (a) 6:1, (b) 4:1, (c) 1.5:1, (d) 1.2:1, (e)
1:1.75, (f) 0:1.
Marked upfield shift (red dotted lines) and broadening of
both aromatic and aliphatic signals can be observed for (Z )-
1a on decreasing the fraction of CD3OD (see spectra aed of
Fig. 4).
1
both (Z )-1a and the relevant triazole 2a, H NMR spectra
were recorded by using solutions containing increasing frac-
tions of CD3OD (see Figs. 4 and 5, respectively), a solvent
in which the intermolecular interactions are minimized.
As a matter of fact a different effect of the structure of the
side chains on the aggregation process could be expected. The
substituent bound to the C(5) of the 1,2,4-oxadiazole ring in
(Z )-1a is a linear alkyl chain, which can give only van der
Waals interactions. In contrast in 2a the alkyl chain is bound
to the C(4) of the 1,2,3-triazole ring via an amide group, which
can give polar (hydrogen bonds and dipoleedipole) interac-
tions with other 2a molecules or with the solvent. Spectra of
the two compounds were also measured in pure CD3OD in
order to obtain data for the non-aggregated molecule (upper
traces of Figs. 4 and 5).
The behaviour of 2a is quite different (Fig. 5): its aromatic
and aliphatic signals appear more and more resolved on going
from spectrum a to e, while neither marked shifts nor broaden-
ing of the peaks are observed. The signals maintain their
spinespin couplings even at a high concentration of D2O
where the signal-to-noise ratio is less favourable (spectrum
a). It is quite evident that triazole 2a does not self-assemble
in solution in these experimental conditions, notwithstanding
the presence of the alkyl chain. The polar amido group favours
the solvation with water or methanol (via hydrogen bonds7)
over substrateesubstrate interactions.
Our findings concerning (Z )-1a and 2a (R¼C4H9) could be
reasonably extended at least to short chain derivatives. More-
over our experiments do not exclude the self-assembly in so-
lution of triazoles with longer alkyl chains as it has been
detected in the gas phase where the absence of solvent mole-
cules allows only substrateesubstrate interactions (see below).
2.2.2. Spectrofluorimetric measurements
The tendency of (Z )-1b and (Z )-1e (R¼C6H13 and C12H25,
respectively) to self-assemble was tested also through spectro-
fluorimetry by using 9-diethylamino-5H-benzo[a]phenoxazin-
5-one (Nile Red) as the fluorescent probe. The solvatochromic
fluorescent probe Nile Red has already been proved to be very
Figure 3. Spectrum of a 1.0ꢁ10ꢂ4 M solution of (Z )-1a in D2O/CD3OD (97:3)
at: (a) 298 K; (b) 333 K.

736
A. Fontana et al. / Tetrahedron 64 (2008) 733e740
efficient in its capability to identify different aggregate mor-
phologies formed by amphiphilic mixtures.8
of magnitude. This effect reveals that the observed phenome-
non is not a simple aggregation process from monomers to mi-
celles but a rather more complex transformation of substrate
into different aggregates (i.e., dimers, trimers and eventually
small clusters).
As a matter of fact, classical techniques, used for the cmc
(critical micellar concentration) determination, such as tensio-
metry9 and solubilization of water insoluble dye-molecules,9
were not able to provide evidence for the formation of true mi-
celles (data not shown). It is generally accepted that cmc
strongly depends on the length of the hydrophobic group.9
In contrast the C6H13-substituted (Z )-1b appears to have com-
parable cac values. This suggests that aggregates of different
type and dimensions are obtained from the two substrates.
Spectra were recorded at different concentrations of the
investigated (Z )-phenylhydrazones (from 1.9ꢁ10ꢂ8 up to
5.5ꢁ10ꢂ4 M) at 298 K (data at increasing concentrations of
(Z )-1b are reported in Fig. S1 of Supplementary data). The
fluorescence intensity rises with increasing [(Z )-1b] and the
maximum emission wavelength, lmax, exhibits a remarkable
blue shift [from 648 to 582 nm for (Z )-1b (Fig. 6a and
Fig. S1 of Supplementary data) and from 648 to 575 nm for
(Z )-1e (Fig. S2 of Supplementary data)].
The cac (critical aggregation concentration) can be evalu-
ated either at the onset of the fluorescence intensity increase
in the plot of fluorescence intensity emission versus log [sub-
strate]8a or at the inflection point in the plot of lmax versus log
[substrate]8b (Fig. 6a and b, respectively).
