The multicomponent Hantzsch reaction: Comprehensive mass spectrometry monitoring using charge-tagged reagents

Article abstract of DOI:10.1002/chem.201303065

A novel strategy for the ESI-MS monitoring of reaction solutions involving the alternate use of permanently charge-tagged reagents has been used for comprehensive mass spectrometry monitoring of the multicomponent Hantzsch 1,4-dihydropyridine reaction. By placing a charge tag on either, or both, of the two key reactants, ion suppression effects for ESI were eliminated or minimized, and comprehensive detection of charge-tagged intermediates was achieved. The strategy allowed the trapping and characterization of the important intermediates in the mechanism for 1,4-dihydropyridine formation.

Full text of DOI:10.1002/chem.201303065

DOI: 10.1002/chem.201303065
Full Paper
&
Reaction Mechanisms
The Multicomponent Hantzsch Reaction: Comprehensive Mass
Spectrometry Monitoring Using Charge-Tagged Reagents
[
a]
[a]
[a]
[b]
Vanessa G. Santos, Marla N. Godoi, Thaꢀs Regiani, Fernando H. S. Gama,
[a]
[b]
[a]
Mirela B. Coelho, Rodrigo O. M. A. de Souza, Marcos N. Eberlin,* and
[
b]
Simon J. Garden*
Abstract: A novel strategy for the ESI-MS monitoring of re-
action solutions involving the alternate use of permanently
charge-tagged reagents has been used for comprehensive
mass spectrometry monitoring of the multicomponent
Hantzsch 1,4-dihydropyridine reaction. By placing a charge
tag on either, or both, of the two key reactants, ion suppres-
sion effects for ESI were eliminated or minimized, and com-
prehensive detection of charge-tagged intermediates was
achieved. The strategy allowed the trapping and characteri-
zation of the important intermediates in the mechanism for
1,4-dihydropyridine formation.
Introduction
nation of aldehydes and some ketones, as well as the reduc-
[18]
tion of preformed imines, in the presence of Lewis acids or
[
1]
[19]
The Hantzsch reaction is one of the oldest and most effective
Brønstead acids, and more recently by using asymmetric or-
[
2]
multicomponent reactions. Despite the multitude of alterna-
tive reaction routes, it still proceeds very selectively by the de-
hydrative coupling of ammonia, two equivalents of a 1,3-dicar-
bonyl compound and an aldehyde (Scheme 1) forming 1,4-di-
hydro-2,3,5,6-substituted pyridines (1,4-DHP) in an “all roads
leads to Rome” fashion. Depending upon the reagents and or
reaction conditions, low yields or unexpected products can be
ganocatalytic procedures giving moderate to excellent levels
[20]
of enantioselectivity. Electron-deficient alkenes are also read-
[21]
ily reduced with an equivalent of 1,4-DHP. Transition metals
and their organometallic complexes can be used in conjunc-
tion with 1,4-DHP to reduce functional groups by transfer
[22]
hydrogenations.
The classical Hantzsch reaction was modified by Beyer,
[23]
[
3]
[4]
obtained, including isomeric 1,2-DHP, hence the need to
fully understand its intricate mechanism. The 1,4-DHP have
been found to posses many pharmacological activities, such
and later by Knoevenagel and co-workers to prepare unsym-
metrically substituted 1,4-DHP by using (alkyl-) aryl-idene-1,3-
dicarbonyls or b-aminocrotonates, and these modifications
[
5]
[6]
[24]
as: antitumorals for multidrug resistant (MDR) tumours,
have been applied by others. Rabe and co-workers have also
[
7]
[8]
[9]
[25]
bronchodilating, antidiabetic, neurotropic, HIV protease
replaced the acetoacetate by 1,5-diketones. These modifica-
[
10]
inhibitors; although they are perhaps best known as calcium
tions have extended the scope and shed light on the intricate
mechanism of the Hantzsch reaction, for which at least five
major different pathways (A–E) have been proposed
(Scheme 2).
[
11]
[12]
channel blockers,
for example: aranidipine,
lercanidi-
[
13]
[14]
pine,
nifedipine
and the multibillion dollar drug
[
15]
amlodipine.
