Microbial hydroxylation of hydroxyprogesterones and α-glucosidase inhibition activity of their metabolites

Article abstract of DOI:10.1515/znb-2007-0419

Microbial transformation of 11α-hydroxyprogesterone (1) with Cunninghamella elegans, Gibberella fujikuroi, Fusarium lini, and Candida albicans yielded 11α,15α,16α-trihydroxypregn-4-ene-3,20-dione (3), 11α-hydroxy-5α-pregnane-3,20-dione (4), 6β,11α- dihydroxypregn-4-ene-3,20-dione(5), 11α-hydroxypregna-1,4-diene-3,20-dione (6), 11α,17β-dihydroxyandrost-4-en-3-one (7), and 11α,15α-dihydroxypregn-4-ene-3,20-dione (8). On the other hand, microbial transformation of 17α-hydroxyprogesterone (2) with Cunninghamella elegans and Fusarium Uni yielded 11α,17α- dihydroxypregn-4-ene-3,20-dione (9), and 17α-hydroxypregna-1,4-diene-3,20- dione (10). The structures of the metabolites 3-10 were deduced on the basis of spectroscopic methods. Compound 3 was identified as a new metabolite, which exhibited a promising inhibitory activity against the α-glucosidase enzyme.

Full text of DOI:10.1515/znb-2007-0419

Synthesis, Spectral and Thermal Properties, and Crystal Structure of
Bis(ethylenediamine)(aqua)copper(II) (Bis)syringate Ethylenediamine
Dihydrate [Cu(en)2(H2O)](sy)2(en)(H2O)2
Zerrin Herena, Hu¨meyra Pas¸aog˘lub, Go¨khan Kas¸tas¸b, Leyla Vurucua, and
Orhan Bu¨yu¨kgu¨ngo¨rb
a Ondokuz Mayis University, Faculty of Arts and Sciences, Department of Chemistry,
TR-55139, Kurupelit, Samsun, Turkey b Ondokuz Mayis University, Faculty of Arts and Sciences,
Department of Physics,
TR-55139, Kurupelit, Samsun, Turkey
Reprint requests to Dr. H. Pas¸aog˘lu. E-mail: hpasa@omu.edu.tr
Z. Naturforsch. 61b, 287 – 291 (2006); received October 5, 2005
The complex [Cu(en)2(H2O)](sy)2(en)(H2O)2 has been synthesized and characterized by its elec-
tronic and vibrational spectra. The molecular structure of the complex has been determined by X-ray
diffraction methods. The complex crystallizes in the orthorhombic space group Pnma with unit-cell
˚
parameters a = 10.7236(5), b = 20.4660(10), c = 14.4523(11)A and Z = 4. In the cation, the Cu(II)
ion has a distorted square pyramidal coordination with two bidendate (en) ligands forming the basal
plane and a H2O molecule in the apical position. The complex cations and syringate anions consti-
tute chains along the b axis in –A–B–A– fashion. The members of the chains are linked by through
N–H···O hydrogen bonds. The (en) molecules are responsible for connecting adjacent layers.
Key words: Syringic Acid, Ethylenediamine, Copper (II) Complex, Thermal Properties
Introduction
structure. In this study, we report the preparation and
structural characterization of the new square pyrami-
dal Cu(II) complex [Cu(en)2(H2O)](sy)2(en)(H2O)2.
Major types of natural antioxidants are the pheno-
lic acids which are derivatives of benzoic and cinamic
acids [1]. There is a definite relationship between the
structures of the phenolic acids and their antioxidant
activities. The inhibitory effectiveness of monophe-
nols is substantially increased by one or two methoxy
groups. Syringic acid, 4-hydroxy-3,5-dimethoxybenz-
oic acid [syH], is one of the most active antioxidants
with two methoxy roups [2, 3]. Furthermore, syringic
acid has been identified as an active ingredient which
contributes to the prevention of cancer, inhibiting tu-
mour initiation, promotion and progression and heart
disease [3, 4]. The salts of 4-hydroxy-3,5-dimethoxy-
benzoic acid with various cations and mixed ligand
complexes of d-block elements have not been studied.
