Oxidative aromatization of 1,4-dihydropyridines and pyrazolines using HbA-H2O2: An efficient biomimetic catalyst system providing metabolites of drug candidates

Article abstract of DOI:10.1016/j.bmcl.2009.05.056

Human hemoglobin (HbA) efficiently catalyses the oxidative aromatization of 1,4-dihydropyridines (1,4-DHPs) and pyrazolines with hydrogen peroxide in phosphate buffer. The results of the study reveal that the rates of oxidative aromatization of 1,4-DHPs a

Full text of DOI:10.1016/j.bmcl.2009.05.056

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4432–4436
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Oxidative aromatization of 1,4-dihydropyridines and pyrazolines using
HbA–H2O2: An efficient biomimetic catalyst system providing metabolites
of drug candidates
*
Atul Kumar , Ram Awatar Maurya, Siddharth Sharma
Medicinal and Process Chemistry Division, Central Drug Research Institute , Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 February 2009
Revised 1 May 2009
Accepted 14 May 2009
Available online 19 May 2009
Human hemoglobin (HbA) efficiently catalyses the oxidative aromatization of 1,4-dihydropyridines (1,4-
DHPs) and pyrazolines with hydrogen peroxide in phosphate buffer. The results of the study reveal that
the rates of oxidative aromatization of 1,4-DHPs are substituent dependent. Thus, the present study is
very useful in understanding the metabolism of 1,4-DHP drugs in liver by cytochrome P450 and designing
novel drugs as well as modifying the existing drugs for better pharmacokinetic profile.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
1,4-Dihydropyridines
Human hemoglobin (HbA)
Biomimetic catalyst
1,4-Dihydropyridines (1,4-DHPs)1 have been established as one
of the first line drugs for treatment of hypertension because of
their promising depressor effect and relatively good tolerability.2
Felodipine, amlodipine, nifedipine, and nicardipine (Fig. 1) are
among the best selling drugs. These drugs act by modulating cal-
cium channel (Ca2+) currents.3 1,4-DHPs have been extensively
studied because of the biological significance of these compounds
in the NADH redox process4 as well as their therapeutic functions
for treatment of a variety of diseases, such as cardiovascular disor-
ders, cancer, and AIDS.5
mammals, family of cytochrome P450 are primarily responsible for
the oxidative metabolism.14 Cytochrome P450 is a very large and
diverse superfamily of hemoproteins found in all domains of life.
Cytochrome P450 uses an excess of both exogenous and endogenous
compounds as substrates in enzymatic reactions. The most common
reaction catalyzed by cytochrome P450 is a monooxygenase, for
example, insertion of one atom of oxygen into an organic substrate
(RH) while the other oxygen atom is reduced to water:
RH þ O2 þ 2Hþ þ 2eꢀ ! ROH þ H2O
Metabolic studies of 1,4-DHP drugs in the human body have
shown that these compounds are oxidized to pyridine derivatives
by the action of cytochrome P450 in the liver.6 The oxidation of
easily available 1,4-DHPs7 to the corresponding pyridine deriva-
tives constitutes principal metabolic route in biological systems,
as well as provides a facile access to the corresponding pyridine
derivatives, which show anti-hypoxic and anti-ischemic activities.8
The main representative of oxidized 1,4-DHPs is cerivastatin which
has found its application in the treatment of atherosclerosis and
other coronary deseases.9 Therefore, oxidative aromatization of
1,4-DHPs has attracted continuing interests of organic and medic-
inal chemists and a plethora of protocols has been developed.10–12
In the course of drug discovery, features such as absorption, dis-
tribution, metabolism, and excretion (ADME) are determinant
parameters that need to be studied at the earliest stages.13 In
Cytochrome P-450 (P-450) has been utilized for catalytic dehydro-
genation of 1,4-dihydropyridines.15 The active site of cytochrome
P450 contains a heme iron center (Fig. 2). The iron is tethered to
the P450 protein via a thiolate ligand derived from a cysteine resi-
due. The iron ion in the metalloporphyrins interconverts between
several oxidation states (+2, +3, +4, and +6) and executes the redox
reactions.16 This prompted us to utilized human hemoglobin–H2O2
(HbA–H2O2)17 as a biomimetic catalyst system for oxidative aroma-
tization of 1,4-DHPs.
Herein we report biocatalytic activity of human hemoglobin
(HbA) for the oxidative aromatization of 1,4-dihydropyridines with
H2O2. We preferred hydrogen peroxide as oxidant over other avail-
able oxidizing agent since it is cheap, operationally safe, environ-
mentally friendly, easy to handle and produces only water as a
by product, which reduces purification requirements.
HbA is an iron porphyrin metalloprotein and acts as an oxygen
carrier in biological system. The commercially available HbA con-
tains approximately 80% of the methaemoglobin (Met Hb). MET-
hemoglobin is an oxygen carrying protein hemoglobin having
* Corresponding author. Tel.: +91 522 2612411; fax: +91 522 2623405.
E-mail address: dratulsax@gmail.com (A. Kumar).
CDRI Communication No. 7680.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.056