2.2.3. ESI-MS measurements
The average cac calculated are 3ꢁ10ꢂ5 and 5ꢁ10ꢂ5 M for
(Z )-1b and (Z )-1e, respectively. Figure 6b shows a not very
steep transition from the lmax of the monomer to that of the
aggregate covering an interval of [(Z )-1b] of about 2 orders
Interestingly, direct evidence for self-assembly for all the
investigated compounds [(Z )-1aee and 2aee] have been ob-
tained by electrospray ionization mass spectrometry (ESI-
MS). Several recent literature examples have been reported
on the use of ESI-MS measurements to detect and evaluate
self-association processes in the gas phase.10e12 In an ESI-
MS analysis, charged species (hosteguest complexes or self-
assembly systems) are transferred from the small droplets of
solution into gas phase under relatively mild conditions, gen-
erally without fragmentation.13 The principal problem in using
ESI-MS in supramolecular chemistry is to determine whether
the peaks present in the spectra correspond to species present
in solution, or whether these peaks are the result of processes
occurring in the mass spectrometer. If this problem is solved,
for example, by comparing data obtained with ESI-MS and
data obtained with other independent techniques, ESI-MS
data can give new information on the supramolecular behav-
iour of the system.
The ESI-MS spectra were recorded in positive ion mode
with a variation of the concentration of the substrate, S,
from 1.0ꢁ10ꢂ6 up to 2.0ꢁ10ꢂ4 M [S dissolved in water/ace-
tonitrile (1:1, v/v)]. All the spectra showed the molecular
ion peak and a high-intensity signal corresponding to the
dimer (S)2. On increasing [S] a weak, double charged signal
corresponding to the trimer (S)3 was also observed.
In Figure 7a and b the spectra of 2b at 3.0ꢁ10ꢂ6 M and
1.1ꢁ10ꢂ4 M, respectively, are reported as examples of the
general behaviour displayed by all the examined compounds.
Figure 7a shows the presence of different peaks ascribable
to the monocharged monomer {m/z¼349, 371, 412 corre-
sponding to [2bþH]þ, [2bþNa]þ and [2bþacetonitrileþNa]þ,
respectively} and dimer {m/z¼719 corresponding to
[(2b)2þNa]þ}. At higher substrate concentrations the peak
of the double charged trimer [(2b)3þKþH]2þ (at m/z¼542)
is also detected.
(a)
60
50
40
30
20
1.6 x 10-5
M
10
0
10-7
10-6
10-5
10-4
[(Z)-1b] (M)
(b)
650
640
630
620
610
600
590
580
-5 M
4.4 x 10
The monotonic increase of (S)2 with increasing [S] {the
peak corresponding to the dimer going from 25% at
[2b]¼3.0ꢁ10ꢂ6 M (Fig. 7a) up to 100% at [2b]¼1.1ꢁ
10ꢂ4 M (Fig. 7b)}, the significant detection of trimers only
at the highest [S] (Fig. 7b) and the appearance of the same
peaks at all the investigated cone voltage (from 20 to 80 V,
10-6
10-7
10-8
10-5
10-4
10-3
[(Z)-1b] (M)
Figure 6. (a) Plot of the fluorescence intensity (arbitrary units) versus the con-
centration of (Z )-1b. (b) Plot of the lmax (nm) versus the concentration of
(Z )-1b.

A. Fontana et al. / Tetrahedron 64 (2008) 733e740
737
Table 1
Major peaks and relative abundance of the ESI-MS spectra of 2b in the pres-
ence of cetyltrimethylammonium bromide, CTAB
Molar ratio
CTAB:2b
Major peak observeda
Relative abundance (%)
1:0.2
1:0.5
1:1
632.4 [CTA$2b]þ
649.4 [CTABþCTA]þ
719.4 [(2b)2þNa]þ
735.3 [(2b)2þK]þ
632.4 [CTA$2b]þ
649.4 [CTABþCTA]þ
719.2 [(2b)2þNa]þ
735.3 [(2b)2þK]þ
632.4 [CTA$2b]þ
649.4 [CTABþCTA]þ
719.3 [(2b)2þNa]þ
735.3 [(2b)2þK]þ
50
100
Trace
Trace
100
48
Trace
Trace
100
46
Trace
Trace
a
The peak corresponding to the heterodimers lacks a bromide ion, accord-
ingly the signals of the dimer of the surfactant correspond to species with only
one bromide ion (in table, CTA: cetyltrimethylammonium ion).
constants of glycopeptide antibiotics, which form both homo
and heterodimers.14,15 The validity of this method relies on
the assumption that the ionization probability of the monomer
should be similar to that of the dimer for every compound.
