Synthesis of novel spin-labeled derivatives of podophyllotoxin as potential antineoplastic agents

Article abstract of DOI:10.1080/00397910500287983

Five novel nitroxyl spin-labeled ester derivatives of podophyllotoxin have been prepared by reacting the corresponding N-(1-oxyl-2,2,6,6-tetramethyl-4- piperidinyloxy-carbonyl) amino acids with the hydroxy group of podophyllotoxin in the presence of dimethylaminopyridine and N,N-dicyclohexylcarbodiimide and evaluated as potential antitumor agents. All of the target compounds showed more significant cytotoxicity against P-388 murine leukemia and A-549 human lung carcinoma in vitro than etoposide. Copyright Taylor & Francis, Inc.

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Synthesis of Novel Spin‐Labeled
Derivatives of Podophyllotoxin
as Potential Antineoplastic
Agents
Ying‐Qian Liu a & Xuan Tian a
a State Key Laboratory of Applied Organic
Chemistry , Lanzhou University , Lanzhou, P.R. China
Published online: 22 Aug 2006.
To cite this article: Ying‐Qian Liu & Xuan Tian (2005) Synthesis of Novel Spin‐Labeled
Derivatives of Podophyllotoxin as Potential Antineoplastic Agents, Synthetic
Communications: An International Journal for Rapid Communication of Synthetic
Organic Chemistry, 35:21, 2749-2758
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Synthetic Communicationsw, 35: 2749–2758, 2005
Copyright # Taylor & Francis, Inc.
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910500287983
Synthesis of Novel Spin-Labeled Derivatives
of Podophyllotoxin as Potential
Antineoplastic Agents
Ying-Qian Liu and Xuan Tian
State Key Laboratory of Applied Organic Chemistry, Lanzhou
University, Lanzhou, P.R. China
Abstract: Five novel nitroxyl spin-labeledester derivatives ofpodophyllotoxinhave been
prepared by reacting the corresponding N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyloxy-
carbonyl) amino acids with the hydroxy group of podophyllotoxin in the presence of
dimethylaminopyridine and N,N-dicyclohexylcarbodiimide and evaluated as potential
antitumor agents. All of the target compounds showed more significant cytotoxicity
against P-388 murine leukemia and A-549 human lung carcinoma in vitro than etoposide.
Keywords: Antineoplastic properties, podophyllotoxin derivatives, spin-labeled
INTRODUCTION
Podophyllotoxin (1) and its many related derivatives are well known to have
pronounced antineoplastic and antiviral properties.[1] Its semisynthetic
analogues of etoposide (2) and teniposide (3) are currently used in chemothe-
rapy for various types of cancer, including small-cell lung cancer, acute
leukemia, lymphoma, testicular carcinoma, and Kaposi’s sarcoma.[2–4]
Interestingly, these semisynthetic derivatives and the parent compound,
podophyllotoxin, showed different mechanisms of action. Podophyllotoxin
inhibits tubulin polymerization through its interaction at the colchicines
binding site,[5] whereas etoposide and congeners involve the inhibition of
topoisomerase II–mediated DNA ligation by reversible cleavable complex
Received in Japan April 23, 2005
Address correspondence to Xuan Tian, State Key Laboratory of Applied Organic
Chemistry, Lanzhou University, Lanzhou, 730000, P.R. China. Tel.: þ 86-931-
8912410; Fax: þ 86-931-8912582; E-mail: liuyq02@st.lzu.edu.cn
2749