Additionally, the 1,4-DHP have great potential as hydride (or
hydrogen) transfer reagents due to their similarity with the
natural product enzyme co-factors NAD(P)H and the oxidized
In the aminocrotonate/aldehyde path A, the major key
player is the aminocrotonate 5, which condenses with 2 to
form 14 via 6. In the aminocrotonate/Knoevenagel path B, 5 is
again a key player but reacts with the Knoevenagel intermedi-
ate 10 to form 7. Via path C, 14 is also formed as in path A,
but via an alternative route. For the diketo/NH3 path D, 10
reacts with a second molecule of 1 to form 11, which also con-
[
16]
form NAD(P). In addition to the reduction of aldehydes and
[
17]
ketones, progress has also been made on the reductive ami-
[
a] V. G. Santos, M. N. Godoi, T. Regiani, M. B. Coelho, Prof. Dr. M. N. Eberlin
ThoMSon Mass Spectrometry Laboratory, Instituto de Quꢀmica
State University of Campinas, 13084-971, Campinas-SP (Brazil)
E-mail: eberlin@iqm.unicamp.br
[
b] F. H. S. Gama, Prof. Dr. R. O. M. A. de Souza, Prof. Dr. S. J. Garden
Department of Organic Chemistry, Instituto de Quꢀmica
Universidade Federal do Rio de Janeiro, Cidade Universitꢁria, CT
Bloco A, Ilha do Fund¼o, RJ 21941-909 (Brazil)
E-mail: garden@iq.ufrj.br
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201303065.
Scheme 1. The classical Hantzsch reaction and the synthesis of 1,4-dihydro-
pyridines.
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Scheme 2. Possible mechanistic paths (A–F) for the Hantzsch reaction. For a definition of the reactions a, b, c, and d to which the substituent information ap-
plies see Scheme 4.
verges to 7. In the aminobiscrotonate path E, 5 is again in-
voked, but it now reacts with 1 and later with 2 to form 4.
Path F is suggested in this study, but seems to be a dead-end
route that equilibrates with the reactants.
[
26]
Hinkel and co-workers have studied substituent effects for
[27]
2
, whereas Haley and Maitland investigated pH effects. They
made no claim to favor a specific reaction mechanism but
[
28]
cited the work of Phillips who had observed that aromatic
aldehydes with two equivalents of ethyl acetoacetate and two
equivalents of concentrated aqueous ammonia in ethanol fol-
lowed by heating gave better results than using the aldehyde
in combination with two equivalents of aminocrotonate or
with one equivalent of both ethyl acetoacetate and the ethyl
aminocrotonate, therefore supporting mechanism D. Insights
on the Hantzsch DHP reaction mechanism can therefore be
obtained by changing the addition order of reagents or by
using preformed intermediates. In a similar strategy, Zhou and
[
29]
Scheme 3. One-pot Hantzsch reaction studied by Zhou and co-workers.
[30]
13
15
Katritzky and co-workers using C and N NMR spectros-
copy observed that 10 and 5 were always formed, which was
taken as evidence for B. They also observed the formation of
18, but assigned it to a metastable side product. Since 13
from D was not observed, B was assumed to be the most
likely path.
[
29]
co-workers (Scheme 3) observed no reaction upon treating
preformed 5 with a-cyanobenzocinnamate (analogous to 10),
which was taken as evidence against path B, but the one-pot
reaction formed the desired pyridine supporting either C or D
Electrospray ionization mass spectrometry (ESI-MS) has
become a major tool for investigating reaction mechanisms
serving as a bridge between gas phase and solution chemis-
(Scheme 2). Path C is an interesting deviation from A, where
the formation of 5 is unimportant for 1,4-DHP synthesis.
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medium (1.0 mL) were taken, diluted in acidified acetonitrile
and subjected to direct infusion ESI in the positive-ion mode.
Spectra were collected at different time intervals but those at
1
0 and 120 min as well as 48 h were the most relevant.
