Remarkably fast and selective aromatization of Hantzsch esters with MoOCl4 and MoCl5: A chemical model for possible biologically relevant properties of molybdenum-containing enzymes

Article abstract of DOI:10.1016/j.bmcl.2012.04.043

Mo(VI) and Mo(V) salts both react selectively with Hantzsch esters to produce substitute pyridines in good-to-excellent yield (75-99%). The remarkable reactivity and selectivity of MoOCl₄ under reflux of acetonitrile and MoCl₅ in dichloromethane at room temperature encouraged us to propose that molybdenum-containing enzymes (such as xanthine or aldehyde oxidase) also participate to some degree in the metabolism of 1,4-dihydropyridine drugs in the liver analogous to NADH in the respiratory chain.

Full text of DOI:10.1016/j.bmcl.2012.04.043

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3676–3681
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Remarkably fast and selective aromatization of Hantzsch esters
with MoOCl4 and MoCl5: A chemical model for possible biologically relevant
properties of molybdenum-containing enzymes
⇑
´
´
´
´
Mladen Litvic , Maja Regovic, Karolina Šmic, Marija Lovric , Mirela Filipan-Litvic
BELUPO Pharmaceuticals, Inc., R&D, Danica 5, 48000 Koprivnica, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history:
Mo(VI) and Mo(V) salts both react selectively with Hantzsch esters to produce substitute pyridines in
good-to-excellent yield (75–99%). The remarkable reactivity and selectivity of MoOCl4 under reflux of
acetonitrile and MoCl5 in dichloromethane at room temperature encouraged us to propose that molyb-
denum-containing enzymes (such as xanthine or aldehyde oxidase) also participate to some degree in
the metabolism of 1,4-dihydropyridine drugs in the liver analogous to NADH in the respiratory chain.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 27 January 2012
Revised 6 April 2012
Accepted 7 April 2012
Available online 13 April 2012
Keywords:
1,4-Dihydropyridines
Aromatization
Dealkylation
Molybdenum pentachloride
Molybdenum oxytetrachloride
As one of the most important transition elements, molybdenum
(and in some cases tungsten) is an essential microelement for
plants, animals and microorganisms.1,2 However, the use of molyb-
denum by biological systems was not recognized until the 1930s
when Bortels first reported that molybdenum (and vanadium)
acted as a catalyst in the fixation of nitrogen by Arthrobacter chroo-
coccum.3 Almost 60 years ago,4 molybdenum was identified as the
‘xanthine oxidase factor’ and today it has been discovered in a vari-
ety of enzymes.2a Each enzyme contains a mononuclear Mo site
with one or two unusual pterin-ene-dithiolate ligands and, usually,
one or more oxo groups in the molybdenum co-ordination sphere.5
The molybdenum center in these enzymes, coordinated by the
variety of structurally similar ligands,1,6 is designated as Moco
(molybdenum-containing cofactor) in Figure 1.
act as drug-metabolizing enzymes9 and are key enzymes in bio-
transformations of xenobiotics.10
The typical substrates for xanthine as well as aldehyde oxidases
are organic compounds with electron-deficient sp2-hybridized car-
bon atoms mostly present in heterocycles such as purines, pyrimi-
dines, imines, etc.11 This is in sharp contrast to cyctochrome P450
(iron-containing heme cofactor, Fig. 1) whose substrates are mainly
compounds with electron rich carbon atoms such as alkanes,
amines, esters, ethers etc.12 Therefore, it is believed that good
substrates for molybdenum-containing enzymes tend to be poor
substrates for cytochrome P450 and vice versa.13 The organic
compounds derived from the six membered nitrogen-containing
heterocycles, 1,4-dihydropyridines (1,4-DHPs), have been recog-
nized as valuable drugs for the treatment of hypertension and other
coronary diseases.14 On the molecular level, 1,4-DHPs cause vasore-
laxation by blocking voltage-operated calcium channels in smooth
muscle cells and also by increasing NO release from intact endothe-
lial cells.15 Newer generations of substituted 1,4-DHPs possess
other pharmacological activities such as: antitumor,16 bronchodi-
lating,17 antidiabetic,18 neurotropic19 and antianginal,20 amongst
others.21 The oxidation (aromatization) of 1,4-DHPs into the
corresponding pyridines is one of the main metabolic pathways of
these drugs. This process is catalyzed by the cytochrome P450
(CYP) 3A4 isoform.22 Moreover, during the storage of drugs contain-
ing 1,4-DHPs, slow oxidation takes place by the action of air oxygen
or UV irradiation23 to furnish the same product—substituted
pyridine.
In humans, only four families7 of molybdenum-containing en-
zymes are known and they are classified by the type of biochemical
transformations they execute. Aldehyde oxidase (EC 1.2.3.1), xan-
thine oxidase (EC 1.2.3.1), xanthine dehydrogenase (EC 1.2.1.37)
and sulphite oxidase (EC 1.8.3.1) are some of the most important
enzymes in organisms with molybdenum-enzyme defficient bio-
logical systems.8 Of all enzymes, those from xanthine oxidase-
dehydrogenase families are particularly important because they
⇑
Corresponding author. Tel.: +385 48 652 457; fax: +385 48 652 894.
E-mail addresses: mladen.litvic@belupo.hr, mlitvic@gmail.com (M. Litvic´).
Present address: Galapagos Research Center Ltd, Prilaz baruna Filipovic´a 29,
10000 Zagreb, Croatia.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.043

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M. Litvic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3676–3681
CH3
O
O
H3C
Mo OH/OH2
O
N
S
N
N
N
N
H
N
Fe
S
HN
CH3
H3C
2-
OPO3
H2N
N
H
O
COOH
COOH
b
a
Figure 1. Molybdenum-containing cofactor (Moco) in xanthine oxidase (a) and iron-containing cofactor (Heme b) in cytochrome enzymes.
