Transformation of 1,2,5,6-tetrahydropyridines with mycellar fungi

Article abstract of DOI:10.1023/A:1026142220384

It has been shown that on biotransformation of a series of 1,2,5,6-tetrahydropyridines with strains of the mycellar fungi Cunninghamella verticillata VKPM F-430, Beauveria bassiana ATCC 7159, and Penicillium simplicissimum KM-16, the culture of Cunninghamella verticillata possesses the greatest transforming activity and selectivity. With the aid of the latter practically quantitative oxidation of 1,2,5,6-tetrahydropyridines occurs into the corresponding trans-diol. The structure and spatial disposition of trans-1-benzyl-3,4-dihydroxypiperidine was demonstrated by data of chromato-mass spectrometric analysis and high resolution NMR spectra and was confirmed by comparison with an authentic sample obtained by an alternate synthesis using the oxidation of 1-benzyl-1,2,5,6-tetrahydropyridine with trifluoroperacetic acid.

Full text of DOI:10.1023/A:1026142220384

Chemistry of Heterocyclic Compounds, Vol. 39, No. 7, 2003
TRANSFORMATION OF 1,2,5,6-TETRA-
HYDROPYRIDINES WITH MYCELLAR FUNGI
P. B. Terentiev, T. M. Zilberstein, A. A. Borisenko, V. A. Shmorgunov, N. F. Piskunkova,
and G. V. Grishina
It has been shown that on biotransformation of a series of 1,2,5,6-tetrahydropyridines with strains of
the mycellar fungi Cunninghamella verticillata VKPM F-430, Beauveria bassiana ATCC 7159, and
Penicillium simplicissimum KM-16, the culture of Cunninghamella verticillata possesses the greatest
transforming activity and selectivity. With the aid of the latter practically quantitative oxidation of
1,2,5,6-tetrahydropyridines occurs into the corresponding trans-diol. The structure and spatial
disposition of trans-1-benzyl-3,4-dihydroxypiperidine was demonstrated by data of chromato-mass
spectrometric analysis and high resolution NMR spectra and was confirmed by comparison with an
authentic sample obtained by an alternate synthesis using the oxidation of 1-benzyl-1,2,5,6-
tetrahydropyridine with trifluoroperacetic acid.
Keywords: piperidinediol, 1,2,5,6-tetrahydropyridine, mycellar fungi, biotransformation,
hydroxylation.
The functional derivatives of piperidine belong to a class of natural and synthetic substances with an
extremely wide spectrum of biological activity [1]. Recently polyhydroxylated derivatives of piperidine
(azasugars) have attracted particular attention, such as castanospermine 1 and its analogs, in which high anti-
HIV activity has been found [2,3].
OH
OH
OH
OH
H
HO
OH
OH
OH
OH
HO
HO
OH
N
N
H
N
R
1
R = Me, Et, Bu
Data are presented in the present work on an investigation of fermentation routes for the synthesis and
the stereochemistry of the structurally and stereochemically simpler analogs of castanospermine, the trans-3,4-
dihydroxypiperidines, by the biotransformation of a series of 1,2,5,6-tetrahydropyridines (THP) with growing
cultures of cells of strains of the mycellar fungi Cunninghamella verticillata VKPM F-430 (C), Beauveria
bassiana ATCC 7159 (B), and Penicillium simplicissimum KM-16 (P). As substrates 1-benzyl-1,2,5,6- (2a),
1-(1-phenylethyl)-1,2,5,6- (2b), and 3-methyl-1-propyl-1,2,5,6-THP (2c) were selected since fungi of the
__________________________________________________________________________________________
Moscow M. V. Lomonosov State University, Moscow 119899, Russia; e-mail:
grishina@org.chem.msu.su. Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 7, pp. 1027-1032,
July, 2003. Original article submitted January 12, 2001.
0009-3122/03/3907-0885$25.00©2003 Plenum Publishing Corporation
885

Beauveria, Cunninghamella, and Penicillium, etc. genus are capable of hydroxylating [4,5] or hydrating [6] the
multiple bonds of THP.
Chromato-mass spectral analysis of extracts obtained after incubation with cultures of each of the fungi
C, B, and P enabled it to be established that all the N-substituted THP 2a-c underwent different conversions
(Scheme 1).
Scheme 1
R2
N
R1
2a–c
R2
HO
O
OH
R2
R2
R2
N
OH
6c
N
N
N
R1
R1
R1
R1
5a–c
3a–c
4a,c
R2
CHO
NH
R1
7c
a R1 = Bn, R2 = H, b R1 = Ph(CH)Me, R2 = H, c R1 = Pr, R2 = Me
As is seen from the data given in Table 1, maximum transforming activity and selectivity were observed
for strain C, which practically completely transformed the three THP 2a-c. Both compounds 2a and 2b,
containing a N-aralkyl substituent, were transformed by a culture of fungus C practically completely and
regioselectively into the trans-3,4-dihydroxy derivatives 5a and 5b.
TABLE 1. Composition in the Mixture of the Products of Biotransformation
of Compounds 2a-c by Cultures of Fungi P, B, and C (retention time, min)
Compound, %
Fungus
3a
3b
3c
4a
4c
5a
5b
5c
1.0
(7.34)
4.0
7.33
19.0
6c
Penicillium (P)
Beauveria (B)
—
—
1.0
(3.41)
7.0
—
—
—
—
—
2.4
2.0
0.6
—
—
—
—
(5.88) (5.88) (3.41) (7.11)
Cunninghamella
—
—
—
—
22.0
97.6
100
59.0
(C)
(4.50) (8.34) (8.46) (7.34) (4.90)
886