Figure 1, spectra A and B, show illustrative ESI(+)-MS after
0 and 120 min, respectively. The spectra reveal several of the
1
key players proposed for the Hantzsch reaction (Scheme 2).
The same set of intermediates with varying proportions and
fading abundances were detected as the reaction progressed
to completion. Protonated 1a of m/z 131 could be detected as
an abundant ion after 10 min of reaction (Figure 1A) but after
120 min it had been nearly completely transformed into 5a or
consumed. The ion of m/z 218 (14a) was intercepted as one of
the most abundant intermediates, which became even more
predominant after 120 min of reaction (Figure 1B). Note that
14a is clearly detected with no signs of 15a or 16a.
In Figure 1 (reaction a) the ion of m/z 324 (21a) was also
abundant, particularly after 10 min (Figure 1A), but it started
to fade after about 120 min (Figure 1B), as was also the case
for 14a. Since most of the reactants are transformed into the
final 1,4-DHP 4, and since it is hard to rationalize a path from
2
1a to 4, we assume that 21a is a “dead-end” intermediate
Scheme 4. The Hantzsch reaction performed with neutral (a) as well as reac-
tions with charge-tagged reagents (b–d).
(path F) that reverts to reactants. Another key intermediate in
Figure 1A is that of m/z 348 (7a). Interestingly, if 7a is indeed
detected, its isomerization to 8a could be monitored. Hence,
the ESI(+)-MS/MS of this ion was acquired periodically
(Figure 2). Note the drastic change in dissociation chemistry
from the early stages of the reaction (30 min, Figure 2A) to
that after 48 h (Figure 2B), which is indicative of the 7a!8a
isomerization (Scheme 2). Additionally, Figures S1–S3 in the
Supporting Information show the ESI-MS/MS for the intercept-
ed species, in which the observed dissociation could be prop-
erly correlated with the proposed structures.
[
31]
[32]
tries. ESI “fishes” intermediates directly to the gas phase in
which MS and MS/MS characterization are performed, provid-
ing therefore continuous snapshots of the dynamic ionic com-
[
33]
position of reaction solutions. ESI-MS is, however, blind to
neutral species and the detection of such species is dependent
upon adventitious equilibria with either protons or metal ions.
To overcome this ESI-MS limitation, an elegant strategy based
[
34]
on charge tags added to reagents or catalysts has therefore
[
35]
been developed.
The final product 4a in its protonated form of m/z 330 was
also detected, even at the early stages of reaction (10 min),
but always as a minor ion, accompanied by the ion of m/z 328
corresponding to an aromatic pyridine Hantzsch product 23a
or 23a’, which at the end of the reaction became nearly the
exclusive ESI(+)-MS detectable ion (Figure 1C). Given this
seemingly anomalous finding, the Hantzsch reaction a was re-
peated with stoichiometric quantities of reagents that were
stirred at room temperature in a septum-sealed flask. The reac-
tion was periodically analyzed by GC-MS and the analyses re-
vealed the formation of the principal product 4a as well as
very minor quantities of the 1,2-DHP isomer (4a’) and its oxi-
dation product (23a’); for more details see the Supporting In-
formation. But when the Hantzsch reaction a was repeated
In this study, we have investigated the mechanism of the
Hantzsch reaction with ESI-MS monitoring using a strategy in-
volving alternate reactions with charge-tagged reagents
(Scheme 4). Since a multitude of reaction intermediates
formed by various but convergent reaction paths (Scheme 2)
have been proposed, the Hantzsch DHP reaction provides
a challenging test for the charge-tag approach to ESI-MS as
a reaction monitoring tool. Specifically, charge tagging was
used for none, either one or both of the two key reactants
1
and 2 (Scheme 4).
Alternate tagging of the reagents in different reactions
should facilitate detection of any intermediate formed by reac-
tion of the specifically tagged reactant, and therefore ion sup-
pression effects should be minimized or eliminated, thus pro-
viding comprehensive monitoring of intermediates. For the
neutral untagged reaction, ESI “fishing” is normally effective
but relies on the adventitious occurrence of favorable equili-
using an excess of NH OAc (4 equiv), substantial quantities of
4
the 1,2-DHP isomer and its oxidation product were readily
formed. The oxidation of 4a’ to 23a’ occurs notably more
easily than the respective oxidation of 4a (Scheme 5).