2750
Y.-Q. Liu and X. Tian
stabilization resulting in tumor-cell death and its corresponding ortho-quinone
via oxidative demethylation.[6] However, because of the typical adverse effects
common to most antineoplastics, such as anemia, hair loss, and severe gastro-
intestinal disturbances, the clinical application of them has been limited to a
certain extent. In this regard, it is necessary to continuously research and syn-
thesize podophyllotoxin and etoposide analogues that encompass a broad
spectrum of antineoplastic activities and overcome several clinical limitations.
Early efforts by clinicians and research scientists to reduce the side effects
of podophyllotoxin and maintain or enhance activity were directed along the
lines of ester synthesis,[7,8] especially because these derivatives showed
strong activity against P-388 lymphocytic leukemia inoculated into mice.
Although several of these esters showed significant activity, these derivatives
themselves have side effects because of general toxicity. In previous studies
we found that the introduction of a stable nitroxyl radical into the molecule
of podophyllotoxin could result in new compounds, which have significant
antitumor activity with marked decrease in toxicity when compared with the
parent compounds.[9–12] A series of spin-labeled derivatives of thio-
TEPA,[13] nitrosourea,[14] rubomycin,[15] and 6-mercaptopurine[16] were also
reported to have pharmacological properties superior to their parent compo-
unds. Recently, it has been found that the nitroxyl radicals can normalize the
level of the oxidized form of P-450 cytochrome, which has been brought
down by the injection of cytostatic agents of lethal doses, and the nitroxyl
moiety serves as a transport vehicle through cell membranes.[17] Therefore,
nitroxyl radicals can be considered as biological response modifiers.
Based onthis, aswellasthe factthat ‘L-aminoacidsare activelytransplanted
into mammalian tissue, have good water solubility, and are often used as carrier
vehicles for some drugs, we designed five novel spin-labeled derivatives with the
hope that C-4-OH group esterified by L-amino acids containing a nitroxyl radical
moiety would simultaneously circumvent the several limitations and develop
superior pharmacological profiles. Herein, we report the synthesis, biological
activity, and structures of five novel spin-labeled compounds.

Spin-Labeled Podophyllotoxin Derivatives
CHEMISTRY
2751
Podophyllotoxin (1) was isolated from a Chinese medicinal herb Podophyllum
emodi Wall var Chinesis Sprague and served as the starting material for the
preparations of all the derivatives.
4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (5) has been prepared
by means of catalytic oxidation of 4-hydroxy-2,2,6,6-tetramethylpiperidine
(4) with sodium tungstate-hydrogen peroxide–EDTA.[18] The reaction of
4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (5) with N,N0-carbonyldiimid-
azole procceds to give the intermediate N-(1-oxyl-2,2,6,6-tetramethylpiperidinyl-
oxycarbonyl)-imidazole (6) by the modified method that Staab described for
the synthesis of N-t-butoxycarbonylimidazole.[19] The intermediate (6),
without isolation, was further reacted with p-toluenesulfonic acid monohydrate
to give its higher reactive tosylate (7). This salt is so reactive that it is instan-
taneously converted into compound 8 when dissolved in an aqueous solution
of sodium azide. Compounds 9a–9e can be obtained in good yield by
reaction of the alkoxycarbonyl azide (8) with free amino acids.[20] Synthesis
of the desired compounds 10a–10e was accompanished by reaction of
compounds 9a–9e with podophyllotoxin in the presence of N,N-dicyclohexyl-
carbodiimide (DCC) and catalytic amount of dimethylaminopyridine (DMAP)
to form an ester linkage under an atmosphere of nitrogen. The rate of reduction
of the representative compound 10e in CDCl3 into the corresponding
N-hydroxy amine compound 11 using phenylhydrazine was monitored
visually by the disappearance of the characteristic red-orange color of the
nitroxyl derivatives (Scheme 1).
The synthetic methodology for the preparation of spin-labeled podophyl-
lotoxin compounds 10a–10e is depicted in Schemes 2 and 3.
BIOLOGICAL EVALUATION
Cytotoxicities of the target compounds 10a–10e against cell cultures of P-388
murine leukemia and A-549 human lung carcinoma were tested in vitro
Scheme 1. Reagents and conditions: i, phenylhydrazine/CDCl3, 25 8C, 10 min.

