The absolute configuration of a nitroxide spin labeled derivative of podophyllotoxin determined by the crystal structure

Article abstract of DOI:10.1139/V06-167

The crystal structure of the nitroxide spin labeled derivative of podophyllotoxin was first reported. X-ray analysis demonstrated that four contiguous chiral centers in the molecule, C1, C2, C3, and C4, adopt cis- (1:2), trans-(2:3), and cis- (3:4) arrang

Full text of DOI:10.1139/V06-167

1603
The absolute configuration of a nitroxide spin
labeled derivative of podophyllotoxin determined
by the crystal structure
Baohan Zhou, Guodong Yin, Xianggao Meng, Yitao Li, and Anxin Wu
Abstract: The crystal structure of the nitroxide spin labeled derivative of podophyllotoxin was first reported. X-ray
analysis demonstrated that four contiguous chiral centers in the molecule, C1, C2, C3, and C4, adopt cis- (1:2), trans-
(2:3), and cis- (3:4) arrangement.
Key words: crystal structure, synthesis, nitroxyl radical, podophyllotoxin.
Résumé : On a déterminé pour la première fois la structure cristalline d’un dérivé de la podophyllotoxine portant un
groupe nitroxyde comme marqueur de spin. Les analyses par diffraction des rayons X ont permis de démontrer que
quatre centres chiraux contigus de la molécule, C1, C2, C3 et C4, adoptent des arrangements cis- (1,2), trans- (2,3) et
cis- (3,4).
Mots clés : structure cristalline, synthèse, radical nitroxyle, podophyllotoxine.
[Traduit par la Rédaction] Zhou et al.
Introduction
Scheme 1 (8). Podophyllotoxin (1), commercial agent (J &
K Chemical Ltd., Shanghai, China), was used as a starting
material.
Podophyllotoxin (1) and its many related derivatives are
well known to possess pronounced antitumor and antiviral
properties. However, the clinical application of podophyl-
lotoxin and its analogues in the treatment of cancer has been
limited by severe toxic side effects during the administration
of the drugs (1–4). It was previously found that the introduc-
tion of a stable nitroxyl radical into the molecule of
podophyllotoxin could result in novel compounds that have
significant antitumor activity with marked decrease in toxic-
ity compared with podophyllotoxin itself (5–7). Though the
structure of spin-labeled podophyllotoxin derivatives can be
indirectly characterized by mp, ESR, IR, MS, and HR-MS
spectral analyses, its elaborate structure can’t be confirmed
The demethylation and bromination of 1 gave 4-bromo-4′-
demethyl-epipodophyllotoxin (2). 33 g of 1 was suspended
in 300 mL of 1,2-dichloroethane and 30 mL of ether and
cooled to 0 °C. A current of dry hydrobromide was passed
in until an increased mass of 82.5 g was obtained. After
standing at 0–2 °C for 20 h, the solvent was removed under
vacuum. The residue was recrystallized from acetone to give
12 g of 2: 36% yield; mp 180–190 °C (dec.). A solution of 2
(2.77 g, 6 mmol) and 2,2,6,6-tetramethyl-1-piperidinyloxy-
4-amine (2.57 g, 15 mmol) in 35 mL of dry THF and 2
drops of dry pyridine was refluxed under N2 for 4 h. After
filtering off the insoluble solids and removing the solvent
under reduced pressure, the crude product was chromato-
graphed twice through silica gel using CH2Cl2–acetone–
Et2NH (40:1:1, v/v) as an eluent to obtain 1.07 g (32%
yield). Crystallization from acetone–hexane afforded the
spin-labeled derivative of podophyllotoxin (3): red solid; mp
218–220 °C. UV (methanol) λmax (nm): 283, 242, 220. IR
(cm–1): 3338 υ(NH), 1757 υ(C=O), 1609 υ(C=C), 1516
υ(C=C), 1479 δS(CH3). ESR: 3 Lines, An = 16.2 G, ∆Ho =
2.66 G, go = 2.0061. MS–FAB m/z (%): 554 (13), 553 (16),
537 (7), 383 (100). Anal. calcd. (%): C 65.08, H 6.74, N
5.06; found: C 65.36, H 6.505, N 4.035.
1
by H NMR because of its paramagnetic properties. More-
over, there are four contiguous chiral centers in the mole-
cule, and its absolute configuration was not determined until
now. In this paper, we synthesized one nitroxide spin labeled
derivative of podophyllotoxin (3), first reported the crystal
structure, and confirmed its absolute configuration.
Experimental section
4-[4′′-(2′′,2′′,6′′,6′′-tetramethyl-1′′-piperidinyloxy)amino]-4′-
demethylepipodophyllotoxin (3) was prepared as shown in
A red crystal of the synthesized compound 3 was mounted
on a glass fiber in a random orientation at 292(2) K. The de-
termination of the unit cell and the data collection were per-
formed with Mo Kα radiation (λ = 0.71073 D) on a Bruker
SMART APEX-CCD diffactometer with a ψ-ω scan mode.
The structure was solved by direct methods with SHELXS-
97 program and expanded by Fourier technique. The non-
hydrogen atoms were refined anisotropically, and the hydro-
gen atoms were determined by theoretical calculations.
Received 28 February 2006. Accepted 18 September 2006.
Published on the NRC Research Press Web site at
http://canjchem.nrc.ca on 1 December 2006.
B. Zhou, G. Yin, X. Meng, Y. Li, and A. Wu.1 Key
Laboratory of Pesticide and Chemical Biology, Ministry of
Education, College of Chemistry, Central China Normal
University, Wuhan 430079, People’s Republic of China.
1Corresponding author (e-mail: chwuax@mail.ccnu.edu.cn).
Can. J. Chem. 84: 1603–1606 (2006)
doi:10.1139/V06-167
© 2006 NRC Canada