Moreover the published15 good agreement between dimeriza-
tion constants determined by ESI-MS and those acquired in
solution by other independent and complementary methods
1
(e.g., H NMR) demonstrates that the ESI-MS spectra reflect,
at least qualitatively, the real stability of the complex in
solution.
Figure 7. (a) ESI-MS spectrum of a 3.0ꢁ10ꢂ6 M solution of 2b in water/ace-
tonitrile (1:1, v/v); ACN: acetonitrile. (b) ESI-MS spectrum of a 1.1ꢁ10ꢂ4 M
solution of 2b in water/acetonitrile (1:1, v/v); ACN: acetonitrile.
Nevertheless we determined the dimerization constants for
S from only ESI-MS measurement with the main aim of com-
paring the stability of the dimer for the different investigated
compounds and aware of the fact that the obtained equilibrium
constants refer to the experimental conditions adopted for the
ESI-MS measurement, the studied equilibrium being:
data not shown) suggest that the detected species are not
artifacts formed in the mass spectrometer during the measure-
ment but rather correspond to species already present in
solution.14,15
The absence of any evidence of aggregation in the instance
of the parallelly studied 2a from 1H NMR measurements could
be due therefore to the solvation ability towards 2b of the
CD3OD/D2O mixtures with respect to the situation observed
by ESI-MS in the gas phase (i.e., in the absence of solvents).
In order to confirm the ability of the studied S to self-
associate, the ESI-MS spectra of 2b were recorded in the pres-
ence of CTAB (at concentration below its cmc) in ion positive
mode at increasing [2b]. The peak intensity at m/z¼632 (de-
riving from a 1:1 association between the cationic surfactant
and 2b) increases with increasing [2b] (data in Table 1) and
eventually becomes the base peak. Thus, 2b shows a high ten-
dency to aggregate only (see the above reported 1H NMR data
concerning 2a) with molecules with a remarkable lipophilic
domain (e.g., CTAB contains a linear alkylic chain of 16 car-
bon atoms).
S þ S#ðSÞ2
ð1Þ
The equilibrium concentrations of S and (S)2 were calculated
from the sum of the ion abundances (measured by peak areas)
of all the monomeric and dimeric forms, respectively. The ap-
parent dimerization constants, Kdim, were calculated by Eq. 2.
½ðSÞꢃ2
Kdim
¼
ð2Þ
2
½Sꢃ
The Kdim values for (Z )-1aee and 2aee were estimated at low
concentration of S (1.0ꢁ10ꢂ5 M for 1,2,4-oxadiazoles and
2.0ꢁ10ꢂ6 M for 1,2,3-triazoles) in order to avoid the forma-
tion of trimers: their values are reported in Table 2 and give
information concerning the trend of dimer stability as a func-
tion of the structure of S.
Interestingly, the triazoles 2aee showed a greater ability to
give dimers, with association constants 6e15 times higher
than those of the oxadiazoles (Z )-1aee. As expected, Kdim
values of (Z )-1 and 2 increase on increasing the linear alkyl
chain length. The effect is particularly strong for the longest
Recently, the binding constants of both small organic hoste
guest complexes or large biological complexes have been de-
termined16e18 by ESI-MS as the relative abundances of the
complexes detected by this technique have been proved16 to
correlate with the equilibrium distribution in solution. For ex-
ample ESI-MS has been used to measure the association

738
A. Fontana et al. / Tetrahedron 64 (2008) 733e740
1H NMR, spectrofluorimetry, and ESI-MS measurements. As-
Table 2
Dimerization constantsa of (Z )-1aee and 2aee
sociation constants, obtained by ESI-MS analysis, provide
qualitative information about the specific interactions, not eas-
ily detectable in solution, involved in the dimerization pro-
cesses of the different (Z )-1aee and 2aee and allow to
differentiate the contributions of the linear alkyl chains and
of the amide group to the aggregation phenomena.
