One-pot synthesis and antimicrobial evaluation of novel 3-cyanopyridine derivatives of (?)-β-pinene

Article abstract of DOI:10.1016/j.bmcl.2016.02.024

A series of novel 3-cyanopyridine derivatives of (?)-β-pinene were designed and synthesized by one-pot four-component domino reactions. The targeted compounds were evaluated for their antimicrobial activity against four bacteria (Klebsiella pneumoniae, Enterobacter aerogenes, Staphylococcus aureus, Staphylococcus epidermidis) and a fungus (Candida albicans). The results showed that most of the minimal inhibitory concentrations (MICs) of these 3-cyanopyridine derivatives against the tested strains was in the range of 15.6–125?mg/L. Among these 3-cyanopyridine derivatives, the MICs of compound 5h against S. epidermidis and C. albicans were 15.6?mg/L, which revealed that compound 5h featured double fluoro substituents at meta- and para-position was the most active compound. In addition, the preliminary structure–activity relationship analysis indicated that the change of substituents on the pyridine ring and benzene ring of 3-cyanopyridine derivatives was an important factor for inducing antimicrobial activity. This research would promote the development of heterocyclic derivatives of β-pinene with antimicrobial activity.

Full text of DOI:10.1016/j.bmcl.2016.02.024

Accepted Manuscript
One-pot synthesis and antimicrobial evaluation of novel 3-cyanopyridine de-
rivatives of (-)-β-pinene
Shengliang Liao, Shibin Shang, Minggui Shen, Xiaoping Rao, Hongyan Si, Jie
song, Zhanqian Song
PII:
S0960-894X(16)30136-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.02.024
BMCL 23575
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
10 December 2015
6 February 2016
10 February 2016
Please cite this article as: Liao, S., Shang, S., Shen, M., Rao, X., Si, H., song, J., Song, Z., One-pot synthesis and
antimicrobial evaluation of novel 3-cyanopyridine derivatives of (-)-β-pinene, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.02.024
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One-pot synthesis and antimicrobial evaluation of novel
3-cyanopyridine derivatives of (-)-β-pinene
Shengliang Liaoa, Shibin Shanga*, Minggui Shena, Xiaoping Raoa, Hongyan Sia, Jie songb and
Zhanqian Songa*
aInstitute of Chemical Industry of Forest Products, China Academy of Forestry; National
Engineering Laboratory for Biomass Chemical Utilization; State Forestry Administration’s Key
and Open Laboratory for Forest Chemical Engineering; Jiangsu Provincial Key Laboratory for
Biomass Energy and Material, Nanjing 210042, Jiangsu, China
bDepartment of Chemistry and Biochemistry, University of Michigan-Flint, 303E Kearsley Street,
Flint, MI 48502, USA
* Corresponding author: shangsb@hotmail.com & lhssxly@hotmail.com.
Abstract: A series of novel 3-cyanopyridine derivatives of (-)-β-pinene were designed and
synthesized by one-pot four-component domino reactions. The targeted compounds were
evaluated for their antimicrobial activity against four bacteria (Klebsiella pneumoniae,
Enterobacter aerogenes, Staphylococcus aureus, Staphylococcus epidermidis) and a fungus
(Candida albicans). The results showed that most of the minimal inhibitory concentrations (MICs)
of these 3-cyanopyridine derivatives against the tested strains was in the range of 15.6-125 mg/L.
Among these 3-cyanopyridine derivatives, the MICs of compound 5h against S. epidermidis and C.

albicans were 15.6 mg/L, which revealed that compound 5h featured double fluro substituents at
meta- and para-position was the most active compound. In addition, the preliminary
structure-activity relationship analysis indicated that the change of substituents on the pyridine
ring and benzene ring of 3-cyanopyridine derivatives was an important factor for inducing
antimicrobial activity. This research would promote the development of heterocyclic derivatives of
β-pinene with antimicrobial activity.
Keywords: β-pinene, nopinone, synthesis, 3-cyanopyridine, antimicrobial
Turpentine oil, an important natural resource, had received much attention for its various
biological activity. Using turpentine oil to develop chemical derivatives with biological activity
was of great interest to the researchers in the field 1. β-Pinene was a main component of turpentine
oil, former researches showed that β-pinene obtained a variety of biological activity such as
2
3
4
5
antibacterial , antiviral , anticancer , and antidepressant-like activity , etc. Meanwhile, the
derivatives of β-pinene also exhibited many biological activity, including antibacterial activity 6-9
,
human ORL1 receptors antagonist 10, prostaglandin D2 receptor antagonist 11. Therefore, using
β-pinene to exploit derivatives with biological activity had wide prospects.
Recent researches showed that the derivatives of pinene with heterocycle groups exhibited good
antimicrobial activity. Wei et al found that some pinanyl-2-aminopyrimidines (Fig. 1a) and pinanyl
isoxazolines (Fig. 1b) exhibited good antibacterial activity against E. coli and B. subtilis 12. Dhar
et al synthesized a pinanyl-β-lactam derivative (Fig. 1c) which obtained excellent antimicrobial
activity against C. albicans and S. aureus 13. However, most of the heterocyclic derivatives of
pinene were synthesized from α-pinene, rather than from β-pinene. Hence, the synthesis and