[
4a]
[
33]
bria for (de)protonation or ion association.
Therefore, the seemingly anomalous result that a pyridine
derivative is the only detectable product, can be readily under-
stood by consideration of the enhanced basicity of the pyri-
dines 23a and 23a’ relative to 4a and/or 4a’ whereby the
former are readily detected by ESI(+)-MS due to more favora-
ble protonation. The suppression of 4a/4a’ demonstrates a lim-
Results and Discussion
Our investigation began with the monitoring of the neutral re-
action a (Scheme 4) in ethanol at 608C. Aliquots of the reaction
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Figure 1. ESI(+)-MS of the reaction solutions for reaction “a” after: A) 10 min, B) 120 min, and C) 48 h; and reaction “b” after: D) 10 min, E) 120 min, and
F) 48 h.
ditives. Reaction b apparently
proceeded faster than the neu-
tral reaction a as seen from the
+
abundant product ion 4b of
m/z 387 detected after 120 min
(
Figure 1, spectrum E). However,
this apparent difference can be
attributed to either the en-
+
hanced reactivity of 2 (an elec-
tron deficient benzaldehyde de-
rivative) or to the fact that the
presence of the charge tag in
+
4
b overcomes the problems as-
sociated with monitoring reac-
tion a in which the Hantzsch
product 4a is nonbasic and not
readily detectable, or to both of
the effects. Additionally, Figure 1
(
spectrum F) reveals that the
final charged-tagged 1,4-DHP
+
ion 4b of m/z 387 was detect-
Figure 2. ESI(+)-MS/MS of: A) 7a (m/z 348) after 30 min of reaction, and B) 8a (m/z 348) after 48 h of reaction.
The contrasting spectra corroborate the 7a!8a isomerization. Other mesomeric structures can also be proposed
for the intermediate structures.
ed almost as the exclusive ion
after 48 h. Again for reaction b,
essentially the same set of inter-
mediates with varying abundan-
itation of the use of ESI-MS for reaction monitoring when
using untagged reactants.
ces was detected along the 48 h period. Among these inter-
mediates, as Figure 1 (spectrum E) illustrates for the reaction
after 120 min, three major and sequential ions of m/z 294
Given the apparent limitations of monitoring the neutral re-
agent reaction a, reaction b (Scheme 4) employing the charge-
+
+
+
(9b ), m/z 276 (10b ) and m/z 405 (7b ) were detected, as
+
+
tagged 2 was then monitored without the addition of ESI ad-
well as the tagged reactant 2 of m/z 164 (see Figures S4–S6
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Scheme 5. Formation of 1,4-DHP (4a) and 1,2-DHP (4a’) products and their
respective pyridine structures due to oxidative aromatization.
in the Supporting Information for the ESI-MS/MS characteriza-
tion). If we reasonably assume that ion discrimination is elimi-
nated or substantially reduced by the preloaded charge, and
that reaction b should facilitate the detection of intermediates
Figure 4. V-EASI monitoring of reaction b. Relative intensities of ions as
a function of time. A color version of the Figure is provided in the Support-
ing Information.
+
+
+
+
involving 2 , the detection of 9b , 10b and 7b and the
+
dominance of 10b points to the participation of the Knoeve-
nagel intermediate 10 in the rate-limiting step, as also pro-
[30]
posed by Katritzky and co-workers from NMR measurements,
and that 10 is transformed into 7. The ion of m/z 220
Figure 1, spectra D and E) seems again to be the dead-end in-
[
3f,3k,37]
uct—as also seen in a number of analogues of 8
—13 is
(
most likely in the present case to be the product of a dead-
end equilibrium and is therefore consumed by the transforma-
tion of 12 into 7 and the latter isomerizing into 8 (Figure 2).