In order to better understand the mechanism of the aromatiza-
Table 1
tion of 1,4-DHPs, a plethora of chemical oxidants have been stud-
ied such as metallic salts,24 non-metallic reagents25 and catalytic
methods26 amongst others.27 Some methods have been designed
to mimic the natural biological processes26f,28 while others have
been developed mainly for the preparation of substituted pyridines
used as the reference standard to study the metabolism of new
drug candidates or as the standard to determine the purity of com-
mercial 1,4-DHP drugs.24e–g
In continuation of our ongoing research program directed to-
ward the development of milder and more selective models for
the aromatization of 1,4-DHPs,24e–g,26e,26f we decided to study tran-
sition metals in higher oxidation states selected from groups 3–12
of the periodic table of elements. This was due to the fact that me-
tal ions such as iron, molybdenum, tungsten, vanadium, etc., are
usually found in the active site of many enzymes.
Herein, we wish to report a comprehensive study of the reaction
performed with some representatives of group 6 (Mo(VI), W(VI))
and 7 (Mn(VII), Re(VII)) elements of the periodic table and the dis-
covery of new, rapid and selective aromatization of 1,4-DHPs.
Although chromium(VI) salts such as CrO3 and a variety of chro-
mates29 produced good results in the aromatization of 1,4-DHPs,
the fact that most of the Cr(VI) salts are highly carcinogenic makes
these methods rather unattractive and therefore, Cr(VI) salts (eq
CrO2Cl2) were not included in our study. To our knowledge, the
salts of the other two members of group 6 (Mo and W) have not
been used so far for the aromatization of 1,4-DHPs whilst use of
Mo and W salts in other types of transformations in organic syn-
thesis is well described in literature.30 This is particularly the case
with MoCl5 whose synthetic potential was recognized by Waldvo-
gel and coworkers in the oxidative dimerization of arenes to
substituted biaryls and thiaanthracenes.31
Aromatization of 1,4-DHP 1 by stoichiometric amount of different metallic oxidants in
various solvents
Entry
Oxidant
Solvent
Time (h)
Conv.a (%)
92
1
2
3
4
5
6
7
8
9
10
11
12
13
MoOCl4
MoOCl4
MoOCl4
MoOCl4
MoCl5
MoCl5
MoCl5
MoO3
WCl6
WCl6
WCl6
KMnO4
Re2O7
CH2Cl2
24
24
24
24
48
b
Toluene
CH3COOH
CH3CN
CH3CN
CH2Cl2
CH3COOH
CH3COOH
CH2Cl2
—
—
b,c
93
100
100
96
b
168
240
72
96
22
—
0 (90)c
0
d
CH3CN
—
CH3COOH
CH3COOH
CH3COOH
97
0.25
24
98
0 (0)c
a
b
c
Determined by TLC analysis.
Low selectivity.
The reaction was carried out at reflux temperature during 96 h.
Traces of the product.
d
a 1,4-DHP molecule. Similar behavior in chemical reactions of
other metal oxides such as V2O5, MoO3, CeO2, HfO2 and GeO2 have
recently been described.24g
The reaction with 2 equiv of MoOCl4 or MoCl5 smoothly fur-
nished product 2g at room temperature with acetonitrile or dichlo-
romethane as solvents (Table 1, entries 1, 4–6) whereas, in toluene
and acetic acid, the reaction was sluggish and non-selective. Inter-
estingly, tungsten hexachloride (WCl6) proved to be much a weak-
er oxidant than Mo(VI) and Mo(V), with reaction only taking place
under reflux of acetic acid (Table 1, entry 11). A similar result was
obtained with MoO3 which slowly furnished 2g in 90% yield after
96 h (Table 1, entry 8) and this was predominantly due to its poly-
meric structure similar to Re2O7 as well as other metallic oxides.
The obtained results encouraged us to further explore MoOCl4
and MoCl5 as oxidants for the efficient aromatization of 1,4-DHPs,
Scheme 2. To increase the conversion rate of reactants, a slight ex-
cess of oxidants were used (2.1 equiv compared to 2 equiv in pre-
liminary work).33
We began our study with MoOCl4, MoO3, MoCl5, WCl6, KMnO4
and Re2O7 on a simple model reaction (Scheme 1) and the obtained
results are outlined in Table 1.
Unexpectedly, the reaction with Re2O7 did not furnish traces of
expected product even after prolonged heating under reflux of ace-
tic acid (Table 1, entry 13). This observation shows a sharp contrast
to the reaction with KMnO4, which resulted in 100% conversion
after only 15 min at room temperature (Table 1, entry 12). This dif-
ference in reactivity can be attributed to the structure of these
salts. Rhenium oxide (Re2O7), unlike KMnO4, exists in polymeric
structure (infinite array of alternating tetrahedral ReO4 and ReO6
octahedra)32 thus preventing the contact of the metal cation with
The reaction of 1g with MoOCl4 was significantly accelerated
under reflux of acetonitrile and was completed within a few min-
utes (Table 2, entry 7) compared to 24 h at rt (Table 1, entry 4). The
other representatives of the substituted 1,4-DHPs, bearing a vari-
ety of substituents on positions 4 in the 1,4-DHP ring (1a–r), were
Ph
Ph
R
R
N
MeO2C
H3C
CO2Me
CH3
MeO2C
H3C
CO2Me
CH3
[O]
MeO2C
H3C
CO2Me
CH3
MeO2C
H3C
CO2Me
CH3
a
N
N
N
H
H
2g
1g
2a-r
1a-r
Scheme 1. The aromatization of 1,4-DHP 1 by variety of oxidants in different
solvents.
Scheme 2. Reagents and conditions: a MoOCl4/CH3CN/reflux or MoCl5/CH2Cl2/st.

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M. Litvic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3676–3681
3678
Table 2
substituent afforded a dealkylated product 1a whereas 1e fur-
nished a mixture of dealkylated and non-dealkylated products
(Table 3, entries 2 and 3).