antimicrobial activity evaluation of heterocyclic derivatives of β-pinene were interesting and
needed.
Fig. 1. Heterocyclic derivatives of pinene
Among the heterocyclic compounds, 3-cyanopyridine was known to have multiple biological
15
activity, such as antibacterial 14, antiviral and anti-inflammatory activity 16. Considering that
both the β-pinene and 3-cyanopyridine exhibited good biological activity, it was of great
significance to synthesize the 3-cyanopyridine derivatives of β-pinene. To the best of our
knowledge, the study on the synthesis and antimicrobial activity of 3-cyanopyridine derivatives of
β-pinene had not been reported before.
In this study, a series of novel 3-cyanopyridine derivatives of (-)-β-pinene were synthesized by
one-pot four-component domino reactions. The structures of these synthesized compounds were
1
characterized by FT-IR, H NMR, 13C NMR and ESI-MS. The antimicrobial activity of these
3-cyanopyridine derivatives were evaluated against four bacteria Klebsiella pneumoniae,
Enterobacter aerogenes, Staphylococcus aureus, Staphylococcus epidermidis and a fungus
Candida albicans.
(-)-β-Pinene was an olefin which could not be directly applied to the mentioned four-component
domino reaction. Hence, (-)-β-pinene was in advance transferred into (+)-nopinone, which was
tolerated to the four-component domino reaction. Although several synthetic methods were
available for the transformation of (-)-β-pinene into (+)-nopinone 17-19, oxidation using KMnO4 as

oxidant under solvent condition was one of the commonly used method 18. Compared with other
methods, the oxidation with KMnO4 had many merits, including mild reaction conditions,
considerable yield and high selectivity.
In this work, the oxidation of (-)-β-pinene was performed with KMnO4 in acetone. The reaction
sequence for the synthesis of (+)-nopinone (2) was presented in Fig. 2. (-)-β-Pinene (1) was firstly
oxidized into nopinic acid (1-a) by KMnO4, then nopinic acid (1-a) was further oxidized by
KMnO4 to form the (+)-nopinone (2). The IR spectrum of (+)-nopinone showed that the υ(C=C-H),
1
υ(C=C) and υ(C=CH2) bands of (-)-β-pinene disappeared and the υ(C=O) band appeared; the H
NMR spectrum showed that the signals at 4.59-4.66 ppm attributed to the olefinic protons
disappeared, while a signal at 2.56 ppm assigned to the protons in the carbons next to the carbonyl
19
group appeared. These characterization results, which agreed with earlier reported data
,
indicated that (+)-nopinone (2) was synthesized by the oxidation with KMnO4 in acetone.
Fig.2. Synthesis of (+)-nopinone (2) from (-)-β-pinene (1) by oxidation with KMnO4;
Reaction conditions: 3 equivalent KMnO4, acetone, r.t., 8 h.
One-pot four-component domino reaction between ketone, aldehyde, ammonium acetate and
ethyl cyanoacetate (or malononitrile) was an efficient method for the synthesis of 3-cyanopyridine
compounds. The one-pot four component domino reactions for the synthesis of 3-cyanopyridines
could be carried out under various reaction conditions 20-23. The catalytic reaction condition using
rare earth as catalysts was commonly applied for the synthesis of 3-cyanopyridines, due to its mild
reaction condition and considerable yield of product 23. In this work, Twenty-five 3-cyanopyridine