2
+
termediate 21b of path F. Expansion of spectrum E (Figure 1)
at 120 min along the m/z 395–430 range (Figure 3) shows that
ESI(+)-MS was also able to intercept two intermediates from
+
Reaction c (Scheme 4) employing the charge-tagged 1 was
+
+
path D, that is, 11 b of m/z 406 and 12b (likely in equilibri-
then monitored (Figure S7a–c in the Supporting Information).
The reaction apparently occurred at a similar rate to the neu-
tral reaction a but after 48 h, the ESI(+)-MS was quite noisy
showing that intermediates were mainly consumed. Further,
+
um with 13b ) of m/z 423.
The periodic analysis of reaction b allowed the observation
of a large number of intermediates consistent with path D.
+
2+
The reaction with the charge-tagged aldehyde 2 was there-
the final product 4c was not detected (Figure S7c) despite
fore also monitored by Venturi easy ambient sonic-spray ioni-
being observed at shorter reaction times. The failure to detect
[
36]
2+
zation (V-EASI; Figure 4). The experiment revealed the rapid
the product 4c at the end of the reaction is perhaps due to
+
consumption of 2 to form the Knoevenagel intermediate
the high reactivity or low stability promoted by the presence
of multiple permanent charge sites under the reaction or ana-
lytical conditions. As Figure 5A illustrates for reaction c after
+
+
1
0b and the concomitant consumption of 10b with the for-
+
mation of the 1,4-DHP ion 4b . A low steady-state concentra-
tion of the ions of m/z 405 (7b ), 406 (11 b ) and 423 (12b )
+
+
+
+
120 min, two major intermediates were detected: 5c of m/z
+
was observed over the timescale in which the reaction was
234 (the enamine) and 10c of m/z 323, the Knoevenagel
+
2+
monitored. The low steady-state concentrations of 11 b and
product. Note that the final product 4c of m/z 270, the reac-
+
+
+
1
2b support path D for the formation of 7. Unlike in the
tant 1 of m/z 235 and the dead-end intermediate 21c of
m/z 428 from F were also detected at this time interval. In con-
[
3j]
study of Singh, in which 13 was isolated as the reaction
product, due to the presence of the highly electron-withdraw-
ing CF3 substituents that stabilize the bishemiaminal prod-
+
trast to the neutral reagent reaction a, the intermediate 14c
of m/z 322 is a minor ion at early stages of the reaction but is
one of the principal ions after
48 h (Figure S7c).
Reactions b and
c illustrate
how the complimentary charge
tagging of the reagents 1 and 2
can allow the detection of im-
portant reaction intermediates in
the absence of any purposefully
incremented ESI additive that
could artificially shift equilibria
of species in solution. Reaction c
also reveals, however, a limitation
associated with the detection of
multiply charged species. The
Figure 3. ESI(+)-MS in the m/z 395–430 range after a reaction time of 120 min for the reaction solution b.
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The analogous reaction of 5a
and 10a in the presence of an
equivalent of 1e resulted in
a 6:1 mixture of the 1,4-DHPs 4a
and 4e. At early stages during
the reaction the formation of
10e was evident and at a later
stage the presence of 5e was
also detected. The increased
acidity of 1e compared with
either 1a or 1 f may facilitate
both the transamination and
transbenzylidination
reactions
even in the absence of AcOH.
Both the reactions of 1e and 1 f
resulted in the formation of con-
siderable quantities of 1a. There-
fore, under neutral or slightly
basic conditions (for example,
Figure 5. ESI(+)-MS of: A) reaction solution “c” after 120 min, and B) reaction solution “d” after 120 min.
reasons for this limitation are not clear at present and require
further examination.
2
+
As 7c (an intermediate that could be formed through the
diverse possibility of mechanisms) could not be detected and
due to the difficulty in following the reaction of charge-tagged
+
1
using the V-EASI technique, an investigation of the reaction
of 5a with 10a was conducted. Both of these intermediates
were prepared by improvising upon literature methods and
subjected to reaction in the presence or absence of a molar
equivalent of AcOH in 95% EtOH at room temperature (for fur-
ther details see the Supporting Information). Unlike the use of
Scheme 6. Transamination of 5a and 1(e,f) under acidic conditions and the
formation of unsymmetrically substituted 1,4-DHPs 4e and 4 f as well as
symmetrical 4a.