2752
Y.-Q. Liu and X. Tian
Scheme 2. Reagents and conditions: i, Na2WO4/H2O2/EDTA; ii, N,N0-carbonyldi-
midazole/THF, stir; iii, p-toluenesulfonic acid monohydrate; iv, NaN3/water, stir;
v, amino acids/MgO, stir, 24 h.
following the methods described by Bergeron.[21] The prototypical inhibitor
etoposide was included as a reference standard; the results of these assays
were used to obtain the corresponding inhibition rates shown in Table 1.
RESULTS AND DISCUSSION
The reaction of the hydroxy group at C-4 of podophyllotoxin with the various
N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl) amino acids gave
the corresponding podophyllotoxin ester derivatives in excellent yields.
Owing to the paramagnetic nature of the nitroxyl spin labels, however, it is
difficult to conveniently get the valuable structural information on these
1
molecules using H NMR spectroscopy. It occurred to us that essentially
the same structural information could be obtained from the 1H NMR
spectra of the corresponding N-hydroxy amines. In addition, because all
N-hydroxypiperidines are air- and moisture-sensitive molecules, these
compounds also are air-oxidizable back to the parent nitroxyl free radical,
especially in solution. Therefore, it is essential that all work with these
compounds in solution be performed as rapidly as possible. To assign the con-
figuration at C-4 position for compounds 10a–10e, we applied the in situ
reduction of the representive compound 10e in CDCl3 with a solution of phe-
nylhydrazine in CDCl3, which led smoothly to the corresponding N-hydroxy
amine 11,[22] in the 1H NMR spectrum of the corresponding N-hydroxy amine
11, The 4-H proton (of carbon forming the ester linkage) is found at dH 5.87
Scheme 3. Reagents and conditions: i, DCC/DMAP, stir, 2 h.

Spin-Labeled Podophyllotoxin Derivatives
Table 1. Cytotoxicity activity of 10a–10e in vitro against
P-388 murine leukemia and A-549 human lung carcinoma
2753
Inhibition rate (%)
Concentration
Compound
(mol/L)
P-388a
A-549b
10a
1024
1025
1026
1027
1028
96.3
91.0
89.1
87.8
77.5
87.3
87.9
82.8
80.1
75.5
10b
1024
1025
1026
1027
1028
99.9
89.2
88.3
81.1
70.8
94.7
86.1
83.5
81.6
50.6
10c
1024
1025
1026
1027
1028
94.9
88.8
86.3
73.8
43.0
89.5
83.5
83.3
73.2
45.9
10d
1024
1025
1026
1027
1028
95.8
94.3
73.9
14.0
0
87.0
83.6
72.8
39.0
10.7
10e
1024
1025
1026
1027
1028
93.5
92.6
80.7
68.6
52.3
87.9
87.0
81.9
79.9
60.0
Etoposide (2)
1024
1025
1026
1027
1028
99.2
70.6
25.5
6.0
97.5
50.7
45.0
0
1.8
0
aMTT methods, drug exposure was for 72 h.
bSRB methods, drug exposure was for 72 h.
(1H, d, J3,4 ¼ 9.2 Hz, 4-H). The data is in agreement with those previously
reported for C-4a-substituted-podophyllotoxin derivatives[23,24]. The assign-
ment of the configuration at the C-4 position for compound 11 was based
on its J3,4 coupling constants (J3,4 ¼ 9.2 Hz). The C-4a-substituted
compounds have a J3,4 ꢀ 8.5 Hz as H-3 is trans to H-4, whereas C-4b-