derivatives (5a-y) were synthesized by the one-pot four-component domino reactions between
(+)-nopinone (2), benzaldehydes (3a-n), ethyl cyanoacetate (or malononitrile) (4) and ammonium
acetate under Yb(OTf)3 catalysis (Fig. 3). When the ethyl cyanoacetate was chose as starting
material, the products were 2-hydroxy-3-cyanopyridines (5a-5m), and when the malononitrile was
used, the products were 2-amino-3-cyanopyridines (5n-5y).
Fig 3. Synthesis of 3-cyanopyridine derivatives of (-)-β-pinene (5a-y)
Accoding to the literature [23], the possible mechanism of this four-component domino reaction
was proposed. Taking compound 5a as an example, the synthesis mechanism of 3-cyanopyridine
derivatives was depicted in Fig. 4. Firstly, (+)-nopinone (2) might react with ammonium acetate to
form an enamine (2-b), meanwhile, the benzaldehyde (3a) and ethyl cyanoacetate (4) were
condensed to form an ethyl (E)-2-cyano-3-phenylacrylate (3a’). Then, the enamine (2-b) reacted
with the ethyl (E)-2-cyano-3-phenylacrylate (3a’) to give an intermediate (4b’). After

cycloaddition, isomerization, and aromatization, the compound 5a was obtained.
Fig. 4. Possible synthesis mechanism of compound 5a
1
The structures of these 3-cyanopyridine derivatives were characterized by IR, H NMR, 13C
NMR and ESI-MS. Taking compound 5j as an example, in IR spectrum the absorption band at
3352 cm-1 was assigned to -OH group, the absorption bands at 2914 cm-1 and 2867 cm-1 were
attributed to -CH2-, and -CH-, the absorption bands at 2220 cm-1 and 1650~1460 cm-1 were
1
represented the existence of -CN and aromatic ring, respectively. In H NMR spectrum of
compound 5j, the signal appearing as a singlet at about 13.8 ppm was assigned to the proton of
hydroxyl group in the pyridine ring. The signals attributed to the aromatic protons of benzene
rings with different substituent groups were observed in the range of 6.8-7.8 ppm. The other
signals were assigned to the protons in the pinane skeleton. In 13C NMR spectrum of compound 5j,
the characteristic C=N peaks related to pyridine ring were observed at 170-150 ppm, the signals of
aromatic carbons appeared at 140.00-110.00 ppm, the characteristic C-Cl peaks related to benzene
ring were observed at ~134 ppm. The signals of –CN group appeared at ~116 ppm. The signals of
aliphatic groups appeared between 50.00 ppm and 20.00 ppm. In ESI-MS spectrum of compound
5j, the signals at 325.1 [M+1] + and 347.1 [M+23] + indicated that the molecular weight of 5j was
324.1. These results showed that the characterization data were in full agreement with the
proposed structures.

The minimal inhibition concentrations (MICs) of the 3-cyanopyridine derivatives of
(-)-β-pinene were determined by serial two-fold broth dilution method. The antimicrobial activity
of the 3-cyanopyridine derivatives against four bacteria (two Gram-positive and two
Gram-negative bacteria) and one fungus were summarized in Table 1. For the Gram-negative
bacteria K. neumoniae and E. aerogenes, the MICs of all the 3-cyanopyridine derivatives were
greater than or equal to 62.5 mg/L, while the MICs of kanamycin were 2 mg/L and 3.9 mg/L,
respectively. The results indicated that these 3-cyanopyridine derivatives exhibited weak
antibacterial activity against K. neumoniae and E. aerogenes. For the Gram-positive bacteria S.
aureus and S. epidermidis, the MICs of most of the 3-cyanopyridine derivatives were in the range
between 15.6-125 mg/L, while the MICs of kanamycin were 2 mg/L and 3.9 mg/L, respectively.
These results revealed that these 3-cyanopyridine derivatives showed moderate antibacterial
activity against S. aureus and S. epidermidis. For the fungi C. Albicans, the MICs of the
3-cyanopyridine derivatives were within the range of 15.6-125 mg/L and the MICs of rifampicin
was 3.9 mg/L, which revealed that these derivatives exhibited moderate antifungal activity against
C. Albicans. Furthermore, among these 3-cyanopyridine derivatives, compound 5h with the MICs
of 15.6 mg/L against S. epidermidis and C. albicans was the most active compound.