10’ (Scheme 3) the enamine 5a and the Knoevenagel product
0a readily reacted with one another to give the Hantzsch
1
ester 4a in reasonable yield. Notably, the reaction in the pres-
ence of AcOH readily became yellow colored and precipitated
a solid that when isolated was colorless (51% yield), whilst the
reaction in the absence of AcOH precipitated a colorless solid
employing an excess of NH OH as the ammonia source) mech-
4
anism B is likely to be very important (transamination is rela-
tively unimportant) whilst under slightly acidic conditions (for
(with a slightly better 58% yield) after a few days at room tem-
example, using an excess of NH OAc as the ammonia source)
4
perature. Additional experiments using the enamine 5a, 1,3-di-
carbonyl compounds (1e or 1 f) and the Knoevenagel product
the various mechanisms will contribute to product formation.
Additionally, given that 5a was reportedly inert to the ethyl
arylidene cyanoacetate 10’ (Scheme 3) the reaction of 5a in
10a revealed that in the presence of AcOH the enamine 5a
readily equilibrates with 1a and the additional 1,3-dicarbonyl
compound (1e or 1 f) forms the respective enamine
(
Scheme 6). No transamination to give 5 f was observed to
occur in the absence of AcOH. The final ratio of the products
a/4 f was found to be 5.6:1 and 1.5:1 for 4a/4e for the reac-
4
tions conducted in the presence of AcOH.
In the absence of AcOH (Scheme 7), the reaction of 5a and
10a in the presence of an equivalent of 1 f resulted almost ex-
clusively in the formation of the 1,4-DHP 4a. A very minor
quantity of 1,4-DHP 4 f was readily detectable by GC-MS to-
wards the end of the reaction. Notably, even at early stages of
the reaction it was possible to observe the formation of
a small quantity of tert-butyl benzylidene acetoacetate (10 f)
due to a transbenzylidination reaction. The reaction of 10 f
with 5a could give rise to the small quantity of 4 f that was
formed during the reaction. The final ratio of 4a/4 f was 40:1.
Scheme 7. Formation of unsymmetrically substituted 1,4-DHPs 4e and 4 f in
the absence of added AcOH.
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detected. Therefore, the participation of 5 may be limited to
direct reaction with 10 (mechanism B) to give 7 and to act as
a reservoir of 1 and 3 (Scheme 2) depending upon the reaction
conditions. Evidence for the direct reaction of 5a and 10a was
obtained by conducting the reaction in the presence of
a second 1,3-dicarbonyl compound in which in the absence of
acid, transamination was minimal or did not occur and the
principal product was 4a. Aldehyde 2 seems also to be easily
converted into 21 in the presence of 1 and 3, but 21 acts as
a dead-end intermediate (Scheme 8). Monitoring of the neutral
reaction a suggests that 14 and 7 are key intermediates
whereas in the charge-tagged reactions b--d, ions 7(8), 9, 10,
11 and 12 were intercepted as the most important intermedi-
ates, but there was no sign of 6, 16 or 17. The combined data,
therefore, suggest the predominance of three convergent
paths B, C and D, and the formation of the final product 4 via
cyclization of 7 and dehydration of 8 (Scheme 8). As proposed
[30]
by Katritzky and co-workers from NMR measurements, the
Knoevenagel intermediate 10 seems indeed to participate in
the rate-limiting step of the Hantzsch reaction, being convert-
ed into the convergent intermediate 7 by reaction with 5, as
well as via 11 and possibly 14.
Therefore, the novel approach of alternate charge tagging
of reagents was successfully applied to investigate an intricate
multicomponent reaction mechanism with ESI(+)-MS, facilitat-
ing the interception of most if not all major intermediates.
There was no need for the addition of acidic or basic additives
to promote (de)protonation, and the absence of such additives
implies that the true ionic composition of the reaction under
its normal reaction conditions can be probed. ESI-MS with
charge-tagged reactants potentially offers a comprehensive
technique for reaction monitoring.