The selectivity was infringed only for compound 2c where the formation of three compounds was
observed. These were 3,4-epoxy-3-methyl-1-propylpiperidine (4c), the product of its hydration (compound 5c),
and 2-methyl-5-propylamino-2-pentenal (7c) representing (in the gas phase) the linear tautomeric form of
2-hydroxy-3-methyl-1-propyl-1,2,5,6-THP (6c).
The least active proved to be culture P, which was completely ineffective on compounds 2a and 2b, and
compound 2c was converted to only 1% into trans-3,4-dihydroxy-3-methyl-1-propylpiperidine (5c) and to 1%
into 3-methyl-1-propylpiperidine (3c). These same compounds were found among the transformation products
of substrate 1c by a culture of fungus B, but in the ratio 2c : 5c : 3c of 89 : 4 : 7. Assumption of the trans
structure for the dihydroxy derivative 5c was made in view of the very high stability of its molecular ion [7].
Culture B also effected an analogous reduction of the multiple bond to an insignificant extent in the
transformation of compounds 2a and 2b. The presence of 2% 1-(1-phenylethyl)piperidine (3b) was identified
among the biotransformation products of 2b by culture B. In the case of compound 2a 1-benzylpiperidine (3a)
and also 1-benzyl-3,4-epoxypiperidine (4a) were detected in the culture fluid in addition to the initial substrate.
The detection of that epoxide enables the assumption that the dihydroxy derivative 5c was formed by the
hydration of the analogous epoxide 1,2,5,6-THP 4c.
We emphasize that in structure 2c only aliphatic groups are present and the carbonyl group is absent,
which puts in question the hypothesis that for the successful selective transformation by culture B of
unsaturated heterocycles a carbonyl group and/or an aromatic fragment needs to be present [8-10]. At the same
time the complete indifference of culture B in attempts to transform 1-benzyloxycarbonyl-1,2,5,6-THP was also
established in [6].
Cultures of fungi P and B are capable of effecting the nonselective transformation of such substrates
only to an insignificant extent.
It was found that a culture of Cunninghamella verticillata VKPM F-430 (C) dihydroxylates the multiple
bond in 1,2,5,6-THP 2a-b regioselectively and in practically quantitative yield. This particular culture of fungus
C should therefore be used for the preparative biotransformation of THP.
For the strict determination of the spatial structure of diol 5a we effected an alternate synthesis of trans-
1-benzyl-3,4-dihydroxypiperidine (8a) by the oxidation of compound 2a fluoroborate with trifluoroperacetic
acid. Samples of trans-1-benzyl-3,4-dihydroxypiperidine, obtained by biotransformation and synthetically,
showed the same chromatographic mobility and practically identical mass and 1H NMR spectra. Comparison of
the chemical shifts and their assignment for both samples of the compound are given in Table 2.
The axial orientation of the protons at 3- and 4-H in 3,4-dihydroxypiperidine 5a is established from the
3
3
3
large values of the vicinal coupling constants J2a3 = 10.3, J5a4 = 11.1, and J34 = 8.9 Hz. This determines the
diequatorial orientation of the 3,4-dihydroxy groups which indicates the preparation of just the trans isomer of
synthetic 3,4-dihydroxypiperidine 5a. An analogous conclusion on the trans structure was also made for the
3
biotransformation product 5a, having practically the same values for the coupling constants J2a3 = 10.2,
3J5a4 = 9.6, 3J34 = 9.8 Hz, which demonstrates the identity of both samples.
TABLE 2. 1H NMR Spectra of Samples of Compound 5a
Chemical shifts, δ, ppm
Compound 5а
2-Н
3-Н
3.52
3.56
4-Н
3.32
3.40
5-Н
6-Н
PhCH
3.48
OH
4.4
3.2
Ar–H
7.3
Biotransformation
product
a 1.92
e 2.93
a 1.98
a 1.55 a 2.02
e 1.83 e 2.73
a 1.59 а 2.08 a 3.51
Synthetic sample
7.3
e 2.94
e 1.90 е 2.75 b 3.53
887