Table 1. Antimicrobial activity of the 3-cyanopyridine derivatives of (-)-β-pinene (5a~y)
MIC (mg/L)
Compound
Gran-negative bacteria
K. neumoniae E. aerogenes
>125 >125
Gram-positive bacteria
S. aureus S. epidermidis
fungus
C. albicans
125
125
125
125
125
125
31.3
62.5
31.3
31.3
15.6
15.6
31.3
62.5
125
125
125
125
125
125
125
125
125
125
125
125
125
>125
>125
3.9
5a
125
125
125
125
62.5
62.5
62.5
62.5
62.5
62.5
>125
>125
>125
>125
125
>125
125
125
125
125
125
125
125
>125
>125
2
125
62.5
62.5
125
62.5
62.5
62.5
62.5
62.5
125
>125
125
>125
>125
125
>125
125
125
125
125
125
125
125
>125
>125
3.9
62.5
125
5b
125
5c
62.5
62.5
62.5
62.5
31.3
31.3
31.3
62.5
125
62.5
125
5d
5e
62.5
31.3
15.6
31.3
31.3
31.3
125
5f
5g
5h
5i
5j
5k
5l
125
125
5m
>125
>125
125
125
5n
125
5o
125
5p
>125
125
62.5
125
5q
5r
>125
>125
>125
>125
125
125
5s
125
5t
31.3
62.5
62.5
62.5
125
5u
5v
5w
125
5x
5y
>125
>125
2
>125
-
2
Kanamycin
Rifampicin
-
-
-
-
3.9
The preliminary structure-activity relationship was analyzed to understand the influence of
structures of these 3-cyanopyridine derivatives on their antimicrobial activity. It can be seen from
Table 1 that 3-cyanopyridine derivatives exhibited better antimicrobial activity than (+)-nopinone,
which suggested that the introduction of 3-cyanopyridine scaffold into the (+)-nopinone molecule
improved the antimicrobial activity against the tested strains, in particular against the

Gram-positive bacteria (S. aureus and S. epidermidis). Besides, compared with derivatives with an
amino substituent at C-2 of the pyridine ring (compound 5n-y), most of the derivatives with
hydroxyl substituent at C-2 of the pyridine ring (compound 5a-m) showed better antibacterial
activity against Gram-positive bacteria. This result revealed that the change of substituent at C-2
of the pyridine ring was an important factor for inducing antimicrobial activity. Moreover, for
2-hydroxy-3-cyanopyridines (5a-m), the derivatives bearing halogen substituent in benzene ring
exhibited better antibacterial activity against Gram-positive bacteria. For example, compound 5h
bearing two fluro substituents in benzene ring have the best antibacterial activity. This may be
because the incorporation of the halogen substituents increased the lipophilicity of the derivatives,
which could improve the penetration capacity of the compound in the lipid membranes and tissues
of bacteria 24. In addition, a previous report showed that tetrahydroquinolines were active against
antibacterial targets, including DNA gyrase and methionyl tRNA synthetase 25. Hence, for the
3-cyanopyridine derivatives of β-pinene with tetrahydroquinoline skeleton, the DNA gyrase or
methionyl tRNA synthetase was considered as the possible molecular target.
As a conclusion, twenty-five new 3-cyanopyridine derivatives of (-)-β-pinene (5a~y) were
1
synthesized and characterized by FT-IR, H NMR, 13C NMR and ESI-MS. The antimicrobial
activity of these 3-cyanopyridine derivatives was evaluated against four bacteria K. neumoniae, E.
aerogenes, S. aureus, S. epidermidis and a fungus C. albicans. The results showed that
3-cyanopyridine derivatives of (-)-β-pinene (5a~y) had moderate antibacterial activity against the
Gram-positive bacteria S. aureus, S. epidermidis, weak antibacterial activity against the
Gram-negative bacteria K. neumoniae, E. aerogenes, and moderate antifungal activities against C.
albicans. Compound 5h exhibited the best antimicrobial activities against overall strains. The

preliminary structure-activity relationship analysis showed that the antimicrobial activity of these
3-cyanopyridine derivatives was influenced by the change of substituents in the pyridine ring and
benzene ring. Besides, DNA gyrase or methionyl tRNA synthetase was speculated as the possible
molecular target of the 3-cyanopyridine derivatives of (-)-β-pinene. This research would promote
the understanding of antimicrobial activity of heterocyclic derivatives of β-pinene.
Acknowledgements
This work was supported by the Joint Funds of the National Natural Science Foundation of China
and Yunnan Provincial Government under Grant No. U1202265.
Supplementary data
1
Supplementary data (experimental and copies of the H, 13C NMR and ESI-MS spectra for
3-cyanopyridine derivatives of (-)-β-pinene (5a~y)) associated with this article can be found, in
the online version.
REFERENCES
[1] Mercier, B; Prost, J.; Prost, M. Int. J. Occup. Med. Env., 2009, 22, 331.
[2] Silva, A. C. R.; Lopes, P. M.; Azevedo, M. M. B.; Costa, D. C. M.; Alviano, C. S.; Alviano, D. S. Molecules,
2012, 17, 6305.
[3] Akram, A.; Paul, S. Iranian J. Microbiol., 2014, 6, 149.
[4] Zhang, Z.; Guo, S.; Liu, X.; Gao, X. Drug Res., 2015, 65, 214.