2754
Y.-Q. Liu and X. Tian
substituted compounds have a J3,4 , 4.5 Hz because of the cis relationship
between H-3 and H-4.[25] Moreover, lack of C-4b isomers may be
explained through the reaction mechanism of the help of DCC in the
presence of a catalytic amount of DMAP to form ester linkage reaction;[25]
a-stereoselectivity of C-4 is retained as a-bond between C-4, and the
hydroxyl group does not get involved in the chemical reaction. On the basis
of these facts, we elucidated the configuration at C-4 position in the series.
We could verify that coupling reaction between compounds 9a–9e and podo-
phyllotoxin are stereospecific in nature. Moreover, the high-resolution mass-
spectral results are full agreement with the calculated mass of these deriva-
tives. Cytotoxicities of the target compounds 10a–10e against cell cultures
of P-388 murine leukemia and A-549 human lung carcinoma were tested in
vitro. As illustrated in Table 1, compounds 10a–10e are more active than
etoposide; compound 10a is the most potent against P-388 and A-549 cells.
Examination of the different amino acid linkages and the resulting activity
in the inhibition of P-388 murine leukemia in vitro reveals the following
order of activity: L-glycine . L-alanine . L-phenylalanine . L-methionine .
L-isoleucine, whereas the resulting activity in the inhibition of A-549 human
lung carcinoma in vitro reveals the following order of activity: L-glycine .
L-phenylalanine . L-alanine . L-methionine . L-isoleucine. These results
show that the structures of L-amino acids have potential effects on the bio-
activity of these compounds. Hence, a systemic, predictable correlation
could be made between the nature of amino acids and anticancer activities.
In addition, the results also indicated that the strategy of introducing a
stable nitroxyl radical into the molecule of podophyllotoxin with L-amino
acids may be successful and they probably have synergistic action to tumor
cell lines. Further biological evaluation is in progress to better define the
antitumor activity of these compounds.
EXPERIMENTAL
All melting points were taken on a Kofler melting-point apparatus and are
uncorrected. IR spectra were obtained on NIC-5DX spectra photometer.
1H NMR spectra were obtained by using a Bruker AM400. All chemical
shifts are reported in ppm from TMS. Mass-spectral analysis was taken
on a ZAB-HS and Bruker Daltonics APEXII49e instrument. Optical
rotations were determined on Perkin-Elmer model 341 spectropolarimeter.
ESR spectra were obtained with
spectrometer.
a Bruker ER-200D-SRC X-band
Natural Product Origin
Podophyllotoxin (1) was isolated from the ethanol extract of the Chinese
medicinal herb Podophyllum emodi Wall var Chinesis Sprague via column

Spin-Labeled Podophyllotoxin Derivatives
2755
chromatography and eluting with a CHCl3–MeOH (20 : 1) mobile phase.
Fractions containing the product were evaporated in vacuo and the podophyl-
lotoxin recrystallized from C6H6. The resulting material was of .97% purity,
which was confirmed by direct comparison with an authentic sample and
spectral data reported previously.[26]
Synthesis of N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxy
carbonyl)-imidazolium Tosylate (7)
A solution of4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (5,1.72g,0.01mol)
in dry ether (10mL) is added dropwise to a stirred solution of N,N0-carbonyldimi-
dazole (1.62 g, 0.01 mol) in dry tetrahydrofuran (10 mL) at room temperature.
After the addition is complete, the solution is stirred for 3 h at room temperature
and then extracted with water (20 mL) and the saturated sodium chloride
solution. The organic phase is dried with sodium sulfate and evaporated in vacuo,
giving the intermediate N-(1-oxyl-2,2,6,6-tetramethylpiperidinyloxycarbonyl) –
imidazole (6); then to a solution of N-(1-oxyl-2,2,6,6-tetramethylpiperidinyloxy-
carbonyl)-imidazole (6, 2.66 g, 0.01 mol) in dry acetone (10 mL) is added a
solution of p-toluenesulfonic acid monohydrate (1.90 g, 0.01 mol) in acetone
(10 mL), and stirred for 5 min. Crystallization is completed by adding ether, and
the product isolated by suction; yield: 3.96 g (90%).
Synthesis of 1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl
Azide (8)
N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-imidazolium tosylate
(7, 2.19 g, 0.005 mol) is added at once to a stirred solution of sodium azide
(0.65 g, 0.01 mol) in water (10 mL) at room temperature. The mixture is
stirred for 30 min and then extracted with hexane (20 mL). The extract is
dried with sodium sulfate and evaporated in vacuo and cooled to 2208C.
The product is isolated as red crystals by suction; yield: 1.02 (91%).
Spectral data for 8 are identical to those reported by Hankovszky et al.[20]
General Procedure for Synthesis of N-(1-Oxyl-2,2,6,6-tetramethyl-
4-piperidinyloxycarbon yl) Amino Acids (9a–9e)
A solution of 1-oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl azide (8,
2.41g, 0.01mol) in dioxan (20 mL) is added to a stirred suspension of the
amino acids (0.01 mol) and magnesium oxide (0.8g, 0.02mol) in water
(10mL). The mixture is stirred for 24h at 35–40 8C, diluted with water