Aromatization of 1,4-DHPs by MoOCl4 (2.1 equiv) at reflux in acetonitrile
Entry
1,4-DHP
R
ta (min)
Yieldb (%)
An interesting result was obtained during the aromatization of a
variety of 4-alkyl-1,4-DHPs with MoOCl4 as an oxidant (Table 4).
Only 1f (R = CH2Ph) afforded exclusively 2a as a result of complete
dealkylation whereas 1d (R = i-Pr) furnished a trace of non-dealky-
lated product 2d (Table 4, entry 3). In case of 4-n-propyl-1,4-DHP
1d, an expected mixture of products 2a and 2d was isolated similar
to other literature metallic salts applied in the same reaction.24g
Surprisingly, the predominant removal of the ethyl group was also
obtained (Table 4, entry 2), which was not the case for most of the
literature methods.24 An even more interesting result was obtained
with 4-methyl-1,4-DHP 1b, which in reaction with MoOCl4, affor-
ded a mixture of dealkylated and non-dealkylated products con-
taining 16% of 2a. To our best knowledge, this is the first
example of a partial dealkylation of the 1,4-DHP carrying methyl
group at position 4 of 1,4-DHP ring.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
H
Me
Et
i-Pr
n-Pr
CH2Ph
Ph
p-NO2C6H4
m-NO2C6H4
o-MeOC6H4
m-MeOC6H4
p-ClC6H4
o-ClC6H4
p-MeC6H4
m-MeC6H4
2-Thienyl
2-Furyl
2-(5-Br-thienyl)
1
15
1
15
10
15
3
90
76c
75c
83c
83c
86d
93
99
98
92
83
92
75
77
83
86
90
89
5
1
15
5
10
15
10
1
1
1
20
As a result of the experimental behavior of 1,4-DHP bearing al-
kyl groups, a plausible free-radical mechanism is being proposed
similar to other metallic salts as oxidants24e–g in Scheme 3.
In the first step, the precomplexation of 1,4-DHP 1g with MoO-
Cl4 gives molybdenum(VI) complex 3, which in turn rapidly
decomposes by intramolecular single electron transfer (SET) to
molybdenum(V) complex of nitrogen radical cation 4. The sponta-
neous rearrangement of 4 to more stable benzylic radical 5 is char-
acteristic only for 1,4-DHPs bearing alkyl groups whereas alkyl
substituents dependant upon the stability of alkyl radicals follow
the mechanism recently described for VOCl3 as well as for some
other oxidants.24f In the next step, if a second equivalent of oxidant
(MoOCl4) is present, a rapid transfer of a second electron to the oxi-
dant furnishes product 2g as a molybdenum(V) complex. If only
1 equiv of MoOCl4 is used in reaction, the complete conversion of
1g was obtained only after 24 h under reflux of acetonitrile. This
was probably due to a slow electron transfer within deactivated
(delocalization of oxygen electrons to free orbitals of molybde-
num) hydroxy-molybdenum(V) complex 5. According to the re-
sults obtained with MoCl5 (Table 3), one can propose that Mo(V)
is even more reactive than Mo(VI) due to the fact that reaction is
easily obtained at room temperature. This difference in reactivity
can be attributed to the structures of molybdenum salts. Whereas
a
b
c
Determined by TLC analysis.
Isolated yield.
Isolated as a mixture with dealkylated product 2a.
Isolated as a sole dealkylated product 2a.
d
also efficiently oxidized to give substituted pyridines (2a–r). The
results outlined in Table 2 indicate that the reaction with MoOCl4
is generally very fast (1–20 min) for all 1,4-DHPs and to our knowl-
edge, is one of the fastest for this type of reaction performed under
conventional heating.
The yields of crude products of high purity obtained after
simple extractive work-up ranged between 72–99%.34,35 Both
electron-withdrawing (Table 2, entries 8–9, 12–13) and electron-
donating substituents (Table 2, entries 10–11, 14–15) on the
aromatic ring were well tolerated and had no significant effect
on the reaction rate.
Unlike MoOCl4, the reaction employing MoCl5 was largely
dependent on the electronic nature of substituents on the 1,4-
DHP ring (Table 3). Although, performed at room temperature,
the reaction is less selective and only of practical importance for
acid-insensitive compounds (Table 3, entries 5, 6 and 9). Interest-
ingly, the presence of electron-withdrawing bromine on the thio-
phene ring deactivated the 1,4-DHP 1r to allow selective reaction
and isolation 2b in good yield (Table 3, entry 9). The main reason
for poor selectivity of the reaction with MoCl5 is probably its action
as an oxidant in the oxidative coupling of electron-rich aromatic
compounds to the corresponding biaryls.31h Although we have
not been able to isolate any such side-products in reaction, we be-
lieve that this is one of the main pathways leading to the decom-
position of 1,4-DHP 1f and 1p (Table 3, entries 4 and 7). As
expected, the aromatization of 1,4-DHP 1d bearing isopropyl
MoCl5 exists in dimeric structure (Mo2Cl10)
32a, MoOCl4 forms infi-
nite chains of tetragonal pyramids (MoOCl4)n.32a Thus by the action
of the solvent under reflux of acetonitrile, polymeric MoOCl4 is
activated to form reactive species of oxidants (solvated monomers)
by which the reaction is almost instantly completed with excellent
selectivity (Table 2).