It has therefore been shown that N-substituted 1,2,5,6-THP may be converted regioselectively and
stereoselectively into trans-3,4-diols by biotransformation.
EXPERIMENTAL
The chromato-mass spectra were taken on a HP-5990 instrument with a HP-5972 mass selective
detector, a quartz capillary column 30 m x 0.2 mm with stationary phase HP-5MS and temperature programing
from 70 to 250°C at a rate of 30°/min.
1
The H NMR spectra were recorded on a Varian VXR-400 (400 MHz) instrument in CDCl3, internal
standard was TMS.
1,2,5,6-Tetrahydropyridines 2a,c were synthesized by the sequential quaternization of pyridine and
3-picoline with benzyl chloride and propyl bromide in absolute acetonitrile respectively. The isolated salts, after
recrystallization from ethyl acetate–ether were reduced with sodium borohydride in absolute ethanol at 0°C
[11,12]. The ethanol was evaporated in vacuum, water was added to the residue, and the mixture twice extracted
with ether. The combined extracts were dried with anhydrous MgSO4, filtered, the solvent evaporated, and the
residue distilled in vacuum.
Compound 2a. Yield 60%; bp 101-102°C (2 mm Hg), Rf 0.85 (Silufol, benzene–acetone, 2:1).
IR spectrum (thin film), ν, cm-1: 1670 (C=C). Mass spectrum, m/z (Irel, %) [origin]: 173 (57) [M]+, 96 (60)
[M-C6H5]+, 91 (100) (C7H7]+, 84 (17), 82 (21) [M-C7H7]+, 70 (16), 65 (14), 58 (14), 44 (28), 42 (23), 41 (19)
[13].
Compound 2c. Yield 75%; bp 85-88°C (30 mm Hg), Rf 0.3 (Silufol, benzene–acetone, 2:1).
IR spectrum (thin film), ν, cm-1: 1667 (C=C). Mass spectrum, m/z: 139 (11) [M]+, 124 (5) [M-CH3]+, 110 (100)
[M-C2H5]+, 94 (5), 81 (4), 68 (5), 67 (7), 42 (20), 41 (8).
1-(1-Phenylethyl)-1,2,5,6-tetrahydropyridine (2b). Sodium borohydride (0.9 g, 2.4 mmol) was added
at 0°C in portions to a solution of 1-(1-phenylethyl)pyridinium chloride (2.6 g, 1,18 mmol) (obtained in
70% yield by boiling in butanol equivalent amounts of 1-(2,4-dinitrophenyl)pyridinium chloride (Zincke salt)
with 1-phenylethylamine) in absolute ethanol (30 ml). The mixture was stirred for 30 min, and the ethanol was
evaporated in vacuum. Water (20 ml) was added to the residue, the mixture was extracted with methylene
chloride (3 × 15 ml), the combined organic extracts were dried with anhydrous MgSO4, the solvent evaporated,
and the residue distilled in vacuum. Compound 2b (1.5 g, 65%) was obtained; bp 95-96°C (2 mm Hg).
Hydrochloride has mp 223-224°C. Found, %: C 69.96; H 8.04; N 5.93. C13H18ClN. Calculated, %: C 69.79;
H 8.10; N 6.26. Mass spectrum, m/z: 187 (14) [M]+, 172 (100) [M-CH3]+, 118 (16), 110 (24) [M-C6H5]+, 106 (8),
105 (85) [C8H9]+, 104 (13), 91 (32), 82 (34) [M-C8H9]+, 79 (26).
1-Benzyl-3,4-dihydroxypiperidine (5a). Boron trifluoride etherate (2.98 ml, 3.28 g, 23.1 mmol) was
added to a solution of 1-benzyl-1,2,5,6-THP 2a (4 g, 23.1 mmol) in absolute CH2Cl2 (12 ml) at -50°C. The
mixture was stirred for 5 min at –50°C and 20 min at room temperature. Triethylammonium trifluoroacetate
(0.9 g, 4.4 mmol) was added, and then a 1.7 M solution of trifluoroperacetic acid in methylene chloride (20 ml).
The mixture was boiled for 1 h 30 min. The excess of oxidizing agent was decomposed with dimethyl sulfide
(0.86 g, 13.9 mmol), and the solution neutralized at 0°C with 10% NaOH to pH 11. The organic layer was
separated, and the aqueous layer was extracted with methylene chloride (5 × 15 ml). The organic extracts were
combined, and dried with anhydrous MgSO4. The mixture was filtered, the solvent evaporated, and 1-benzyl-
3,4-dihydroxypiperidine (2.38 g, 50%) was obtained as white crystals; mp 96-97°C (ethyl acetate–hexane, 1:1),
1
Rf 0.6 (Silufol, CHCl3–MeOH, 3:1). IR spectrum (CCl4), ν, cm-1: 3605, 3630 (OH). H NMR spectrum
(400 MHz, CDCl3), δ, ppm (J, Hz): 1.55 (1H, dq, J5a6a = 11.6, J5a5e = 11.9, J4a5a = 11.1, J5a6e = 3.4, 5-H a); 1.83
888