2756
Y.-Q. Liu and X. Tian
(30 mL), and extracted with ethyl acetate. The aqueous phase is acidified with
10% hydrochloric acid at 08C and extracted with ethyl acetate; the organic
phase is dried with sodium sulfate and evaporated. The residue is crystallized
from ether, giving N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)
amino acids (9a–9e). Spectral data for 9a–9e are identical to the reported by
Hankovszky et al.[20]
General Procedure for Synthesis of Target Compounds 10a–10e
A mixture of N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl) amino
acids (0.001 mol), podophyllotoxin (0.414 g, 0.001 mol), and dimethylamino-
pyridine (DMAP, 0.1 g) was stirred in dry dichloromethane (10 mL) for 5 min
at room temperature under nitrogen. N,N-dicyclohexylcarbodimide (DCC,
0.21 g, 0.001 mol) was added and the reaction mixture was stirred for 2 h.
The reaction mixture was filtered, and the filterate was evaporated. The
residue was separated by flask-column chromatography (gradient elution
with mixtures of dichloromethane–acetone) on silica gel and monitored by
TLC. Synthesized target compounds 10a–10e were characterized by mp,
ESR, IR, MS, and HRMS spectral analyses.
Compound 10a: Yield: 92%; mp 135–1378C; [a]2D0 275 (c ¼ 0.5,
CH2Cl2); IR (KBr) (cm21): 3373 (NH), 1780 (lactone), 1723 (NHCO),
1485, 1507, 1589 (aromatic C55C), 930 (OCH2O), 1125, 1179, 1240 (C–
O), 1371 (NO.); ESR: g0 ¼ 2.0058, DHpp ¼ 35.309Gs, AN ¼ 15.81 Gs
(triplet peak in 1 ꢁ 1024M, CH2Cl2); MS(FAB) m/z(int.%): 669 (Mþ, 38),
397(100); HRMS: m/z calcd. for C34H41N2O12: 671.2811[M þ 2H]þ;
Found: 671.2802 [M þ 2H]þ.
Compound 10b: Yield: 90%; mp 138–1408C; [a]2D0 268 (c ¼ 0.5,
CH2Cl2); IR (KBr) (cm21): 3344 (NH), 1781 (lactone), 1716 (NHCO),
1485, 1507, 1589 (aromatic C55C), 930 (OCH2O), 1126, 1175, 1239 (C–
O), 1365 (NOꢂ); ESR: g0 ¼ 2.0058, DHpp ¼ 44.268 Gs, AN ¼ 15.81 Gs
(triplet peak in 1 ꢁ 1024 M, CH2Cl2); MS (FAB) m/z (int.%): 683 (Mþ,
30), 397 (100); HRMS: m/z calcd. for C35H43N2O12: 685.2967 [M þ 2H]þ;
Found: 685.2962 [M þ 2H]þ.
Compound10c: Yield:91%; mp 115–1178C; [a]2D0 265(c ¼ 0.5, CH2Cl2);
IR (KBr) (cm21): 3337 (NH), 1781 (lactone), 1719 (NHCO),1485, 1507, 1589
(aromatic C55C), 930 (OCH2O), 1126, 1175, 1239 (C–O), 1365 (NOꢂ); ESR:
g0 ¼ 2.0058, DHpp ¼ 34.255Gs, AN ¼ 15.81 Gs (triplet peak in 1 ꢁ 1024 M,
CH2Cl2); MS (FAB) m/z (int.%): 743 (Mþ, 68), 397 (100); HRMS: m/z
calcd. for C37H47N2O12S: 745.3001 [M þ 2H]þ; found: 745.3001 [M þ 2H]þ.
Compound 10d: Yield: 83%; mp 117–1188C; [a]2D0 258 (c ¼ 0.5,
CH2Cl2); IR (KBr) (cm21): 3349 (NH), 1782 (lactone), 1718 (NHCO),1485,
1507, 1589 (aromatic C55C), 930 (OCH2O), 1126, 1178, 1239 (C–O), 1366
(NOꢂ); ESR: g0 ¼ 2.0058, DHpp ¼ 32.674Gs, AN ¼ 15.81 Gs (triplet peak in
1 ꢁ 1024M, CH2Cl2); MS (FAB) m/z (int.%): 725 (Mþ, 48), 397 (100);