Ethylenediamine is an N-donor chelator and has been
studied intensively, both structurally and spectroscopi-
cally for many years [5, 6]. Therefore, we selected sy-
ringic acid as primary ligand and prepared Cu(II) eth-
ylenediamine complexes and examined their thermal
stability in air, IR spectral characteristics and crystal
Results and Discussion
Spectral and magnetic properties
The coordination sphere of the complex can be
treated in terms of C2v molecular symmetry. In the IR
spectrum, the region of the NH2 stretching vibrations
is relatively broaden by partial superposition with the
O-H vibrations of the coordinated and uncoordinated
water molecules. The bands at 3370 and 3050 cm−1
are due to the symmetric and asymmetric stretching
vibrations of the NH2 groups of the coordinated and
uncoordinated en ligands. The vibrational frequencies
the CH2 groups of the en ligands appear at 800 cm−1
This is in agreement with literature data [7, 8]. The
.
Cu–N stretching frequency was observed at 750 cm−1
In the IR spectrum of free syringic acid there is a strong
absorption band at 1700 cm−1 for the COOH group,
which is absent for the complex, and the bands due
to the asymmetric and symmetric stretching of car-
boxylate groups occur at 1575 and 1373 cm−1, respec-
tively [9]. The bands of the C-H asymmetric stretching
.
Syringic acid = 4-hydroxy-3,5-dimethoxybenzoic acid.
c
0932–0776 / 06 / 0300–0287 $ 06.00 ꢀ 2006 Verlag der Zeitschrift fu¨r Naturforschung, Tu¨bingen · http://znaturforsch.com
- 10.1515/znb-2006-0308
Downloaded from De Gruyter Online at 09/12/2016 10:40:13AM
via free access

288
Z. Heren et al. · Bis(ethylenediamine)(aqua)copper(II)
◦
˚
Table 1. Crystal data and structure refinement parameters for Table 2. Selected bond lengths (A) and bond angles ( ) for
[Cu(en)2(H2O)](sy)2(en)(H2O)2.
[Cu (en)2(H2O)](sy)2(en)(H2O)2.
˚
Bond lengths (A)
Cu1–N1
Cu1–N2
O5–C11
Empirical formula
Formula weight
Temperature [K]
Wavelength [A]
Crystal system
Space group
Unit cell dimensions [A]
a,
b,
C24H48CuN6O13
692.22
296
0.71073 Mo-Kα
orthorhombic
Pnma
2.014 (3)
2.012 (3)
1.266 (4)
Cu1–O1
O6–C11
2.285 (3)
1.255 (4)
˚
Bond angles (◦)
N2–Cu1–O1
N2–Cu1–N1
Symmetrie Code i x, 1/2−y,z.
92.00 (11)
84.10 (12)
N1–Cu1–O1
N2i–Cu1-N1
95.35 (12)
171.55 (15)
˚
10.7236 (5),
20.466 (1),
14.452 (1)
3171.8 (3)
4
Table 3. Hydrogen bonds (A, ◦) for [Cu(en)2(H2O)](sy)2-
(en)(H2O)2.
˚
c
3
˚
V [A ]
Z
D–HA···A
D–H
H···A
D···A
D–HA
Absorption coefficient [mm−1
]
0.760
1.450
N1–H1···O6i
N2–H8···O2ii
O7–H23···O6iii
N4–H10···O2iv
N4–H10···O4iv
N3–H11···O2v
O1–H9···O5
O8–H24···O5
O2–H22···O3
O2–H22···N3v
0.93 (3)
0.89 (3)
0.83 (3)
2.06 (4)
2.11 (4)
1.84 (3)
2.960 (4) 165 (3)
2.996 (4) 174 (3)
2.663 (3) 167 (3)
Dcalcd. [Mg m−3
Crystal size [mm3]
Theta range for
]
0.310×0.180×0.060
1.41 – 27.14
0.914 (17) 1.860 (19) 2.740 (3) 161 (3)
0.914 (17) 2.40 (3)
0.865 (17) 1.90 (2)
0.92 (4)
0.98 (5)
0.820
data collection [deg]
Measured reflections
Independent reflections
Absorption correction
Refinement method
3.007 (2) 124 (3)
2.730 (4) 160 (3)
2.762 (3) 175 (4)
2.853 (3) 141 (5)
2.659 (3) 114.89
2.730 (4) 153.30
38715
3575
Integration
Full-matrix least-squares on F2
R1 = 0.043, wR2 = 0.078
R1 = 0.112, wR2 = 0.092
0.810
1.85 (4)
2.02 (5)
2.207
Final R indices [F2 > 2σ(F2)]
R Indices (all data)
0.820
1.973
Symmetry codes: i x, 1/2 − y, z; ii 3/2 − x, y − 1/2, 1/2 + z; iii x −
Goodness-of-fit on F2
Largest differ−en3ce peak
1/2, y, 1/2−z; iv 3/2−x, 1−y, z−1/2; v 1−x, 1−y, 1−z.