On the whole the combined kinetic and spectrometric study
has provided evidence of the self-assembly in both (Z )-1aee
and 2aee.
b
S
Kdim (Mꢂ1
)
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
a
5.4ꢁ103
6.0ꢁ103
7.2ꢁ103
1.5ꢁ104
2.2ꢁ104
6.9ꢁ104
8.1ꢁ104
8.9ꢁ104
9.4ꢁ104
3.2ꢁ105
4. Experimental
The values were averaged over 10 experiments recorded at different
concentration of S.
b
The errors were estimated to be approximately 15%.
4.1. General
Synthesis and characterization of 5-alkyl-3-benzoyl-1,2,4-
oxadiazoles (AeE), (E )- and (Z )-phenylhydrazones of 5-alkyl-
3-benzoyl-1,2,4-oxadiazoles (1aee), and the relevant triazoles
(2aee) are reported in Supplementary data. HRMS were
recorded on a Thermo Finnigan Mat95XP apparatus.
alkyl chain derivatives [(Z )-1d, (Z )-1e, and 2e], whereas Kdim
values are very similar for those with shorter alkyl chains [see
Kdim values for (Z )-1aec and 2aed].
The dependence of Kdim on linear alkyl chain length at C(5)
in (Z )-1aee indicates that the van der Waals interactions must
contribute significantly to the self-assembling, particularly for
the derivatives with the longest alkyl chains. However, dimers
from triazoles 2aee turn out to be more stable than oxadiazole
dimers suggesting that intermolecular hydrogen bonds, partic-
4.2. General procedure for the synthesis of 5-alkyl-3-
benzoyl-1,2,4-oxadiazoles
5-Alkyl-3-benzoyl-1,2,4-oxadiazoles (A, CeE) were pre-
pared through the reactions of phenylglyoxyloyl chloride
oxime (u-chloroisonitrosoacetophenone) and the respective
oximes (pentanal, heptanal, nonanal, undecanal, and tridecanal
oximes) in refluxing toluene according to literature.19 The
crude product of reaction was purified by chromatography
on silica gel (elution with a gradient ranging from 98:2 to
90:10 petroleum ether/ethyl acetate). The yields (not opti-
mized) of the pure ketones ranged between 20 and 30%.
ularly strong for amides,7 can significantly contribute to Kdim
.
3. Conclusions
Rate constants for MRH of (Z )-phenylhydrazones of a se-
ries of 5-alkyl-3-benzoyl-1,2,4-oxadiazoles 1aee with linear
alkyl chains of significantly different length (from C4 up to
C12) at the same pSþ are only slightly different, in agreement
with the similar electronic and the negligible steric effects of
linear alkyl chains of different length. It had already been
proved2e that the rearrangement rates of the (Z )-phenylhydra-
zones of some 5-alkyl-3-benzoyl-1,2,4-oxadiazoles (alkyl¼
4.3. General procedure for the synthesis of the (E )- and
(Z )-phenylhydrazones of 5-alkyl-3-benzoyl-1,2,4-
oxadiazoles (1a, cee)
i
t
Me, Et, Pr and Bu) in D/W are only moderately affected by
the substituent of the 1,2,4-oxadiazole ring.
The title compounds were prepared and purified according
to the methods reported.2cef Phenylhydrazine (8 mmol) was
added to a solution of 5-alkyl-3-benzoyl-1,2,4-oxadiazole
(4 mmol) and acetic acid (0.2 mL) in ethanol (80 mL). The
mixture was kept at room temperature in the dark. After
48 h the ethanol solution was concentrated at reduced pressure
to a small volume and the residue extracted with ethyl ether.
The combined ethereal extracts were washed repeatedly with
water, dried with sodium sulfate and evaporated at reduced
pressure. The residue was chromatographed on silica gel
(eluted with a gradient ranging from 98:2 to 90:10 petroleum
ether/ethyl acetate) to give the (Z )- and, in case, the
(E )-isomer.
An interesting outcome obtained from kinetic data is the
significant decrease of the slope values of the lines obtained
by plotting the logarithmic rate constants versus pSþ (from
0.96 down to 0.76) on going from the substrate with the short-
est alkyl chain (C4), (Z )-1a, to the substrate with the longest
alkyl chain (C12) at C(5), (Z )-1e. This decrease appears to
be indicative of a variation in the polarity of the microenviron-
ment in which the rearrangement process takes place and
therefore of the tendency of the (Z )-1aee to self-assemble. In-
deed, slopes significantly lower than unity indicate that the
base-catalyzed rearrangement does occur in a medium less
polar than the hydrophilic D/W mixture.2,4e6
A further confirmation of the formation of aggregated sub-
strates comes from the first time observed dependence of the
kA,R of the investigated MRH on the (Z )-1b concentration.