However, the observed non-typical and new dealkylation of
methyl and ethyl groups in case of the 1,4-DHPs 1b and 1c cannot
be explained by the mechanism outlined in Scheme 3 due to the
fact that methyl and ethyl radicals are not stabilized by delocaliza-
tion as benzyl or isopropyl radicals. Therefore, one of the possibil-
ities is intramolecular nucleophilic attack of chloride anion within
the complex of 1,4-DHP (1b or 1c) and MoOCl4 (Scheme 4) or the
action of anhydrous hydrogen chloride released in reaction. The
acetonitrile acts as a mild base as well as a dipolar aprotic solvent
thus activating HCl to be more susceptible for nucleophilic attack
to nonbulky methyl or ethyl groups (Scheme 4). Literature sup-
ports this claim as it is known that Cl- anion acts as a strong but
very selective nucleophile in the reaction of Krapcho dealkoxycarb-
onylation of malonic esters but only in dipolar aprotic solvents at
elevated temperatures.37
Table 3
Aromatization of 1,4-DHPs by MoCl5 (2.1 equiv) in CH2Cl2 at rt36
Entry
1,4-DHP
R
t (min)
Yield (%)
1
2
3
4
5
6
7
8
9
1a
1d
1e
1f
1g
1m
1p
1q
1r
H
i-Pr
n-Pr
CH2Ph
Ph
o-Cl-C6H4
2-Thienyl
2-Furyl
2-(5-Br-thienyl)
1
5
1080
10
1440
1440
60
75
90
a
—
—
b
97c
92
b
—
c
20
240
—
80
The greater selectivity of Mo(VI) compared to Mo(V) encour-
aged us to propose the participation of molybdenum-containing
enzymes, especially oxydoreductases, in the metabolism of 1,4-
DHP drugs. Although it is commonly agreed that cytochrome
a
b
c
Isolated as a mixture with 2a (8:2).
Decomposition.
Reaction not selective.

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M. Litvic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3676–3681
Table 4
The ratio of dealkylated versus non-dealkylated pyridines obtained during aromatization of 1,4-DHPs by MoOCl4 at reflux in acetonitrile
Entry
1,4-DHP
R
Dealkylated product 2aa (%)
Non-dealkylated producta (%)
1
2
3
4
5
1b
1c
1d
1e
1f
Me
Et
i-Pr
n-Pr
CH2Ph
16
93
99.8
87
84
7
0.2
13
0
100
a
Determined by HPLC analysis.
that metabolism takes place only by the action of cytochromes
p450. Therefore, we are proposing that analogous to coenzyme
NADH, Hantzsch esters are dependant upon their structure in
being metabolized to some degree by the action of molybdenum-
containing oxidoreductase. This prediction is supported by the re-
sults obtained with simple chemical models employing MoOCl4
and MoCl5 as oxidants.
H
H
MeO2C
H3C
CO2Me
CH3
MeO2C
H3C
CO2Me
CH3
intramolecular
SET
.
+
N
N
+6
Cl
+5
Cl
Cl
Cl
Cl
Mo
Mo
Cl
Cl
Cl
The metabolism of 1,4-DHP 1c is characterized by the complete
dealkylation leading to an inactivation of cytochrome39 as a result
of ethylation of the enzyme porphyrine moiety with ethyl radical
formed during the reaction.40 However, according to literature re-
sults obtained by the chemical models of cytochrome,26f,28a–c,41
dealkylation of 1c does not take place suggesting that some other
enzymes might also participate in the metabolism of this type of
substrate. The results obtained employing MoOCl4 (almost com-
plete dealkylation of 1c, Table 4) indicate that 1,4-DHPs could also
be the substrates for some of the molybdenum-containing enzyme
families. Thus, the two plausible mechanisms presented in Scheme
4 are proposed to explain the involvement of the xanthine oxidase
(XO) family in the aromatization of model 1,4-DHP 1g. The direct
analogy to the mechanism of the XO in biological systems includes
the insertion of an oxygen atom from the Moco to the benzylic
group of the 1,4-DHP ring affording 6 via concerted mechanism42
(Pathway A, Scheme 4). In the next step, via intramolecular rear-
rangement of an unstable reduced enzyme and substituted benzyl
alcohol complex, product 2g is released to form fully reduced XO-
Mo(IV).
OH
OH
3
4
MeO2C
H3C
CO2Me
CH3
MeO2C
H3C
.
CO2Me
1equiv. MoOCl4
SET
rearrang.
N
N
CH3
Cl
+5
Cl
+5
Cl
Cl
Cl
Cl
Mo
Mo
Cl
Cl
OH
2g
OH
5
Scheme 3. The proposed mechanism of the aromatization of 1,4-DHP 1g by
MoOCl4.
P450 is the only enzyme performing this biochemical transforma-
tion, the results obtained with 1,4-DHPs bearing alkyl substituents
might also be explained by the action of molybdenum-containing
enzymes. Molybdenum-containing enzymes have a broad specific-
ity for the oxidation of a variety of substrates such as aldehydes,
substituted pyridines, pyrimidines, pyrines, pteridines and in what
is most important, the oxidation of NADH to NAD+, thus acting as
key enzymes in the respiratory chain.38 It should be taken into ac-
count that a key structural fragment of NADH is the 1,4-DHP ring,
which is oxidized by the interaction of enzymes with molybdenum
in its active site, contrary to Hantzsch esters for which it is believed
The second mechanism (Pathway B, Scheme 4) is characterized
by the first SET from 1,4-DHP 1g to Moco to give radical cation 7,
which upon rearrangement is converted to more stable benzylic
radical 8 and in the next step, by second SET and deprotonation to
product 2g. The observed dealkylation of 1,4-DHPs bearing alkyl
substituents is better explained by the latter mechanism, as is the
case of 4-ethyl-1,4-DHP 1c, which is presented in greater detail in
Scheme 5.
R
O
Mo O
O
A
B
O
concerted
mechanism
OH
R
VI
IVMo O
Me
NH
Me
H
S
S
Me
NH
Me
H
H
S
R
S
R
O
IV
OH
1g
Mo OH
S
MeO2C
+
CO2Me
Me
S
2
R = CO2Me
XO
6
Me
N
SET
1g
2g
SET
XO-Mo(IV)
O
O
OH
O
VMo O
V
Mo O
H
S
S
MeO2C
Me
CO2Me
Me
.
MeO2C
Me
CO2Me
H
H
+
S
S
.
+
N
H
N
H
Me
XO-Mo(V)
XO-Mo(V)
8
7
Scheme 4. The plausible mechanism (A-concerted mechanism; B-two step electron transfer mechanism) for the action of xanthine oxidase (XO) in the aromatization of 1,4-
DHP 1g.