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Can. J. Chem. Vol. 84, 2006
Scheme 1.
O
N
OH
Br
O
O
HN
O
O
O
N
O
O
O
H
2N
O
O
O
HBr
O
O
OMe
MeO
OMe
MeO
OMe
MeO
OMe
OH
OH
(2)
(1)
(3)
Table 1. Crystal data and structure refinement for 3.
Empirical formula
C30H37N2O8
Formula weight
Temperature
Wavelength (D)
Crystal system
Space group
553.62
292(2) K
0.71073
Orthorhombic
P212121
a (D)
b (D)
c (D)
α = β= γ
Volume (D3)
Z
Dcalcd (mg/m3)
8.1082(6)
19.3396(13)
42.130(3)
90°
6606.4(8)
4
1.222
Absorption coefficient (µ) (mm–1)
F(000)
0.087
2576
0.30 × 0.20 × 0.20
2.16–25.00
–9 ≤ h ≤ 9, –19 ≤ k ≤ 23, –50 ≤ l ≤ 48
33402
6467 (Rint = 0.0851)
Crystal size (mm3)
θ range for data collection (°)
Limiting indices
Reflections collected
Independent reflections
Refinement method
Full-matrix least-squares on F2
6467/0/853
1.023
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices (I > 2σ(I))
R1 = 0.0614, wR2 = 0.1453
R indices (all data)
R1 = 0.1039, wR2 = 0.1692
Extinction coefficient
Largest diff. Peak and hole (e D–3)
0.0002(4)
0.189 and –0.188
Because the tumor-damaging potency of podophyllotoxin
and related compounds is closely associated with their
stereochemistry (9, 10), the determination of their absolute
configuration might shed further light on the mechanism of
their action, a question of importance in the search for effec-
tive chemotherapeutic agents. Synthetic organic chemists
have been attracted to the stereochemical challenge repre-
sented by the four contiguous chiral centers of podo-
phyllotoxin, the rigid trans-lactone, and the axially locked 1-
aryl substituent. The early syntheses (11–14) generated
Crystal data and structure refinement for compound 3 are
listed in Table 1. The selected bond lengths and bond angles
are given in Table 2 and 3, respectively. The other tables of
CIF have been deposited as supplementary material.2
Results and discussion
Figure 1 shows the molecular structure of compound 3,
and the atomic numbering of the same compound is shown
in Fig. 2.
2 Supplementary data for this article are available on the journal Web site or may be purchased from the Depository of Unpublished Data,
Document Delivery, CISTI, National Research Council Canada, Ottawa, ON K1A 0S2, Canada. DUD 5111. For more information on ob-
taining material, refer to http://cisti-icist.nrc-cnrc.gc.ca/irm/unpub_e.shtml. CCDC 298236 contains the crystallographic data for this manu-
script. These data can be obtained, free of charge, via ww.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax +44 1223 336033; or deposit@ccdc.cam.ac.uk).
© 2006 NRC Canada