Spin-Labeled Podophyllotoxin Derivatives
2757
HRMS: m/z calcd. for C38H49N2O12: 727.3437 [M þ 2H]þ, found: 727.3429
[M þ 2H]þ.
Compound 10e: Yield: 88%; mp 120–1218C; [a]2D0 253 (c ¼ 0.5,
CH2Cl2); IR (KBr) (cm21): 3334 (NH), 1780 (lactone), 1719 (NHCO),
1485, 1506, 1589 (aromatic C55C), 929 (OCH2O), 1125, 1177, 1239 (C–
O), 1365 (NOꢂ); ESR: g0 ¼ 2.0058, DHpp ¼ 27.404 Gs, AN ¼ 15.81 Gs
(triplet peak in 1 ꢁ 1024 M, CH2Cl2); MS (FAB) m/z(int.%): 759 (Mþ,
27), 397 (100); HRMS: m/z calcd. for C41H47N2O12: 761.3280 [M þ 2H]þ;
found: 761.3284 [M þ 2H]þ.
General Procedure for Reduction of the Representative Compound
10e to Compound 11
To a solution of 5 mg of compound 10e in 0.3 ml of CDCl3 in an NMR tube
was added 0.5 ml of 0.03 M phenylhydrazine in CDCl3. After 10 min at
258C under nitrogen, the progress of the reduction could be monitored
visually by the disappearance of the red-orange color of compound 10e. It
gave the corresponding N-hydroxy amine 11. Compound 11 was characterized
1
by IR, H NMR, and MS (FAB) as rapidly as possible.
IR (KBr) (cm21): 3336 (NH, OH, s), 1773 (lactone), 1708 (NHCO), 1485,
1594, 1600 (aromatic C55C), 933 (OCH2O), 1126, 1179, 1241 (C–O);
1H NMR (CDCl3) dH: 1.18 (6H, s, 2CH3 in tetramethylpiperidine ring), 1.21
(6H, s, 2CH3 in tetramethylpiperidine ring), 1.50–2.00 (4H, m, 2CH2 in tetra-
methylpiperidine ring), 2.52 (1H, m, 3-H), 2.66 (1H, dd, J ¼ 14.0Hz, 4.4 Hz,
2-H), 3.05 (2H, m, Ph-CH2), 3.70 (6H, s, 30,50-OCH3), 3.79 (3H, s, 40-OCH3),
3.94 (6H, d, J ¼ 8.8 Hz, 11-H), 4.01 (1H, m, CH in tetramethylpiperidine
ring), 4.49 (1H, d, J ¼ 4.4 Hz, 1-H), 4.67 (1H, dd, J ¼ 10.2Hz, 6.6 Hz, 1100-
H), 4.96 (1H, m, Ph-CH2-CH), 5.87 (1H, d, J ¼ 9.2 Hz, 4-H), 5.98 (2H, d,
J ¼ 6.0 Hz, OCH2O), 6.32 (2H, s, 20,60-H), 6.47(1H, s, 8-H), 6.67(1H, s, 5-H),
7.11(5H, s, Ph-CH2); MS(FAB) m/z (int.%): 761 ([M þ 1]þ, 27), 397 (100).
Biological Assay
Compounds 10a–10e were dissolved in Me2SO at a concentration of 20 mM, as
the stock solution was diluted before use with H2O to the desired concentration
of each compound. The cells were grown in RPMI 1640 medium with 10%
fetal-calf serum, penicillinG (100 IU/ml) and streptomycin (100 mg/ml) at
378C under a humidified 95% air þ5% CO2 atmosphere. Assays for the cyto-
toxicity in P-388 murine leukemia and A-549 human lung carcinoma cells were
carried out according to the procedures described by Bergeron.The prototypical
inhibitor etoposide was included as reference standard; the results of these
assays were used to obtain the corresponding inhibition rates.

2758
Y.-Q. Liu and X. Tian
ACKNOWLEDGMENT
This work was financially supported by the Natural Science Foundation of
Gansu Province Anti-SARS Foundation (ZGS-033-A-43-013).
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