0.420; −0.573
˚
and hole (e A
)
netic moment value of the complex is 1.22 BM corre-
sponding to one unpaired electron.
Thermal properties
The crystals contain water of crystallization and
copper-coordinated water. The thermal dehydration
◦
occurred in two steps with endothermic effects at 40 C
◦
and 132 C (Fig. 1). One mole of coordinated water
and two moles of crystallization water are removed
at these stages (exp. 8.89%; calcd. 7.81%). A color
change from black to brown was noted on removal
of the water molecules. Further heating causes evap-
oration of one of the ethylenediamine molecules, re-
◦
sulting in an endothermic DTA peak at 154 C (exp.
12.89%; calcd. 12.18%). The product is very unsta-
ble and undergoes further decomposition by release of
the remaining ethylenediamine and decomposition ◦of
the syringiato ligands. The exothermic peak at 481 C
can be explained by the burning of the organic residue
formed in the previous stages. The final solid product
of thermal decomposition was identified as CuO (exp.
86.85%; calcd. 88.50%).
Fig. 1. TG and DTA curves of the [Cu(en)2(H2O)](sy)2(en)
(H2O)2 complex.
of the CH3 groups appear at similar frequencies as in
free syringic acid (2850 cm−1).
The electronic d-d transition spectrum of Cu(en)2
(H2O)](sy)2(en)(H2O)2 is compatible with the dis-
torted square pyramidal configuration. The maximum
absorption is at 567 with ε = 61.0 Lmol−1cm−1
.
Crystal structure
This band is assigned to the a1 → b1 d-d transition
[10– 14]. The band observed around 299 nm (ε =
The crystallographic analysis confirmed that the
1362 Lmol−1cm−1) is attributed to MLCT. The mag- complex consists of discrete [Cu(en)2H2O]2+ cations,
- 10.1515/znb-2006-0308
Downloaded from De Gruyter Online at 09/12/2016 10:40:13AM
via free access

Z. Heren et al. · Bis(ethylenediamine)(aqua)copper(II)
289
Fig. 2. Perspective view and atom label-
ing scheme of [Cu(en)2(H2O)](sy)2(en)
(H2O)2. Atomic displacement ellipsoids
are drawn at the 50% probability level.
Symmetry transformation used to gener-
ate equivalent atoms (i) x, −y+1/2, z.
One of the interesting features of the structure is
that the en molecules exhibit chemically different func-
tions. While in one form they coordinates to metal
cation, in the other form they are molecules of sol-
vation (Fig. 3). In the latter, they provide bifurcated
(three-centered) hydrogen bonds (Table 3). The sy-
ringate anions [sy]− do not act as ligands, but are incor-
porated into the crystal structure by hydrogen bonds.
The bond distances in the carboxylate group of de-
protonated [sy]− are nearly equal (Table 2). This is
responsible for the shifting and splitting of the vibra-
tional bands of the carboxylate group.
˚
The Cu–N distances are 2.014(3) and 2.012(3) A,
similar to those found in related structures [15– 17].
The trans angles N–Cu–N at the Cu(II) ion devi-
ate from linearity with a value of 171.55(15)◦. The
˚
Cu1–O1 distance is 2.285(3) A, and thus in agree-
ment with the reported values for square pyramidal
structures [18 – 20]. It is found that the apical Cu–O
bond is larger than the basal Cu–N bonds. This is be-
cause the short Cu–N bonds prevent the water mole-
cules from approaching the copper atom, and keep it
Fig. 3. A view of the complex, [Cu(en)2(H2O)](sy)2(en)
(H2O)2, normal to (100), showing different types of hydro-
gen bonding interactions responsible for the layered struc-
ture. Refer to Table 3 for symmetry codes.
˚
at a distance of 2.285 (3) A. The (en) chelate ring
(Cu1 N2 N1 C2 C1) is close to planar, with an r. m. s
˚
deviation of 0.1918 A. The Cu(II) cation departs from
two deprotonated syringate anions, one neutral (en)
molecule and two water molecules of solvation. The
Cu(II) ion has a distorted square-pyramidal coordina-
tion. The basal coordination plane is formed by two
symmetry-related, chelating (en) groups. The coordi-
nation is completed by a water molecule located at the
pyramid apex (Fig. 2).
the mean plane defined by the four nitrogen atoms of
˚
the pyramidal basis by 0.0358(18) A towards the axial
water ligand.