Direct evidence for self-assembly of the examined (Z )-phe-
nylhydrazones (1aee) of 3-benzoyl-5-alkyl-1,2,4-oxadiazoles
both in solution and in the gas phase have been obtained by
4.4. Heat-induced rearrangement of the (Z )-
phenylhydrazones (1a, cee) into triazoles (2a, cee)
Thermally induced rearrangement of (Z )-1a, cee gave 2a,
cee by heating the samples (0.2 g) in a preheated oil-bath

A. Fontana et al. / Tetrahedron 64 (2008) 733e740
739
(150 ꢄC) for a few minutes. By purification on a silica gel
column (petroleum ether/ethyl acetate 85:15) triazoles were
obtained in practically quantitative yields.
preferentially solubilizes, was confirmed also by the blue shift
of the lmax of Nile Red observed at similar oxadiazole
concentrations.
4.5. Kinetic measurements
4.8. ESI-MS measurements
The kinetics in D/W (1:1, v/v) were followed spectrophoto-
metrically as previously described2cef by measuring the disap-
pearance of (Z )-1aee at the wavelengths of their absorption
maxima, where the absorptions of the relevant triazoles 2aee
were negligible. An operational pH scale, pSþ,2c was estab-
lished in aqueous dioxan by employing the pKa values of acids
determined by interpolation from the data reported by Harned
and Owen.20 For D/W (1:1, v/v) the pH-meter reading after
calibration against buffers was not significantly different
from pSþ, requiring a correction of only þ0.16.
ESI-MS spectra were acquired with a single quadrupole
mass spectrometer. A syringe driven by a pump was used
for direct injection of the sample into the instrument (ZMD
Micromass single quadrupole operating at m/z¼4000). Chang-
ing the cone voltage from 80 to 20 V did not change signifi-
cantly the absolute intensity of the peaks.
To minimize the influence of variations in instrumental con-
ditions on the reliability of mass spectra, the ESI-MS parameters
(i.e., the pressure of the gas, the desolvation temperature, the
capillary and cone voltages, etc.) were kept rigorously constant
from run to run in each series of experiments. In particular a
capillary voltage of 2.70 kV and a cone voltage of 20 V were
applied; a desolvation temperature of 150 ꢄC was used.
The solutions of (Z )-1aee and 2aee in acetonitrile/water
(1:1, v/v) were introduced at a flow-rate of 15 mL minꢂ1. For
the determination of dimerization constants the errors were es-
timated to be approximately 15%. For the measurements with
surfactant a cone voltage of 30 V was applied. The values of
relative abundance were averaged at least from 10 independent
experiments.
Pseudo-first-order conditions were ensured by using a large
excess of borate buffer (1.25ꢁ10ꢂ2 M); the concentration of
(Z )-1aee was 5.00ꢁ10ꢂ5 M. Kinetic measurements for (Z )-
1b were carried out at variable substrate concentration (from
3.0ꢁ10ꢂ5 to 5.0ꢁ10ꢂ4 M). The spectrophotometric measure-
ments were collected with a double-beam spectrophotometer
(Varian Cary 1E). The rate constants are accurate within ꢀ3%.
4.6. NMR measurements
1H and 13C NMR spectra of all synthesized compounds
were carried out on spectrometers (Varian Gemini) operating
at 300 (proton) and 75.5 MHz (carbon) or at 200 and
50 MHz for proton and carbon, respectively, using the solvent
peak as internal reference. Chemical shifts are reported in
parts per million (d scale).
In the experiments with CTAB the monomeric surfactant
gave very intense signals that could mask the peaks of other
species present in the solution. We have therefore examined
the spectral range m/z¼330e750, renormalizing the relative
abundance. In every case mass spectra were averaged over
typically 50 scans.
1D and 2D NMR spectra were recorded on spectrometers
operating at 600 or at 400 MHz (Varian Mercury). In order
1
to completely solubilize the substrate, the H NMR experi-
Acknowledgements
ments were performed in mixed solutions of D2O/CD3OD us-
ing residual HOD as an internal standard (4.76 ppm at 298 K).