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M. Litvic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3676–3681
O
Mo O
O
References and notes
V
Et
H
O
Mo O
O
S
VI
MeO2C
CO2Me
Me
H
+
S
1. (a) Schwarz, G.; Mendel, R. R.; Ribbe, M. W. Nature 2009, 460, 839; (b) Kisker,
C.; Schindelin, H.; Rees, D. C. Annu. Rev. Biochem. 1997, 66, 233; (c) Mendel, R. R.
Planta 1997, 203, 399; (d) Hille, R. Essays Biochem. 1999, 34, 125; (e) Mendel, R.
R. J. Exp. Bot. 2007, 58, 2289; (f) Mendel, R. R.; Hänsch, R. J. Exp. Bot. 2002, 53,
1689; (g) Campbell, W. H. Plant Physiol. 1996, 111, 355.
2. (a) Hille, R. Chem. Rev. 1996, 96, 2757; (b) Mendel, R. R.; Smith, A. G.; Marquet,
A.; Warren, M. J. Nat. Prod. Rep. 2007, 24, 963; (c) Johnson, J. L.; Hainline, B. E.;
Rajagopalan, K. V.; Arison, B. H. J. Biol. Chem. 1984, 259, 5414.
3. Bortels, H. Arch. Mikrobiol. 1930, 1, 333.
4. De Renzo, E. C.; Kaleita, E.; Heytler, P. G.; Oleson, J. J.; Hutchins, B. L.; Williams,
J. H. Arch. Biochem. Biophys. 1953, 45, 247.
5. Stiefel, E. I. J. Chem. Soc., Dalton Trans. 1 1997, 3915.
6. (a) Johnson, J. L.; Hainline, B. E.; Rajagopalan, K. V. J. Biol. Chem. 1984, 259, 5414;
(b) Young, C. G.; Wedd, A. G. Chem. Commun. 1997, 1251; (c) Gutteridge, S.;
Tanner, S. J.; Bray, R. C. Biochem. J. 1978, 175, 869.
S
1c
SET
H
S
.
+
Me
N
H
XO
XO-Mo(V)
9
alkyl radical
transfer
O
H
OEt
IV
O
OEt
Mo O
S
MeO2C
CO2Me
Me
IV
-2a
Mo OH
S
H
S
S
2
Me
N
H
7. (a) Coughlan, M. P. J. Inherit. Metab. 1983, 6, 70; (b) Kitamura, S.; Sugihara, K.;
Ohta, S. Drug Metab. Pharmacokinet. 2006, 21, 83.
XO-alkylated
XO-alkylated
10
8. (a) Appignani, B. A.; Kaye, E. M.; Wolpert, S. M. AJNR 1996, 17, 317; (b) Roth, A.;
Nogus, C.; Monnet, J. P.; Ogier, H.; Sandubray, J. M. Virchows Arch. [A] 1985, 405,
379; (c) Schwarz, G.; Santamaria-Araujo, J. A.; Wolf, S.; Lee, H.-J.; Adham, I. M.;
Gröne, H.-J.; Schwegler, H.; Sass, J. O.; Otte, T.; Hänzelmann, P.; Mendel, R. R.;
Engel, W.; Reiss, J. Hum. Mol. Gen. 2004, 13, 1249.
Scheme 5. The plausible mechanism for the action of XO in the metabolism of 4-
ethyl-1,4-DHP 1c.
9. (a) Klecker, R. W.; Cysyk, R. L.; Collins, J. M. Bioorg. Med. Chem. 2006, 14, 62; (b)
Gu, C.; Collins, R.; Holsworth, D. D.; Walker, G. S.; Voorman, R. L. Drug Metab.
Dispos. 2006, 34, 2044; (c) Torres, R. A.; Korzekwa, K. R.; McMasters, D. R.;
Fandozzi, C. M.; Jones, J. P. J. Med. Chem. 2007, 50, 4642; (d) Kawashima, K.;
Hosoi, K.; Naruke, T.; Shiba, T.; Kitamura, M.; Watabe, T. Drug Metab. Dispos.
1999, 27, 422.
10. (a) Parkinson, A. Biotransformation of Xenobiotics, in Casarett and Doull’s
Toxicology: The Basic Science of Poison, 6th ed.; McGraw-Hill: New York, 2001.
p 133; (b) Ashikawa, Y.; Fujimoto, Z.; Noguchi, H.; Habe, H.; Omori, T.; Yamane,
H.; Nojiri, H. Structure 2006, 14, 1779.
11. (a) Schepetkin, I. A.; Cherdyntseva, N. V.; Kagiya, V. T. Patophysiology 2001, 8,
119; (b) Terao, M.; Kurosaki, M.; Barzago, M. M.; Varasano, E.; Boldetti, A.;
Bastone, A.; Fratelli, M.; Garattini, E. J. Biol. Chem. 2006, 281, 19748; (c) Itoh, K.;
Masubuchi, A.; Sasaki, T.; Adachi, M.; Watanabe, N.; Nagata, K.; Yamazoe, Y.;
Hiratsuka, M.; Mizugaki, M.; Tanaka, Y. Drug Metab. Dispos. 2007, 35, 734.
12. (a) Atkins, W. M.; Sligar, S. G. J. Biol. Chem. 1988, 263, 18842; (b) Stevenson, J.-
A.; Westlake, A. C. G.; Whittock, C.; Wong, L.-L. J. Am. Chem. Soc. 1996, 118,
12846.