(1H, dq, J4a5e = 3.9, 5-He); 1.92 (1H, t, J2a2e = 10.6, J2a3a = 10.3, 2-Ha); 2.02 (1H, dt, J6a6e = 11.4, J5e6a = 1.9,
6-Ha); 2.73 (1H, d, 6-He); 2.93 (1H, dd, J2e3a = 2.9, 2-He); 3.32 (1H, dt, J3a4a = 8.9, 4-Ha); 3.48 (2H, s, Ph-CH2);
3.52 (1H, td, 3-Ha); 4.4 (2H, br. s, OH); 7.3 (5H, m, Ar-H). Found, %: C 69.72; H 8.24; N 6.42. C12H17NO2.
Calculated, %: C 69.54; H 8.27; N 6.76.
Biotransformation was effected in a growing cell culture of strains P, B, and C at pH 5.0 by the
procedure of [6,8]. Two parallel experiments were carried out for each substrate and culture. The substrate
(compounds 2a-c) for transformation was introduced as the hydrochloride at 100 mg/liter, the volume of culture
fluid for one test was 100 ml. After the end of the process the cells were filtered off, the filtrate was acidified to
pH 3 with hydrochloric acid, evaporated to a volume of 7-10 ml, and extracted with chloroform (3 × 10 ml) to
remove contaminants of a nonbasic nature. The aqueous layer was made alkaline with 10% NaOH solution to
pH 11, and extracted with chloroform (6 × 10 ml). The combined chloroform extracts were dried with
anhydrous MgSO4, evaporated to 2 ml, and analyzed by chromato-mass spectrometry.
Mass spectra of the compounds identified (Scheme 1):
3a. 175 (26) [M]+, 174 (41) [M-H]+, 146 (5), 132 (4), 98 (27) [M-C6H5]+, 91 (100) [C7H7]+, 84 (18),
65 (22), 43 (15), 41 (14).
3b. 189 (7) [M]+, 174 (100) [M-CH3]+, 112 (6), 105 (100) [C8H9]+, 91 (4), 84 (3), 77 (3).
3c. 141 (7) [M]+, 140 (4), 112 (100) [M-C2H5]+, 84 (3) [M-C4H9]+, 70 (3), 43 (4), 42 (5).
4a. 189 (5) [M]+, 146 (3) [M-C2H3O]+, 138 (8), 118 (7) [M-C4H7O]+, 112 (6) [M-C6H5]+, 91 (100)
[C7H7]+, 89 (10), 77 (12), 65 (27), 63 (15), 55 (26), 51 (24), 41 (32).
4c. 155 (12) [M]+, 126 (95) [M-C2H5]+, 116 (34), 115 (39), 98 (100) [M-C3H5O]+, 96 (23), 84 (43)
[M-C4H7O]+, 83 (31), 69 (23), 44 (32), 43 (56), 42 (48), 41 (53).
5a. 207 (22) [M]+, 206 (14), 190 (15) [M-OH]+, 172 (5) [M-OH-H2O]+, 154 (27), 146 (7) [M-C2H5O2]+,
134 (7), 130 (14) [M-C6H5]+, 116 (21) [M-C7H7]+, 98 (8), 91 (100) [C7H7]+, 70 (27), 65 (10), 43 (7), 41 (10).
5b. 221 (3) [M]+, 206 (100) [M-CH3]+, 204 (1) [M-OH]+, 144 (20) [M-C6H5]+, 116 (5) [M-C8H9]+,
106 (6), 105 (51) [C8H9]+, 103 (9), 91 (27), 79 (11), 77 (14) [C6H5]+.
5c. 173 (95) [M]+, 155 (18) [M-H2O]+, 143 (8), 142 (11), 138 (51) [M-H2O-OH]+, 129 (12)
[M-CH3COH]+, 128 (10), 125 (21), 115 (26), 112 (24), 111 (47), 99 (98) [M-C3H6O2]+, 97 (30), 83 (48),
55 (100) [C3H5N]+, 43 (58).
6c. 155 (3) [M]+, 126 (100) [M-C2H5]+, 116 (4), 115 (6), 112 (3), 98 (2), 84 (11) [M-C4H9N]+, 70 (4),
55 (6), 43 (21), 42 (10), 41 (8).
The work was carried out with the financial support of the Russian Fund for Fundamental Investigations
(RFFI), Grant No. 98-03-33026a.
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