ROESY data were collected on a Varian spectrometer oper-
ating at 600 MHz, using a 90ꢄ pulse width of 6.1 ms and a spec-
tral width 6000 Hz in each dimension, respectively. The data
were recorded in the phase sensitive mode using a CW spin-
lock field of 2 KHz, without spinning the sample. Acquisitions
were recorded at mixing times 100e500 ms. Other instrumen-
tal settings are: 128 increments of 2K data points, 32e64
scans per t1, 1.5 s delay time for each scan.
The authors thank M.I.U.R. (Rome) [PRIN 2005
(2005034305) and PRIN 2006 (2006034372)] for the financial
supports. Investigations supported also by the Universities of
Bologna and Palermo (ex-60%, funds for selected research
topics).
Supplementary data
Kinetic constants for MRH of compounds 1aee, fluores-
cence emission spectra of Nile Red on increasing the concen-
tration of (Z )-1b, plot of the fluorescence intensity versus the
concentration of (Z )-1e. Plot of the lmax (nm) versus the con-
4.7. Fluorimetric measurements
1
Steady-state emission spectra were recorded on a spectroflu-
orimeter Jasco FP-6200, with a band-pass of 5 nm and an exci-
tation l¼550 nm. Aqueous solutions (5.0ꢁ10ꢂ7 M) of the
fluorescent probe Nile Red were used for all the measurements.
On addition of different aliquots of a mother solution of the
oxadiazoles (Z )-1b and (Z )-1e in dioxan to the aqueous solu-
tion of Nile Red, fluorescence emission remained very low up
to a critical aggregation concentration (cac).8 The formation of
aggregates from (Z )-1b and (Z )-1e, into which Nile Red
centration of (Z )-1b, characterization and H NMR spectra of
investigated compounds are provided. Supplementary data
associated with this article can be found in the online version, at
doi:10.1016/j.tet.2007.11.014.
References and notes
1. (a) Boulton, A. J.; Katrizky, A. R.; Hamid, A. J. Chem. Soc. C 1967,
2005e2007; (b) Boulton, A. J. Lectures in Heterocyclic Chemistry; Hetero

740
A. Fontana et al. / Tetrahedron 64 (2008) 733e740
Corporation: Provo, UT, July 1973; (c) Afridi, A. S.; Katrizky, A. R.;
8. (a) Coutinho, P. J. G.; Castanheira, E. M. S.; Rei, M. C.; Elisabete, M.;
Oliveira, C. D. R. J. Phys. Chem. B 2002, 106, 12841e12846; (b) Stuart,
M. C. A.; van de Pas, J. C.; Engberts, J. B. F. N. J. Phys. Org. Chem. 2005,
18, 929e934; (c) Seliskar, C. J.; Brand, L. J. Am. Chem. Soc. 1971, 93,
5414e5420.
Ramsden, C. A. J. Chem. Soc., Perkin Trans. 1 1976, 315e320; (d)
Katrizky, A. R.; Gordev, M. F. Heterocycles 1993, 35, 483e518; (e)
Van der Plas, H. C. Ring Transformations of Heterocycles; Academic:
`
London, 1973; Vols. 1 and 2; (f) L’Abbe, G. J. Heterocycl. Chem. 1984,
21, 621e638.
9. Clint, J. H. Surfactant Aggregation; Chapman and Hall: New York, NY,
1992; pp 82e129.
2. (a) Ruccia, M.; Vivona, N.; Spinelli, D. Adv. Heterocycl. Chem. 1981, 29,
141e169; (b) Vivona, N.; Buscemi, S.; Frenna, V.; Cusmano, G. Adv.
Heterocycl. Chem. 1993, 56, 49e154; (c) Cosimelli, B.; Frenna, V.;
Guernelli, S.; Lanza, C. Z.; Macaluso, G.; Petrillo, G.; Spinelli, D.
J. Org. Chem. 2002, 67, 8010e8018; (d) Bottoni, A.; Frenna, V.; Lanza,
C. Z.; Macaluso, G.; Spinelli, D. J. Phys. Chem. A 2004, 108, 1731e1740;
(e) Frenna, V.; Vivona, N.; Consiglio, G.; Spinelli, D. J. Chem. Soc.,
Perkin Trans. 2 1984, 541e545; (f) Guernelli, S.; Lo Meo, P.; Morganti,
S.; Noto, R.; Spinelli, D. Tetrahedron 2007, 63, 10260e10268.