The packing in the crystal is stabilized by strong and
moderate hydrogen bonding. These intermolecular in-
teractions are depicted in Fig. 3, and are included in
- 10.1515/znb-2006-0308
Downloaded from De Gruyter Online at 09/12/2016 10:40:13AM
via free access

290
Z. Heren et al. · Bis(ethylenediamine)(aqua)copper(II)
Synthesis of Cu (II) syringate
The complex was prepared by mixing an aqueous solu-
tion of Cu(II) acetate (1 mmol) with a methanolic solution
of syringic acid (2 mmol). The reaction mixture was stirred
◦
overnight at 50 C and the brown product was isolated by
filtration, washed with cold distilled water and diethyl ether
and dried in air.
Synthesis of the Cu(II) syringate ethylenediamine complex
A solution of ethylenediamine (2 mmol) in distilled wa-
ter (20 ml) was added dropwise with stirring to a solution of
Cu(II) syringate (1 mmol) in distilled water (40 ml). The so-
lution was heated and stirred for 5 h at 70 ◦C and then the
mixture was cooled to room temperature. The black crystals
were filtered and washed with 10 ml of cold distilled water
and acetone and dried in vacuo. UV/vis (H2O): −λ1maxε) =
Fig. 4. The unit-cell packing of the complex, [Cu(en)2(H2O)]
(sy)2(en)(H2O)2, normal to (100).
˜
567 nm (4.11). –IR (KBr): ν = 3370 and 3050 cm (NH2).
Table 3. As shown in Fig. 4, [Cu(en)2H2O]2+ complex
cations and [sy]− anion constitute a chain which is re-
peated along the b axis in a –A–B–A– fashion, with a
dihedral angle of 24.14 (10)◦ between the (en) chelate
ring (Cu1 N2 N1 C2 C1) and the (C3 C4 C5 C6 C7 C8)
plane. The neighbouring chains are linked by uncoor-
dinated en ligands through N3–H11···O2v (v: 1 − x,
1 − y, 1 − z) and N4–H10···O2iv (iv: 3/2 − x, 1 − y,
z − 1/2) hydrogen bonds (Fig. 3). This arrangement
leads to a layered structure. It can be seen from Fig. 4
that the uncoordinated en and water molecules form
bridges between the layers through inter-molecular hy-
drogen bonds. The aqua ligands are linking adjacent
–C24H48CuN6O13 (692.2): calcd. C 41.64, H 6.94, N 12.14;
found C 41.05, H 7.43, N 12.82.
X-ray structure determination
Diffraction experiments were carried out at 296 K on a
Stoe IPDS diffractometer. The structure was solved by di-
rect methods using the program SHELXS97 [21]. All non-
hydrogen atoms were refined anisotropically by full-matrix
least-squares methods [SHELXL-97]. Hydrogen atoms at-
tached to C9, C10 and O2 were placed in geometrically ide-
alized positions and refined as riding atoms. Therefore, the
standart uncertainty (s.u) values for O2–H22···O3 and O2–
H22···N3v hydrogen bondings is not included in Table 3. All
other H atoms were located in a difference map, and their co-
[sy]− anions through O1–H9···O5 hydrogen bonds, ordinates andUiso(H) values were refined freely. The relevant
crystal data and experimental conditions along with the final
parameters are summarised in Table 1. Data collection: X-
Area, cell refinement: X-Area, data reduction: X-RED [21];
program(s) used to refine structure: SHELXL97 [22]; molec-
ular graphics: ORTEP-3 for Windows [23]; software used to
prepare material for publication: WinGX [24].
and are effective in forming a layered structure.
Experimental Section
Materials and measurements
All chemicals used were analytical reagent products.
Copper(II)acetatetetrahydrate and ethylenediamine were ob-
tained from Merck. Syringic acid was purchased from
ACROS Organics. The elemental analysis for C, H and N
were carried out at TUBITAK Marmara Research Centre.
The magnetic susceptibility measurement at room tempera-
ture was performed using a Sherwood Scientific MXI model
Gouy magnetic balance. The UV/vis spectrum was obtained
for the aqueous solutions of the complexes with a Unicam
UV2 spectrometer in the range 900 – 190 nm. The IR spec-
trum was recorded in the 4000 – 400 cm−1 region with a
Mattson 1000 FT-IR spectrometer using KBr pellets. Ther-
Supplementary data
Crystallographic data for the structural analysis have
been deposited with the Cambridge Crystallographic Data
Centre, CCDC No. 271102. Copies of this information
may be obtained free of charge from the Director, CCDC,
12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-
1223-336033; e-mail: deposit@ccdc.cam.ac.uk or www:
http://www.ccdc.cam.ac.uk).