A. Kumar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4432–4436
4433
Cl
NO2
NO2
Cl
Cl
O
O
CH2Ph
N
MeOOC
NH2
COOMe
MeOOC
MeOOC
COOEt
MeOOC
COOEt
O
N
H
N
H
N
H
N
H
Nicardipine
Felodipine
Nifedipine
Amlodipine
F
O
NH2
OH OH
NH2
N
N
O
O
P
N
OH
O
HO
COOH
P
N
O
O
O
N
O
MeO
HO
OH
HO
OH
N
NADH
Cerivastatin
Figure 1.
phate buffer and subsequently eluted over Sephadex G-25 column
to obtain the purified form of oxyhemoglobin (HbAo). The percent-
age of Oxy Hb of the total HbA was determined from the Absor-
bance (A) at 540 nm and at 576 nm. This equation is based on
reported molar absorptivities of oxy-Hb and met-Hb.18
Cys
S
N
Cys
N
Fe3+
N
70A540 ꢀ 117A576
S
X ¼
ꢁ 100%
N
169A576 ꢀ 236A540
In our preliminary experiments 1,4-DHP 1a was chosen as a model
substrate and the reaction was studied by using 30% aqueous
hydrogen peroxide with the catalytic amount of purified oxy-Hb
(method-I). We observed that the purified oxyhemoglobin oxidised
1,4-DHP quite efficiently and furnished the corresponding pyridine
derivative in high yield but this procedure needs extra efforts for
purification of the catalyst. We therefore, developed two other
methods for the oxidative aromatization (method II and III).
The crude oxyhemoglobin (method II) did not work well for oxi-
dative aromatization of 1,4-DHPs. However, method III was found
to be best among three protocols (I, II, and III). The protocol III gave
excellent yields of aromatized 1,4-DHP in short reaction times
(Fig. 3). No reaction was detected when either hemoglobin or
hydrogen peroxide was used alone.
OOC
OOC
Figure 2. Heme group in active site of cytochromes.
Fe3+ state of iron in the heme group and is therefore unable to
transfer oxygen. Thus, our initial strategy was to convert met-
hemoglobin into oxy-hemoglobin which can efficiently transfer
the oxygen. Therefore, we converted commercial human hemoglo-
bin into oxyhemoglobin by the treatment with dithionite in phos-
H2O2 (30% w/v)
Ferryl oxy Hb
Oxy Hb
brown coloured
[Met Hb-OOH]
greenish coloured
Ph
Method-III
MeOOC
COOMe
H2O2 (30% w/v)
stirr, r.t.
N
H
H2O2 (30% w/v)
Phosphate buffer pH-6.5
stirr, r.t.
1a
Ph
MeOOC
COOMe
Ph
MeOOC
COOMe
N
H
1a
in situ Na2S2O4, stirr, r.t.
Crude
Oxy Hb
HbA
H2O2 (30% w/v)
stirr, r.t.
N
Phosphate buffer, pH-6.5
2a
Method-II
Ph
Na2S2O4, stirr, r.t.
MeOOC
COOMe
H2O2 (30% w/v)
stirr, r.t.
Phosphate buffer
pH-6.5
N
H
1a
elution over SephadexG-25column
HbAo
Purified Oxy Hb
Crude Oxy Hb
Method-I
Figure 3. Optimization of the protocol for HbA/H2O2 mediated oxidative aromatization of 1,4-DHPs.