[5] Guzmán-Gutiérrez, S. L.; Bonilla-Jaime, H.; Gómez-Cansino, R.; Reyes-Chilpa, R. Life Sci., 2015, 128, 24.
[6] Nikitina, L. E.; Startseva, V. A.; Vakulenko, I. A.; Khismatulina, I. M.; Lisovskaya, S. A.; Glushko, N. P.;
Fassakhov, R. S. Pharm. Chem. J., 2009, 43, 251.
[7] Gavrilov, V. V.; Startseva, V. A.; Nikitina, L. E.; Lodochnikova, O. A.; Gnezdilov, O. I.; Lisovskaya, S. A.;
Glushko, N. I.; Klimovitskii, E. N. Pharm. Chem. J., 2010, 44, 126.
[8] Gao, Y. Q.; Song, J.; Shang, S. B.; Wang, D.; Li, J. BioResources, 2012, 7, 4150.
[9] Gao, Y. Q.; Shang, S. B.; Wang, D.; Cui, Y. J. Lett. Drug Des. Discov., 2013, 10, 613.
[10] Mentzel, M.; Reiche, D. B.; Bruckner, R.; David, S.; van Steen, B. J.; Schon, U.; Jasserand, D.; Preuschoff,
U. 2007, US 7241770 B2 2007-07-10.
[11] Hida, T.; Mitsumori, S.; Honma, T.; Hiramatsu, Y.; Hashizume, H.; Okada, T.; Kakinuma, M.; Kawata, K.;
Oda, K.; Hasegawa, A.; Masui, T.; Nogusa, H. Org. Process Res. Dev., 2009, 13, 1413.
[12] Wei, B. S.; Gu, W.; Xu, X.; Yang, Y. Q.; Wang, S. F. Chin. J. Org. Chem., 2013, 33, 2196.
[13] Dhar, P.; Chan, P.; Cohen, D. T.; Khawam, F.; Gibbons, S.; Snyder-Leiby, T.; Dickstein, E.; Rai, P. K.; Watal,
G. J. Agr. Food Chem., 2014, 62, 3548.
[14] Vyas, D. H.; Tala, S. D.; Akbari, J. D.; Dhaduk, M. F.; Joshi, K. A.; Joshi, H. Indian J. Chem. B, 2009, 48,
833.
[15] Ibrahim, E. S.; Elgemeie, G. E. H.; Abbasic, M. M.; Abbasd, Y. A.; Elbadawic, M. A.; Attiad, A. M. E.
Nucleos. Nucleot. Nucl., 1995, 14, 1415.
[16] Abdellatif, K. R. A.; Abdelall, E. K. A.; Fadaly, W. A. A.; Kamel G. M. Med. Chem. Res., 2015, 24, 2632.
[17] Stolle, A. Eur. J. Org. Chem., 2013, 12, 2265.
[18] Liu, B.; Ao, W. W.; Yang, Y.; Wang, S. F. Nanjing Linye Daxue Xuebao, Ziran Kexueban, 2010, 34, 89.
[19] Szuppa, T.; Stolle, A.; Ondruschka, B.; Hopfe, W. ChemSusChem 2010, 3, 1181.

[20] Mungra, D. C.; Patel, M. P.; Patel, R. G. Arkivoc, 2009, 14, 64.
[21] Radi, M.; Vallerini, G. P.; Petrelli, A.; Vincetti, P.; Costantino, G. Tetrahedron Lett., 2013, 54, 6905.
[22] Kalaria, P. N.; Satasia, S. P.; Avalani, J. R.; Raval, D. K. Eur. J. Med. Chem., 2014, 83, 655.
[23] Tang, J.; Wang, L. M.; Yao, YF.; Zhang, L.; Wang, W. B. Tetrahedron Lett., 2011, 52, 509.
[24] Jeschke, P. Pest Manag Sci., 2009, 66, 10.
[25] Sridharan, V.; Suryavanshi, P. A.; Menéndez, J. C. Chem. Rev., 2011, 111, 7157.

Graphical Abstract
One-pot synthesis and antimicrobial evaluation of novel 3-cyanopyridine derivatives of
(-)-β-pinene
A series of novel 3-cyanopyridine compounds were synthesized from the natural resource
β-pinene, and their antimicrobial activities were evaluated.