¨
˙
Acknowledgements
The authors wish to acknowledge the Faculty of Arts and
mal analysis curves (TG and DTA) were recorded simultane- Sciences, Ondokuz Mayis University, Turkey, for the use of
ously in a static air atmosphere with a Rigaku TG8110 ther- the Stoe IPDS-II diffractometer (purchased under grant No.
mal analyzer. The heating rate was 10 ◦C min−1
.
F279 of the University Research Fund).
- 10.1515/znb-2006-0308
Downloaded from De Gruyter Online at 09/12/2016 10:40:13AM
via free access

Z. Heren et al. · Bis(ethylenediamine)(aqua)copper(II)
291
[1] S. Z. Dzicdzic, B. J. F. Hudson, Food Chemistry 14, 45
(1984).
[2] M. E. Cuvelier, H. Richard, C. Berst, Bioscience
Biotechnology Biochemistry 56, 324 (1992).
[3] E. M. Marinova, N. N. Yarishlieva, Food Chemistry 81,
189 (2003).
[14] C. Y. Wu, C. C. Su, Polyhedron 163, 383 (1997).
[15] I. M. Procter, B. J. Hathway, P. J. Nicholls, J. Chem.
Soc. A, 1678 (1968).
[16] K. Canan, T. Y. Veysel, K. Y. Turan, Acta Crystallogr.
E60, m593 (2004).
[17] A.-Y. Fu, D.-Q. Wang, F.-M. Wang, Acta Crystallogr.
E60, m1527 (2004).
[18] R. M. K. Deng, K. B. Dillion, A. E. Goeta,
M. Mapolelo, Inorg. Chim. Acta. 315, 245 (2001).
[19] G. Jovanovski, P. Naumov, O. Grupcˇe, Eur. J. Solid
State Inorg. Chem. 35, 231 (1998).
[20] P. Naumov, G. Jovanovski, G. B. D. Michael, W. S. Ng,
Inorganica Chimica Acta 314, 154 (2001).
[21] Stoe & Cie, X-AREA and X-RED. Stoe& Cie, Darm-
stadt, Germany (2001).
[4] E. H. Siemann, L. L. Creasy, Am. J. Enol. Vitic. 43, 49
(1992).
[5] A. A. Bertelli, Int. J. Tiss. React. 17 (1995).
˙
[6] H.Ic¸budak, T. K. Yazıcılar, V. Y. Yılmaz, Thermochim.
Acta 93, 335 (1999).
[7] O. Z. Yes¸ilel, H. Ic¸budak, H. Olmez, P. Naumov, Synth.
¨
˙
React. Inorg. Metal-Org. Chem. 33, 77 (2003).
˙
[8] H. Ic¸budak, V. T. Yılmaz, Synth. React. Inorg. Met-
Org. Chem. 27, 1517 (1997).
[9] S. Oksuz, A. Ulubelen, A. Barla, W. Voelter, Turk. J.
Chem. 26, 457 (2002).
[10] B. J. Hathaway, A. A. G. Tomlison, Coor. Chem. Rev.
5, 1 (1970).
[11] B. J. Hathaway, in Comprehensive Coordination
Chemistry, Vol. 5, p. 594, Pergamon, Oxford (1987).
[12] C. C. Su, J. S. Lin, S. L. Lin, S. L. Wang, F. L. Liao, J.
Coord. Chem. 30, 91 (1993).
[22] G. M. Sheldrick, SHLEXS-97 and SHELXL-97. Pro-
gram for Refinement of Crystal Structures, University
of Go¨ttingen, Germany (1997).
[23] L. J. Farrugia, ORTEP-III. Program For Molecular
Drawing. J. Appl. Crystallogr. 30, 565 (1997).
[24] L. J. Farrugia, WinGX Suite for Single Crystal Small
Molecule Crystallography, J. Appl. Crystallogr. 32,
837 (1999).
[13] F. A. Cotton, Advanced Inorganic Chemistry, pp. 642,
Wiley-Interscience, New York (1980).
- 10.1515/znb-2006-0308
Downloaded from De Gruyter Online at 09/12/2016 10:40:13AM
via free access