4434
A. Kumar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4432–4436
In order to check the feasibility of HbA/H2O2 catalyzed system
as well as electron deficient aromatic substituents at C-4. The reac-
tion is quite general and was successfully applied to 1,4-DHPs hav-
ing aliphatic as well as heteroaromatic substituents at C-4.
However when the C-4 of the 1,4-DHP was substituted with benzyl
or isopropyl group dealkylation occurred (entries 16 and 17). Ben-
zyl alcohol was isolated as a side product for entry (16) but the cor-
responding alcohol was not isolated for entry (17). The reaction
was quite regioselective as only oxidative aromatization of the
1,4-DHPs occurred. Alcohols were not oxidized in our reaction con-
ditions. We attempted to oxidize various alcohols (ethanol, isopro-
pyl alcohol, and benzyl alcohol) in the present reaction conditions
but corresponding oxidized product was not obtained. This indi-
cates that an alcohol present in a side chain of 1,4-DHP should
not oxidize under the reaction conditions.
A careful examination of the results of Table 2 revealed that the
reaction time for each 1,4-DHP was different. This inspired us to
investigate the substituent effect over the rate of oxidative aroma-
tization of 1,4-DHPs. The reactions were performed under protocol
III. The test samples were withdrawn at 30 min intervals and HPLC
was performed to determine the progress of the reaction (% oxida-
tion). The percentage oxidation was calculated by measuring the
AUC of oxidized and unoxidized 1,4-DHP. We studied the effect
of substitution at C-2 and C-6 (methyl vs n-propyl) (Fig. 4a), C-3
and C-5 (methyl, ethyl, isopropyl, and tert butyl esters) (Fig. 4b)
and C-4 (H, phenyl, p-methoxyphenyl, and p-nitrophenyl)
(Fig. 4c) of the 1,4-DHPs over the rate of oxidative aromatization.
The results of this study are summarized in Figure 4.
in organic medium, we performed the oxidative aromatization in
ethanol, acetonitrile, methanol, and CH2Cl2. Although organic med-
ium were found unsuitable for this biomimetic oxidative aromati-
zation, HbA/H2O2 system worked quite efficiently in 15% of
acetonitrile in phosphate buffer. The results of this study are sum-
marized in Table 1.
The oxidative aromatization of 1,4-DHP 1a proceeded smoothly
at room temperature in 15% acetonitrile in phosphate buffer giving
excellent yields of 2a in 5 h (method III). The presence of acetoni-
trile was necessary for the reaction to proceed smoothly. Since the
reaction gave almost quantitative yields of the products in the
presence of 15% acetonitrile in phosphate buffer, we did not study
the affects of adding any tension active compounds to the system
(Micellar system). An attempt to increase the reaction rate by heat-
ing the reaction mixture resulted in decreased yields of 2a. Upon
refluxing the reaction mixture, 2a was isolated in only 40% yield.
Thus, we concluded that stirring the reaction at room temperature
was the optimal condition.
Next, we examined the scope of the HbA–H2O2 mediated oxida-
tive aromatization of 1,4-DHPs (Table 2). A series of 1,4-DHPs were
aromatized using the optimized reaction conditions. The reaction
proceeded smoothly with various 1,4-DHPs bearing electron rich
Table 1
Optimization of solvent for HbA–H2O2 mediated oxidative aromatization of 1aa
The results of Figure 4a shows that size of R1 at C-2 and C-6 of
the 1,4-DHPs affects the rate of oxidative aromatization. With
n-propyl group (entry 5) rate of aromatization was slow in com-
parison to methyl group (entry 2). In contrast to it the rate of aro-
matization increases significantly with increasing the size of ester
group. In the case of methyl ester (entry 1) rate of aromatization
was slower while with tertiary butyl ester (entry 4) rate of aroma-
tization was faster (Fig. 4b). The rate of aromatization was faster
for simple 1,4-DHPs (R3 = H, entry 15). The reaction time was very
little affected by the electronic nature of substituent present in
Entry
Solvent
Time (h)
Yield of 2ab (%)
1
2
3
4
5
6
CH2Cl2
Ethanol
Methanol
Acetonitrile
Phosphate buffer
15% Acetonitrile in phosphate buffer
12
8
8
8
8
30
60
62
72
80
95
5
a
Reaction condition: 1,4-DHP 1a (1 mmol), ferryl oxy Hb (0.1 mmol), H2O2
(2 mmol), stir, rt.
b
Isolated yield.
Table 2
HbA/H2O2 catalyzed oxidative aromatization of Hantzsch 1,4-DHPsa
R3
R
R1OOC
R2
COOR1
R2
R1OOC
R2
COOR1
R2
HbA (0.1 mmol), 30% (w/v) H2O2 (2 mmol)
Phosphate buffer (pH = 6.5)
rt, Stirr
N
H
1
N
2
Entry
R
R1
R2
R3
Productb
Time (h)
Yieldc (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
C6H5
C6H5
C6H5
C6H5
C6H5
4-CH3–C6H5
4-CH3O–C6H5
4-Cl–C6H5
3-NO2–C6H5
4-NO2–C6H5
C6H5CH@CH
2-Furyl
2-Thenyl
CH3
H
CH2C6H5
CH(CH3)2
CH3
CH3
CH3
CH3
CH3
(CH2)2CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C6H5
C6H5
C6H5
C6H5
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2o
2o
5
4.5
3
2
5.5
4
4.5
5
5
5
5
4.5
4.5
3.5
3
4
4
97
96
98
95
96
95
96
95
94
96
97
99
98
95
97
85
80
CH2CH3
CH(CH3)2
C(CH3)3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
C6H5
4-CH3–C6H5
4-CH3O–C6H5
4-Cl–C6H5
3-NO2–C6H5
4-NO2–C6H5
C6H5CH@CH
2-Furyl
2-Thenyl
CH3
H
H
H
a
Reaction conditions: 1,4-DHP (1 mmol), HbA (0.1 mmol), H2O2 (30% w/v, 2 mmol), 15% acetonitrile in phosphate buffer (pH 6.5, 3 ml), stir, rt.
All the products were known and characterized by comparison of their melting points, IR, and 1H NMR spectra with literature.
b