Zhou et al.
1605
Table 2. Selected Bond lengths (D).
Fig. 1. Molecular structure of compound 3.
Bond
Distance
C1—N1
C1—C9
C1—C2
C2—C3
C2—C12
C3—C13
C3—C4
C4—C10
C4—C1′
C5—C6
C5—C10
C6—O2
C7—C8
C7—O1
C8—C9
C9—C10
C11—O1
C11—O2
C12—O3
C13—O4
C13—O3
C6—C7
1.472(6)
1.525(7)
1.541(7)
1.513(7)
1.522(7)
1.497(8)
1.524(7)
1.516(8)
1.523(6)
1.349(9)
1.406(7)
1.388(7)
1.359(8)
1.374(7)
1.424(8)
1.398(7)
1.409(11)
1.434(11)
1.444(8)
1.205(7)
1.362(8)
1.381(9)
Table 3. Selected bond angles (°).
Angle
(°)
Fig. 2. Atomic numbering of compound 3.
N1–C1–C9
N1–C1–C2
C9–C1–C2
C3–C2–C12
C3–C2–C1
C12–C2–C1
C13–C3–C2
C13–C3–C4
C2–C3–C4
C10–C4–C1′
C10–C4–C3
C1′–C4–C3
110.1(4)
110.6(4)
107.8(4)
101.2(4)
108.6(4)
119.7(4)
103.6(4)
120.0(5)
113.3(4)
113.2(4)
108.2(4)
114.0(4)
C(7'')
CH3
C(8'')
CH3
N(1'')
OO(1'')
N
C(3'')
C(2'')
CH3
C(9'')
C(4'')
C(5'')
HNN(1)
C(6'')
O(1)
C(8)
O
CH3
C(10'')
C(12)
C(7)
C(9)
C(1)
C(2)
O O(3)
C(11)
C(3)
C(4)
C(13)
C(6)
C(10)
O
O(2)
much new and interesting chemistry but led to the inactive
cis-lactone.
C(5)
O O(4)
C(1')
C(6')
X-ray analysis demonstrated that four contiguous chiral
centers in the molecule, C1, C2, C3, and C4, adopt cis-
(1:2), trans- (2:3), and cis- (3:4) arrangement (Fig. 1). The
angles of C3–C2–C12, C3–C2–C1, and C13–C3–C2 are
101.2(4)°,108.6(4)°, and 103.6(4)°, respectively, which are
less than the normal 109.5° angle. The angles of C12–C2–
C1, C13–C3–C4, and C2–C3–C4 are 119.7(4)°, 120.0(5)°,
and 113.3(4)°, respectively, which are much larger than the
normal 109.5° angle. These results show that C2 and C3 are
distorted atoms. Perhaps because the trans-lactone is a rigid
ring and can not rotate freely. The six-membered ring con-
taining N–O radical adopts a chair conformation, and the
bond length of N—O is 1.270(5) D, which is in the normal
range of nitroxyl radicals (1.25–1.31 D) (15, 16).
C(2')
C(3')
OO(1')
CH3
C(5')
O
O(3')
C(4')
H3C
OH
O(2')
C(7')
C(8')
Acknowledgement
The Project was sponsored by the Scientific Research
Foundation for the Returned Overseas Chinese Scholars,
State Education Ministry (No. [2005] 383).
© 2006 NRC Canada

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Can. J. Chem. Vol. 84, 2006
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