3. (a) Buscemi, S.; Pace, A.; Pibiri, I.; Vivona, N.; Lanza, C. Z.; Spinelli, D.
Eur. J. Org. Chem. 2004, 974e980; (b) Buscemi, S.; Pace, A.; Pibiri, I.;
Vivona, N. J. Org. Chem. 2002, 67, 6253e6255; (c) Buscemi, S.; Pace,
A.; Pibiri, I.; Vivona, N.; Spinelli, D. J. Org. Chem. 2003, 68, 605e
608; (d) Buscemi, S.; Pace, A.; Pibiri, I.; Vivona, N.; Caronna, T. J. Fluo-
rine Chem. 2004, 125, 165e173; (e) Buscemi, S.; Pace, A.; Palumbo
Piccionello, A.; Macaluso, G.; Vivona, N.; Spinelli, D.; Giorgi, G.
J. Org. Chem. 2005, 70, 3288e3291; (f) Buscemi, S.; Pace, A.; Palumbo
Piccionello, A.; Pibiri, I.; Vivona, N.; Giorgi, G.; Mozzanti, A.; Spinelli,
D. J. Org. Chem. 2006, 71, 8106e8113.
10. Schmuck, C.; Wienand, W. J. Am. Chem. Soc. 2003, 125, 452e459 and
references therein.
11. (a) Manet, I.; Francini, L.; Masiero, S.; Pieraccini, S.; Spada, G. P.;
Gottarelli, G. Helv. Chim. Acta 2001, 84, 2096e2107; (b) Haino, T.; Fujii,
T.; Fukazawa, Y. J. Org. Chem. 2006, 71, 2572e2580.
`
12. Guernelli, S.; Lagana, M. F.; Mezzina, E.; Ferroni, F.; Siani, G.; Spinelli,
D. Eur. J. Org. Chem. 2003, 4765e4776.
13. (a) Cech, N. B.; Enke, G. E. Mass Spectrom. Rev. 2001, 20, 362e387; (b)
Schalley, C. A. Int. J. Mass Spectrom. 2000, 194, 11e39; (c) Kebarle, P.;
Tang, L. Anal. Chem. 1993, 65, 972Ae986A; (d) Kebarle, P. J. Mass
Spectrom. 2000, 35, 804e817.
14. Jørgensen, T. J. D.; Roepstorff, P.; Heck, A. J. R. Anal. Chem. 1998, 70,
4427e4432.
15. Staroske, T.; O’Brien, D. P.; Jørgensen, T. J. D.; Roepstorff, P.; Williams,
D. H.; Heck, A. J. R. Chem.dEur. J. 2000, 6, 504e509.
16. Kempen, E. C.; Brodbelt, J. S. Anal. Chem. 2000, 72, 5411e5416.
17. Oshovsky, G. V.; Verboom, W.; Fokkens, R. H.; Reinhoudt, D. N.
Chem.dEur. J. 2004, 10, 2739e2748.
4. Frenna, V.; Vivona, N.; Consiglio, G.; Corrao, A.; Spinelli, D. J. Chem.
Soc., Perkin Trans. 2 1981, 1325e1328.
18. Zhan, Y.; Meng, C.; Cunyu, Y.; Fengrui, S.; Zhiqiang, L.; Shuying, L.
Rapid Commun. Mass Spectrom. 2007, 21, 683e690.
5. D’Anna, F.; Frenna, V.; Macaluso, G.; Marullo, S.; Morganti, S.; Pace, V.;
Spinelli, D.; Spisani, R.; Tavani, C. J. Org. Chem. 2006, 71, 5616e5624.
6. Laidler, K. J. Chemical Kinetics; McGraw-Hill: London, 1965; pp 450e
463.
19. Sasaki, T.; Yoshioka, T.; Suzuki, Y. Bull. Chem. Soc. Jpn. 1971, 44, 185e
189.
20. (a) Bates, R. G. SoluteeSolvent Interactions; Coetze, J. F., Ritchie, C. D.,
Eds.; Marcel Dekker: New York, NY, 1969; p 46; (b) Harned, H. S.; Owen,
B. B. The Physical Chemistry of Electrolytic Solution, 3rd ed.; ACS
Monograph No. 137; Reinhold: New York, NY, 1970; pp 755, 716.
7. Le Questel, J.-Y.; Laurence, C.; Lechkar, A.; Helbert, M.; Berthelot, M.
J. Chem. Soc., Perkin Trans. 2 1992, 2091e2094.