A. Kumar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4432–4436
4435
Effect of substitution of R2over the rate of
aromatization
Table 3
Scope of the HbA–H2O2 catalyst system for oxidative aromatization of 1,2,3-
trisubstituted pyrazolinesa
120
100
80
60
40
20
0
a
b
R1
HbA-H2O2
R1
R2
R2
Phosphate buffer
rt, stirr
N
N
N
N
Ph
Ph
3
4
Me (Entry-2, Table-2)
Entry
R1
C6H5
4-MeOC6H4
4-NO2C6H4
4-ClC6H4
2-Furyl
R2
Productb
Yieldc (%)
n-Pr (Entry-5, Table-2)
1
2
3
4
5
6
C6H5
C6H5
C6H5
C6H5
4-CH3–C6H4
4-ClC6H4
4a
4b
4c
4d
4e
4f
80
85
86
84
83
78
0
30 60 90 120 150 180 210 240 270 300 330
Time(min)
Effect of substitution of R1 over the rate of aromatization of
1,4-DHPs
2-Thenyl
a
120
100
80
60
40
20
0
Reaction conditions: Pyrazoline 3 (1 mmol), HbA (0.1 mmol), H2O2 (30% w/v,
2 mmol), 15% acetonitrile in phosphate buffer (pH 6.5, 3 ml), stir, rt, 5 h.
b
All the products were known and characterized by comparison of their melting
points, IR, and 1H NMR spectra with literature.
c
Isolated yield.
Me Entry-1, Table-2
iPr Entry-3, Table-2
Et Entry-2, Table-2
tBu Entry-4, Table-2
substituted pyrazoline derivatives with HbA–H2O2 led to the for-
mation of corresponding pyrazoles in high yields within 4–5 h at
room temperature.
A detail study for mechanistic aspect of HbA–H2O2 mediated
oxidation of 1,4-DHPs and pyrazoline is currently underway in
our group. All these results will be published in a separate paper
in near future.
0
30
60
90 120 150 180 210 240 270 300
Time (min)
In conclusion, we have reported an efficient biomimetic oxida-
tive aromatization of 1,4-DHPs and 1,2,3-trisubstituted pyrazolines
catalyzed by HbA/H2O2 in 15% acetonitrile–phosphate buffer. The
present study provides useful correlation between substituent ef-
fect and the rate of aromatization of 1,4-DHPs. Such correlations
are important in designing new drugs, modifying the existing
drugs for better pharmacokinetic profile and understanding
metabolism of 1,4-DHPs based drugs in liver by cytochrome P450
enzymes.
Effect of substitution of R3 (phenyl ring) over the
rate of aromatization
120
100
80
60
40
20
0
c
4-NO2-C6H5 Entry-10, Table-2
4-CH3-C6H4 Entry-6, Table-2
C6H5 Entry-2, Table-2
Acknowledgments
H Entry-15, Table-2
R. A. Maurya and S. Sharma are thankful to CSIR New Delhi for
financial support. Authors also acknowledge SAIF-CDRI for proving
spectral and analytical data.
0
30
60
90 120 150 180 210 240 270 300
Time(min)
R3
Supplementary data
R1OOC
COOR1
4
3
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.05.056.
5
2
R2
N
H
R2
6
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