The first step of the mechanism is similar to aromatization of 1g
and includes SET from 1c to Moco to form radical cation 9 and par-
tially reduced XO-Mo(V), whereas rearrangement of radical cation
9 is different and includes the elimination of the ethyl radical to
produce a dealkylated product in the protonated form 10 and
alkylated enzyme (XO-alkylated). In the last step, deprotonation
of 10 by the enzyme releases product 2a and an ethylated enzyme
in a fully reduced form. The mechanisms presented in Schemes 4
and 5 are in accordance with the general behavior of molybde-
num-containing enzymes and includes the formation of Mo(V)
species that are well described and proved as intermediates in a
mechanistic cycle of this type of enzyme.43
We believe that results obtained with molybdenum salts are
good chemical models for the possible chemical behavior of molyb-
denum-containing enzymes. Results obtained employing tungsten
hexachloride (WCl6), albeit in more vigorous conditions, also
showed great selectivity on the model 1,4-DHP (Table 1, entry
11). Although much less abundant in nature, this might explain
why tungsten (VI) and not molybdenum is predominantly present
in enzymes isolated from hyperthermophilic archaea.44 Due to their
higher chemical reactivity and under these conditions, the moybde-
num-containing enzymes (VI) would probably not be selective
enough to perform the same reactions as enzymes involved in the
metabolism of organsims living in such a harsh environment. This
prediction is based on results obtained by MoOCl4 and MoCl5
(Table 1, entries 3 and 7) that resulted in very poor chemical selec-
tivity, which is in sharp contrast to tungsten(VI) chloride during
which a clean reaction was observed (reflux of acetic acid).
Further research is in progress in order to clarify the role of pure
isolated molybdenum-containing enzymes employed in the aro-
matization of 1,4-DHPs in vitro as well as their real action in living
organisms.
13. Rettie, A. E.; Fisher, M. B. In Handbook of Drug Metabolism; Wolf, T. F., Ed.;
Marcel Dekker: New York, 1999; p 131.
14. (a) Grün, G.; Fleckenstein, A. Arzneimittelforschung (Drug Res.) 1972, 22, 334; (b)
Bossert, F.; Meyer, H.; Wehinger, E. Angew. Chem. 1981, 93, 755; (c) Janis, R. A.;
Triggle, D. J. J. Med. Chem. 1983, 26, 775.
15. Berkels, B.; Roesen, R.; Dhein, S.; Fricke, U.; Klaus, W. Cardiovasc. Drug Rev.
1999, 17, 179.
16. Tsuruo, T.; Iida, H.; Nojiri, M.; Tsukagoshi, S.; Sakurai, Y. Cancer Res. 1983, 43,
2905.
17. Chapman, R. W.; Danko, G.; Siegels, M. I. Pharmacology 1984, 29, 282.
18. Malaise, W. J.; Mathias, P. C. F. Diabetologia 1985, 28, 153.
19. Krauze, A.; Germane, S.; Eberlins, O.; Sturms, I.; Klusa, V.; Duburs, G. Eur. J. Med.
Chem. 1999, 34, 301.
20. Peri, R.; Padmanabhan, S.; Singh, S.; Rutledge, A.; Triggle, D. J. J. Med. Chem.
2000, 43, 2906.
21. Zhou, X.; Zhang, L.; Tseng, E.; Scott-Ramsay, E.; Schentag, J. J.; Coburn, R. A.;
Morris, M. E. Drug Metab. Dispos. 2005, 33, 321.
22. (a) Böcker, R. H.; Guengerich, F. P. J. Med. Chem. 1986, 28, 1596; (b) Kudo, S.;
Okumura, H.; Miyamoto, G.; Ishizaki, T. Drug Metab. Dispos. 1999, 27, 303.
23. Memarian, H. R.; Abdoli-Senejani, M.; Tangestaninejad, S. J. Iran Chem. Soc.
2006, 3, 285.
24. (a) Vanden Eynde, J. J.; D’Ozario, R.; Van Haverbeke, Y. Tetrahedron 1994, 50,
2479; (b) Karami, B.; Montazerozohori, M.; Habibi, M. H.; Zolfigol, M. A.
Heterocycl. Commun. 2006, 11, 513; (c) Vanden Eynde, J. J.; Delfosse, F.;
Mayence, A.; Van Haverbeke, Y. Tetrahedron 1995, 51, 6511; (d) Bagley, M. C.;
Lubinu, M. C. Synthesis 2006, 1283; (e) Litvic´, M.; Cepanec, I.; Filipan, M.; Kos,
In summary, this paper describes a remarkably fast and selec-
tive method for the aromatization of substituted Hantzsch-1,4-
DHPs employing MoOCl4 as an oxidant under reflux in acetonitrile
as a solvent. The products, substituted pyridines, were isolated in
high purity and excellent yield. The 1,4-DHPs bearing alkyl groups
afforded unusual product distribution as a result of the dealkyla-
tion process. According to the obtained results, it is proposed that
molybdenum-containing enzymes participate in the metabolism of
1,4-DHP drugs to some degree.
ˇ ´
´
´
K.; Bartolincic, A.; Druškovic, V.; Tibi, M. M.; Vinkovic, V. Heterocycles 2005, 65,
´
´
´
23; (f) Filipan-Litvic, M.; Litvic, M.; Cepanec, I.; Vinkovic, V. ARKIVOC 2008, xi,
96; (g) Filipan-Litvic´, M.; Litvic´, M.; Vinkovic´, V. Tetrahedron 2008, 64, 10912.
25. (a) Zolfigol, M. A.; Kiany-Borazjani, M.; Sadeghi, M. M.; Memarian, H. R.;
Mohammadpoor-Baltork, I. Synth. Commun. 2000, 30, 2945; (b) Zolfigol, M. A.;
Kiany-Borazjani, M.; Sadeghi, M. M.; Mohammadpoor-Baltork, I.; Memarian, H.
R. Synth. Commun. 2000, 30, 3919; (c) Zolfigol, M. A.; Kiany-Borazjani, M.;
Sadeghi, M. M.; Mohammadpoor-Baltork, I.; Memarian, H. R. Synth. Commun.
2000, 30, 551; (d) Varma, R. S.; Kumar, D. J. Chem. Soc., Perkin Trans. 1 1999,
1755; (e) Chavan, S. P.; Dantale, S. W.; Kalkote, U. R.; Jyothirmai, V. S.; Kharul,
R. K. Synth. Commun. 1998, 28, 2789; (f) Chai, L.; Zhao, Y.; Sheng, Q.; Liu, Z.-Q.
Tetrahedron Lett. 2006, 9283; (g) Panchgalle, S. P.; Choudhary, S. M.; Chavan, S.
P.; Kalkote, U. R. J. Chem. Res. (S) 2004, 550.
Acknowledgment
26. (a) Mashraqui, S. H.; Karnik, M. A. Tetrahedron Lett. 1998, 39, 4896; (b) Kamal,
A.; Ahmad, M.; Mohd, N.; Hamid, A. M. Bull. Chem. Soc. Jpn. 1964, 37, 610; (c)
Nakamichi, N.; Kawashita, Y.; Hayashi, M. Org. Lett. 2002, 4, 3955; (d) Misner, R.
E. Diss. Abstr. 1969, 29B, 2817; (e) Filipan-Litvic´, M.; Litvic´, M.; Vinkovic´, V.
The authors wish to express their gratitude to Belupo Pharma-
ceuticals, Inc. for financial support of this research.

´
3681
M. Litvic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3676–3681
Tetrahedron 2008, 64, 5649; (f) Filipan-Litvic´, M.; Litvic´, M.; Vinkovic´, V. Bioorg.
Med. Chem. 2008, 16, 9276.
27. (a) Itoh, T.; Nagata, K.; Matsuya, Y.; Miyazaki, M.; Ohsawa, A. J. Org. Chem. 1997,
62, 3582; (b) Zhu, X. Q.; Zhao, B. J.; Cheng, J. P. J. Org. Chem. 2000, 65, 8158; (c)
Zolfigol, M. A.; Zebarjadian, M. H.; Sadegh, M. M.; Mohammadpoor-Baltork, I.;
Memarian, H. R.; Shamsipur, M. Synth. Commun. 2001, 31, 929; (d) Ko, K. J.;
Kim, J. Y. Tetrahedron Lett. 1999, 40, 3207.
a few portions. After stirring for 15 min. Organic extract was separated and
water layer was additionally extracted with dichloromethane (2 Â 10 mL).
Combined organic extracts were dried over anhydrous Na2SO4, filtered and
evaporated. The crude product was purified by column chromatography on
silica gel (CH2Cl2/EtOAc, 9:1) to yield the product of purity >98%. The identities
of products were confirmed by mp, IR, 1H and 13C NMR spectral data and
compared to the authentic samples of literature products.
28. (a) Nasr-Esfahani, M.; Moghadam, M.; Tangestaninejad, S.; Mirkhani, V. Bioorg.
Med. Chem. Lett. 2005, 15, 3276; (b) Moghadam, M.; Nasr-Esfahani, M.;
Tangestaninejad, S.; Mirkhani, V. Bioorg. Med. Chem. Lett. 2006, 16, 2026; (c)
Karami, B.; Montazerozohori, M.; Nasr-Esfahani, M. Heterocycles 2005, 65,
2181; (d) Nasr-Esfahani, M.; Moghdam, M.; Valipour, G. J. Iran Chem. Soc. 2008,
5, 244.
35. Analytical dana for selected product (2r): Pale yellow needles; mp: 110.0–
112.0 °C; Rf (CH2Cl2/EtOAc, 9:1) = 0.42; IR (KBr):
m
max: 2953, 1732, 1559, 1530,
1433, 1400, 1374, 1337, 1238, 1208, 1125, 1102, 1037 cmÀ1
;
1H NMR
(600 MHz, CDCl3): d 2.57 (s, 6H, Me), 3.73 (s, 6H, OCH3), 6.81–6.82 (d, 2H,
J = 3.8 Hz), 7.01 (d, 2H, J = 3.8 Hz); 13C NMR (600 MHz, CDCl3): d 22.8, 52.5,
114.6, 126.9, 128.8, 130.0, 137.2, 137.5, 155.6, 167.9; Anal. Calcd for
29. (a) Khadilkar, B.; Jaisinghani, H.; Khare, A. Ind. J. Chem. 1998, 37B, 817; (b) Ko, K.
Y.; Park, J. Y. Bull. Korean Chem. Soc. 1995, 16, 200; (c) Sadeghi, M. M.;
Mohammadpoor-Baltork, I.; Memarian, H. R.; Sobhani, S. Synth. Commun. 2000,
30, 1661; (d) Zolfigol, M. A.; Salehi, P.; Ghorbani-Choghamarani, A.; Safaiee, M.;
Shahamirian, M. Synth. Commun. 2007, 37, 1817.
C15H14BrNO4S: C 46.89, H 3.67, N 3.65. Found: C 46.7, H 3.5, N 3.6.
36. General procedure for the aromatization of 1,4-DHPs by MoCl5: To a solution of
1,4-DHP (1.0 mmol) in dichloromethane (10 mL), MoCl5 (0.57 g, 2.1 mmol) was
added and the reaction mixture was stired at room temperature for the time
indicated in Table 3. After that to the reaction mixture were added water
(20 mL), dichloromethane (10 mL) and solid NaHCO3 (2 g) in a few portions.
After stirring for 15 min. organic extract was separated and water layer was
additionally extracted with dichloromethane (2 Â 10 mL). Combined organic
extracts were dried over anhydrous Na2SO4, filtered and evaporated. The crude
product was purified by column chromatography on silica gel (CH2Cl2/EtOAc,
9:1) to yield the product of purity >98%. The identities of products were
confirmed by mp, IR, 1H and 13C NMR spectral data and compared to the
authentic samples of literature products.
30. (a) Jin, S.-H.; Jin, J.-Y.; Kim, Y.-I. Macromol. Res. 2003, 11, 501; (b) Firouzabadi,
H.; Jamalian, A.; Karimi, B. Bull. Chem. Soc. Jpn. 2002, 75, 1761.
31. (a) Boshta, N. M.; Bomkamp, M.; Schnakenburg, G.; Waldvogel, S. R. Chem. Eur.
J. 2010, 16, 3459; (b) Waldvogel, S. R.; Aits, E.; Holst, C.; Fröhlich, R. Chem.
Commun. 2002, 1278; (c) Kramer, B.; Averhoff, A.; Waldvogel, S. R. Angew.
Chem. Int. Ed. 2002, 41, 2981; (d) Waldvogel, S. R. Synlett 2002, 622; (e) Mirk,
D.; Kataeva, O.; Fröhlich, R.; Waldvogel, S. R. Synthesis 2003, 2410; (f) Kramer,
B.; Fröhlich, R.; Bergander, K.; Waldvogel, S. R. Synthesis 2003, 91; (g) Kramer,
B.; Waldvogel, S. R. Angew. Chem., Int. Ed. 2004, 43, 2446; (h) Mirk, D.;
Wibbeling, B.; Fröhlich, R.; Waldvogel, S. R. Synlett 2004, 1970; (i) Bomkamp,
M.; Artiukhov, A.; Kataeva, O.; Waldvogel, S. R. Synthesis 2007, 1107; (j) Spurg,
A.; Schnakenburg, G.; Waldvogel, S. R. Chem. Eur. J. 2009, 15, 13313.
32. (a) Cotton, F. A.; Wilkinson, G. Advanced Inorganic Chemistry, 5th ed.; John
Wiley & Sons, Inc.: New York, 1988; (b) Saito, T. J. Chem. Soc., Dalton Trans. 1
1999, 97.
37. Krapcho, A. P. ARKIVOC 2007, ii, 1.
38. (a) Krenitsky, T. A.; Neil, S. M.; Elion, G. M.; Hitchings, G. H. Arch. Biochem.
Biophys. 1972, 150, 585; (b) Maia, L.; Duarte, R. O.; Ponces-Freire, A.; Moura, J. J.
G.; Mira, L. J. Biol. Inorg. Chem. 2007, 12, 777; (c) Yoshisue, K.; Masuda, H.;
Matsushima, E.; Ikeda, K.; Nagayama, S.; Kawaguchi, Y. Drug Metab. Dispos.
2000, 28, 1162.
39. Davies, H. W.; Britt, S. G.; Pohl, L. R. Chem. Biol. Interact. 1986, 58, 345.
40. (a) Ortiz de Montellano, P. R.; Beilan, H. S.; Kunze, K. L. J. Biol. Chem. 1981, 256,
6708; (b) Guengerich, F. P.; Böcker, R. H. J. Biol. Chem. 1988, 263, 8168; (c)
Kennedy, C. H.; Mason, R. P. J. Biol. Chem. 1990, 265, 11425.
41. Aliyan, H.; Fazaeli, R.; Momeni, A. R.; Massah, A. R.; Naghash, H. J.; Khosravi, F.
Heterocycles 2007, 71, 2027.
42. Alfaro, J. F.; Jones, J. P. J. Org. Chem. 2008, 73, 9469.
43. (a) Benson, N.; Farrar, J. A.; McEwan, A. G.; Tomson, A. J. FEBS 1992, 307, 169;
(b) Okamoto, K.; Matsumoto, K.; Eger, B. T.; Pai, E. F.; Nishino, T. PNAS 2004,
101, 7931.
44. L’vov, N. P.; Nosikov, A. N.; Antipov, A. N. Biochemistry (Moscow) 2002, 67,
196.
33. IR spectra were recorded on a Perkin–Elmer Spectrum One spectrometer. 1H
NMR and 13C NMR were recorded on a Bruker 600 for CDCl3 solutions, shifts are
given in ppm downfield from TMS as an internal standard. HPLC analyses for
determination of the amount of dealkylation product 2a were performed with
a Thermo Separation Products (San Jose, USA) instrument equipped with
vacuum degasser SCM 1000, quaternary gradient pump P 4000, autosampler
AS 3000, scanning UV/vis detector UV 3000 HR and ChromQuest 251 software.
TLC analyses were performed on Merck’s (Darmstadt, Germany) DC-alufolien
with Kieselgel 60254. Melting points were determined using a Büchi B540
instrument. The 1,4-DHPs were prepared by modified Hantzsch procedures45
and fully characterised (mp, IR, 1H, 13C, NMR, spectras, and elemental analysis)
in our recent papers.24f,24g,26e The literature known aromatization products
were characterized by a comparison with authentic samples (melting point)
and their NMR (1H, 13C) and IR spectra.24e,25g,26e,29b,29d,46
45. (a) Loev, B.; Goodman, M. M.; Snader, K. M.; Tedeschi, R.; Macko, E. J. Med.
Chem. 1974, 17, 956; (b) Meyer, H.; Bossert, F.; Horstmann, H. Liebigs Ann.
Chem. 1977, 1888; (c) Phillips, A. P. J. Am. Chem. Soc. 1950, 72, 2780; (d) Litvic´,
´
´
34. General procedure for the aromatization of 1,4-DHPs by MoOCl4: To a solution of
corresponding 1,4-DHP (1.0 mmol) in CH3CN (10 mL), MoOCl4 (0.53 g,
2.1 mmol) was added and the reaction mixture was stirred for the time
indicated in Table 2. After cooling to room temperature to the reaction mixture
were added water (20 mL), dichloromethane (20 mL) and solid NaHCO3 (2 g) in
M.; Cepanec, I.; Vinkovic, V. Heterocycl. Commun. 2003, 9, 385; (e) Litvic, M.;
Filipan, M.; Pogorelic´, I.; Cepanec, I. Green Chem. 2005, 7, 771.
46. (a) Memarian, H. R.; Mohammadpoor-Baltork, I.; Javahery, M. J. Chin. Chem. Soc.
2006, 53, 511; (b) Zeynizadeh, B.; Dilmaghani, K. A.; Roozijoy, A. J. Chin. Chem.
Soc. 2005, 52, 1001.