Figure 6. ESI(+)-MS for reaction solution d in the: A) m/z 196–204 range
after 120 min, and B) m/z 202–209 range after 48 h.
the presence of ethyl benzylidene cyanoacetate with and with-
out AcOH was also investigated. However, in both cases no re-
action was observed to occur, confirming the inertness of this
substrate type under the reaction conditions employed.
In principle, reaction d (Scheme 4) could seem ideal at first
in terms of charge tagging as two reactants are tagged, but
could be intrinsically challenging since it requires the interac-
+
+
tion of two positively charged ions: 2 and 1 . The reaction,
however, if properly assisted by counter-anions, could favor
detection of all intermediates displaying net charges varying
Experimental Section
+
+
from 1 to 3 (Scheme 2). Reaction d was indeed much slower
+
+
and the reactants 1 of m/z 235 and 2 of m/z 164 were
always detected as the predominant ions (Figure S8a–c in the
Supporting Information), but after 120 min (Figure 5, spec-
trum B) and particularly at 48 h (Figure S8c), two intermediates
ESI-MS(/MS) monitoring of the Hantzsch reaction
In all four experiments studied (a–d) the reactions were performed
as follows: in a flask (10 mL) with ethanol (2 mL), 0.1 mmol of each
reagent: aldehyde, acetoacetate (in the case of a and b) or aceto-
acetamide (in case of c and d) and ammonium acetate were mixed
and stirred at 608C for 48 h. Aliquots of the reaction medium
+
could be detected: 5d of m/z 234 and the dead-end inter-
2
+
3+
mediate 21d of m/z 181, as well as the final product 4 of
m/z 199. In addition, spectrum expansion around m/z 200 re-
(5.0 mL in the case of reaction a and 1.0 mL in the case of the ex-
2
+
periments with charged tags b–d) were taken, diluted in acetoni-
trile (1 mL) with formic acid (0.1%) in experiment a due to the lack
of charge in the reagents, or without additive in the case of all
other experiments (b–d). The aliquots were directly infused into
the ESI source using a syringe pump (Harvard Apparatus) at a flow
vealed the presence of 9d of m/z 199.63 at 120 min (Fig-
ure 6A). The key intermediate 7d of m/z 205.14 was also de-
3
+
tected at 48 h (Figure 6B).
ꢀ1
rate of 10 mLmin . In the case of the V-EASI experiments the reac-
tion solution was directly analyzed as the ionization method pro-
motes the self-pumping of the reaction solution by the Venturi
Conclusion
By using ESI(+)-MS(/MS) monitoring and the alternate charge-
tagging strategy most, if not all, relevant intermediates for the
Hantzsch DHP reaction were intercepted and characterized.
Combined with experimental data using preformed intermedi-
ates, the evidence supports the overall mechanistic view of
Scheme 8. The intermediate 5 was detected by ESI-MS, as has
[36]
effect. All experiments were performed on a tandem Q-Tof mass
spectrometer (Micromass, UK) equipped with an electrospray (ESI)
source in positive ion mode. The main ESI conditions were capillary
voltage: 3000 eV; cone voltage: 30 eV; source and desolvation
temperature: 1008C. The spectra were acquired along the 100–500
m/z range. ESI-MS/MS of the cationic species was performed by
mass selecting the ion of interest using the first quadrupole Q1.
The selected ion was in turn subjected to 4–40 eV collision-induced
[
30]
also been the case by NMR spectroscopy, but the intermedi-
ate 6 as well as the sequential intermediates 17–19 were not
Chem. Eur. J. 2014, 20, 12808 – 12816
www.chemeurj.org
12814
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
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[38]
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[
39]
[
4-(dimethylamino)phenyl]acetoacetamide.
Spectroscopic data:
H NMR (500 MHz,
1
N-[4-(dimethylamino)phenyl]acetoacetamide
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3
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electrospray ionization
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mechanisms
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multicomponent reactions
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Received: August 2, 2013
Revised: July 3, 2014
Published online on September 1, 2014
Chem. Eur. J. 2014, 20, 12808 – 12816
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ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim