Spectroscopic studies and PM5 semiempirical calculations of new hydrazone of gossypol with 3,6,9-trioxadecylhydrazine

Article abstract of DOI:10.1016/j.molstruc.2006.06.034

A new hydrazone of gossypol with 3,6,9-trioxadecylhydrazine (GHTO) has been synthesised and its structure has been studied by ¹H NMR, ¹³C NMR, FT-IR spectroscopy and PM5 semiempirical methods. The results have shown that the newly synthesised hydrazone exists in solution in the N-imine-N-imine tautomeric form, stabilized by several intramolecular hydrogen bonds among which the O₇H ^?N₁₆ intramolecular hydrogen bond is the strongest. The structure of GHTO is visualized by the PM5 semiempirical calculations.

Full text of DOI:10.1016/j.molstruc.2006.06.034

Journal of Molecular Structure 830 (2007) 72–77
www.elsevier.com/locate/molstruc
Spectroscopic studies and PM5 semiempirical calculations of
new hydrazone of gossypol with 3,6,9-trioxadecylhydrazine
*
Grzegorz Bejcar, Piotr Przybylski, Monika Walkowiak, Bogumil Brzezinski
Faculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland
Received 5 June 2006; accepted 30 June 2006
Available online 14 August 2006
Abstract
A new hydrazone of gossypol with 3,6,9-trioxadecylhydrazine (GHTO) has been synthesised and its structure has been studied by
1H NMR, 13C NMR, FT-IR spectroscopy and PM5 semiempirical methods. The results have shown that the newly synthesised hydra-
zone exists in solution in the N-imine–N-imine tautomeric form, stabilized by several intramolecular hydrogen bonds among which the
O7H ꢀ ꢀ ꢀN16 intramolecular hydrogen bond is the strongest. The structure of GHTO is visualized by the PM5 semiempirical calculations.
ꢀ 2006 Elsevier B.V. All rights reserved.
Keywords: Gossypol; Hydrazones; 3,6,9-Trioxadecylhydrazine; FT-IR; 1H NMR; 13C NMR; PM5 calculations; Hydrogen bonds
1. Introduction
spectroscopic as well as semiempirical PM5 studies of a
new hydrazone of gossypol with 3,6,9-trioxadecylhydr-
azine. The tautomeric structure of this new compound is
discussed in detail.
Gossypol 2,20-bis(8-formyl-1,6,7-trihydroxy-5-isopro-
pyl-3-methylnaphthalene) is a well-known disesquiterpene
showing great biological activity [1–10]. Very important
groups of gossypol derivatives are its Schiff bases and
hydrazones, which are characterized by significantly lower
toxicity than gossypol [11,12]. It is also well known that
gossypol can occur in three symmetrical tautomeric forms
shown in Scheme 1. In contrast, Schiff bases and hydraz-
ones of gossypol (Scheme 2) can be present in only two
symmetrical tautomeric forms: imine–imine (N-imine–N-
imine) and enamine–enamine (N-enamine–N-enamine)
forms which are analogous to the aldehyde–aldehyde and
ketol–ketol tautomeric forms of gossypol, respectively.
Recently, we have studied the structures of several Schiff
bases [13–22] and hydrazones [23–28] of gossypol. All these
papers reported the occurrence of the enamine–enamine
tautomeric forms of gossypol Schiff bases and N-imine–
N-imine tautomeric forms of gossypol hydrazones both in
solution and in the solid state. As a continuation of these
studies, in the present paper we report the synthesis and
2. Experimental
2.1. Synthesis of 3,6,9-trioxadecylhydrazine
3,6,9-Trioxadecylchloride was synthesized from triethyl-
ene glycol monomethyl ether and PCl3 in presence of pyr-
idine (procedure from Ref. [29]). The purity of this
1
compound was controlled by H and 13C NMR spectra.
3,6,9-Trioxadecylchloride [15.15 g (0.083 mol)], hydra-
zine monohydrate [41.5 g (0.83 mol)] and 15 cm3 of ethyl
alcohol were refluxed 6 h. After this time the solvent was
evaporated under a reduced pressure from the reaction
mixture. The residue was several times extracted by ethyl
ether. The ether solutions collected were dried by freshly
roast calcium oxide, the solvent was evaporated and the
residue was distilled under reduced pressure. Pure 3,6,9-tri-
oxadecylhydrazine is a colourless liquid with a characteris-
tic amine-like odour. Boiling point 130–135 ꢁC/3 mmHg.
1
*
The purity of this compound was controlled by H and
Corresponding author. Tel.: +48 61 829 1330; fax: +48 61 865 8008.
13C NMR spectra.
E-mail address: bbrzez@main.amu.edu.pl (B. Brzezinski).
0022-2860/$ - see front matter ꢀ 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2006.06.034

G. Bejcar et al. / Journal of Molecular Structure 830 (2007) 72–77
73
O
H
H
O
H
O
H
O
O
H
H O
H
O
O
H
aldehyde-aldehyde
H
O
H
O
H
O
H
O
H
O
O
H
O
H
O
H
O
H
O
O
H
O
H
O
H
H
H
O
O
H
O
H
lactol-lactol
ketol-ketol
Scheme 1. Tautomeric forms of gossypol.
H
N
H
17
N
18
19
O
O
O
O
O
16N
H
OH
N
H
O
11
20
21
H
OH
H
O
OH
H
8
1
O
O
9
O
O
O
O
O
H
O
H
2
7
6
22
H
HO
HO
23
10
3
H
N
H
N
N
HO
O
5
4
12
O
O
15
14
13
N
24
H
H
GHTO
GHTO
N-imine - N-imine
N-enamine - N-enamine
Scheme 2. Tautomeric forms of GHTO.
˚
2.2. Synthesis of 3,6,9-trioxadecylhydrazone of gossypol
(GHTO)
spectral-grade solvents were stored over 3 A molecular
sieves for several days.
A cell with Si windows and wedge-shaped layers was
used to avoid interferences (mean layer thickness
170 lm). The spectra were taken with an IFS 66v/s FT-
IR spectrophotometer (Bruker, Karlsruhe) equipped with
a DTGS detector; resolution 2 cmꢁ1, NSS = 125. The
Happ–Genzel apodization function was used.
GHTO was synthesised by addition of a solution of
206.18 mg (1.15 · 10ꢁ3 mol) of 3,6,9-trioxadecylhydrazine
in 5 cm3 absolute ethanol to a solution of 300 mg of gossy-
pol (5.78 · 10ꢁ4 mol) in 15 cm3 absolute ethanol. The mix-
ture was stirred under reflux for 5 h under argon
atmosphere. After cooling of the mixture, the reaction
product precipitated as a brown-yellow powder. The prod-
uct was recrystallized from absolute ethanol and finally
dried under reduced pressure. All manipulations were per-
formed under argon atmosphere. Yield: 392 mg (80.7%).
Melting point: 171–173 ꢁC.
All manipulations with the substances were performed
in a carefully dried and oxygen-free glove box.
2.4. NMR measurements
The NMR spectra of GHTO were recorded in CDCl3
using a Varian Gemini 300 MHz spectrometer. All spectra
were locked to deuterium resonance of CD3OD or CDCl3,
respectively. The error in ppm values was 0.01.
Elementary
analysis
C44H62N4O12:
calculated,
C = 62.99%, H = 7.45%, N = 6.68%; found: C = 62.95%,
H = 7.42%, N = 6.71%.
All 1H NMR measurements were carried out at the
operating frequency 300.075 MHz; flip angle, pw = 45ꢁ;
spectral width, sw = 4500 Hz; acquisition time, at = 2.0 s;
relaxation delay, d1 = 1.0 s; T = 293.0 K and TMS as the
internal standard. No window function or zero filling was
used. Digital resolution = 0.2 Hz/point.
2.3. FT-IR measurements
The FT-IR spectra of gossypol and GHTO were recorded
in methylenechloride and chloroform (0.05 mol dmꢁ3),
respectively at 300 K. Chloroform and methylenechloride

74
G. Bejcar et al. / Journal of Molecular Structure 830 (2007) 72–77
13C NMR spectra were recorded at the operating fre-
quency 75.454 MHz; pw = 60ꢁ; sw = 19,000 Hz;
found at 6.76 ppm, and its position is almost the same as
in the spectra of all other hydrazones studied because this
proton is engaged in the O6Hꢀ ꢀ ꢀO7 weak intramolecular
hydrogen bond. The position of the O1H proton signal at
5.78 ppm is slightly shifted toward higher frequencies rela-
tive to those of the respective signals observed in the spec-
tra of other hydrazones. This shift is probably evoked by
the formation of a weak intramolecular hydrogen bond
between the O1H proton and O23–24 oxygen atom from
the oxaalkyl chain, which is in agreement with the 13C
NMR chemical shit of the C1 atom as well as with the
PM5 semiempirical calculations discussed below.
at = 1.8 s; d1 = 1.0 s; T = 293.0 K and TMS as the internal
standard. Line broadening parameters were 0.5 or 1 Hz.
The 1H and 13C NMR signals have been assigned indepen-
dently for each species using one- or two-dimensional
(COSY, HETCOR) spectra. The 1H NMR spectrum of
GHTO was also measured after an addition of two drops
of CD3OD to identify the signals of OH and NH protons.
2.5. PM5 semiempirical calculations
PM5 semiempirical calculations were performed using
the Win Mopac 2003 program [30–33]. For calculated
hydrazone of gossypol full geometry optimization was car-
ried out without any symmetry constraints.
In the 13C NMR spectrum of GHTO (Table 2) the signal
of the C7 atom is observed at 150.2 ppm. This chemical
shift is comparable to that observed in the spectra of gos-
sypol hydrazones studied previously (150.2 ppm [23–25])
and completely different from those of gossypol Schiff
bases (172.3–173.0 ppm) [13–22]. Furthermore, the value
of the chemical shift of about 150 ppm is characteristic of
the CAOH carbon atoms of phenol molecules [34,35].
2.6. Elementary analysis
The elementary analysis of GHTO was carried out on
Vario ELIII (Elementar, Germany).
Thus, these results have clearly demonstrated that the pro-
ꢀ ꢀ ꢀ
ton in the O7H
N
intramolecular hydrogen bond is
16
3. Results and discussion
localized at the O7 oxygen atom and for this reason the
GHTO molecule exists in the solution as the N-imine–N-
imine tautomeric form.
The formula and the atom numbering of the hydrazone
of gossypol with 3,6,9-trioxadecylhydrazine (GHTO) are
shown in Scheme 2.
3.2. FT-IR studies
3.1. NMR studies
The FT-IR spectra of GHTO in chloroform and of gos-
sypol in methylenechloride solutions are compared in
Fig. 1a. The same spectra on the extended scales in the
regions of the m(OH) and m(C@O, C@N) stretching vibra-
tions are also shown (Fig. 13b and c, respectively). In all
figures, the solid lines represent the spectra of GHTO
and dashed-dotted lines the spectra of gossypol, for
comparison.
The chemical shifts of the signals observed in the 1H and
13C NMR spectra of GHTO are shown in Tables 1–3,
respectively. These signals were assigned using one- and
two-dimensional COSY and HETCOR spectra, respective-
ly, as well as after the addition of CD3OD to the probe.
1
In the H NMR spectrum of GHTO (Table 1) the sig-
nals of the protons of OH and that of NAH groups are
observed separately. After the addition of CD3OD these
In the spectrum of GHTO (Fig. 1b, solid line) the band
assigned to the m(OH) stretching vibrations is only slightly
shifted to 3494 cmꢁ1 relative to the band at 3502 cmꢁ1 in
the spectrum of gossypol. This indicates that the OH
groups in the positions 1, 10 in both compounds are
involved in similarly strong hydrogen bonds. The broad-
ened band assigned to the stretching vibrations of OH
groups at the 6, 60 positions and to the stretching vibrations
signals vanish completely. The highest chemical shift of
ꢀ ꢀ ꢀ
the O7H
N
16
intramolecular hydrogen-bonded proton is
observed at 14.50 ppm. This chemical shift is up to now
the highest when compared to that of the respective intra-
1
molecular hydrogen bond observed in the H NMR spec-
trum of earlier studied gossypol hydrazones (14.43, 14.34
and 14.47 ppm) [23–25]. The signal of the O6H proton is
0
of N17AH and N17 AH groups arises in the range
Table 1
1H NMR chemical shifts (ppm) of GHTO in CDCl3
Compound
Chemical shift (ppm)
O1H C4H O6H O7H C11
H
C12
H
C13
H
C14H C15
H
N17
H
C18
H
C19
H
C20
3.46a 3.58a 3.58
(t) (t) (t)
3.46a 3.58a 3.58
(t) (t) (t)
H
C21
H
C22
H
C23
H
C24H
GHTO
5.78 7.68 6.76 14.50 9.58
2.10
(s)
2.10
(s)
3.86
1.54 (d)
5.40
(s)
–
3.34
(t)
3.34
(t)
3.68
(t)
3.68
(t)
3.46
(t)
3.46
(t)
3.26
(s)
3.27
(s)
(bs)
–
(s)
7.68
(s)
(bs)
–
(bs)
–
(s)
9.61
(s)
(sept) 1.55 (d)
3.86 1.53 (d)
(sept) 1.56 (d)
GHTO + CD3OD
s, singlet; bs, broad singlet; d, doublet; t, triplet; sept, septet.
a
Assignment can be changed.

G. Bejcar et al. / Journal of Molecular Structure 830 (2007) 72–77
75
Table 2
13C NMR chemical shifts (ppm) of carbon atoms of GHTO in CDCl3 (gossypol moiety)
Compound
Chemical shifts (ppm)
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
GHTO
149.4
114.4
132.1
117.3
126.0
144.4
150.2
107.1
114.2
129.5
152.0
20.4
27.4
20.7
20.7
Table 3
13C NMR chemical shifts (ppm) of carbon atoms of GHTO in CDCl3 (oxalkyl moiety)
Compound
GHTO
Chemical shifts (ppm)
C18
C19
C20
C21
C22
70.4
C23
71.8
C24
49.6
68.3
70.4
70.5
58.9
a
100
tion of the N-imine–N-imine tautomeric form (analogous
to the imine–imine form of gossypol Schiff base) within
the GHTO molecule. The most important result confirming
this conclusion is the presence of the band of m(C@C)
vibrations characteristic of naphthalene ring, in the spec-
trum of gossypol at 1573 cmꢁ1. In the spectrum of GHTO
molecule, this vibration is shifted to 1561 cmꢁ1 due to the
coupling of a lone electron pair on N17 atom with the p
electron system.
50
0
4000
3500
3000
2500
2000
1500
1000
500
100
50
0
b
3.3. PM5 calculations
The calculated heats of formation for N-imine–N-imine
and N-enamine–N-enamine tautomeric forms of GHTO
molecule are ꢁ1445.89 kJ molꢁ1 and ꢁ1410.79 kJ molꢁ1
,
3494
3502
respectively, indicating that the first tautomer is energeti-
cally favourable.
3750
3500
3250
1561
3000
The hydrogen bond lengths and angles stabilising the N-
imine–N-imine tautomeric form of GHTO are collected in
Table 4 and its structure is shown in Scheme 3. The hydro-
gen bonds existing in this structure are marked by dots. The
strongest hydrogen bond is the O7ꢀ ꢀ ꢀHAN16 intramolecular
c
100
50
0
1615
˚
one. The length of this bond is about 2.45 A and its hydro-
1624
1573
gen bond angle is 153.6ꢁ, indicating that it belongs to the
hydrogen bonds of medium strength. This fact is also in
good agreement with the FT-IR and 1H NMR spectroscopic
results. Within the structure of GHTO two additional intra-
molecular hydrogen bonds are formed between the N17AH
and O1AH proton donors and the oxygen atoms of the oxa-
alkyl chains. The parameters of these intramolecular hydro-
gen bonds indicate that they are of low strength. This finding
is in very good agreement with the spectroscopic data dis-
cussed above. A comparison of the GHTO structure with
that of the respective Schiff base of gossypol with 3,6,9-tri-
oxadecylamine [15] demonstrates that the O7ꢀ ꢀ ꢀHAN16
intramolecular hydrogen bond becomes stronger in the
GHTO molecule and the oxaalkyl chains are hydrogen
bonded in different ways due to the presence of the
N17AH proton donor group in the structure of hydrazone.
The dihedral angle in the calculated structure of (S)-
GHTO, shown in Scheme 3 is +96.9 and this value is typ-
ical of all Schiff bases and hydrazones of gossypol studied.
1700
1675
1650
1625
1600
1575
1550
1525
1500
Wavenumber [cm-1]
Fig. 1. FT-IR spectrum of (——) GHTO in CHCl3, and for comparison
of (- - -) gossypol in CH2Cl2; (a) 4000–400 cmꢁ1, (b) 3750–3000cmꢁ1, (c)
1700–1500 cmꢁ1
.
ꢀ3ꢀ4ꢀ 00–3200 cmꢁ1. The vibrations of the strongest O7H
N16 hydrogen-bonded proton are relatively well observed
in the FT-IR spectrum in the region below 3000 cmꢁ1
.
Details of the tautomeric structure of GHTO can be
concluded from the region 1700–1500 cmꢁ1. In the spec-
trum of gossypol the m(C@O) vibration of the aldehyde
group is observed at 1624 cmꢁ1. In the spectrum of GHTO
this band vanishes completely and only one broad band,
mainly caused by the overlaying of the m(C11@N),
m(C@C) and d(NAH) vibrations, with a maximum at about
1615 cmꢁ1 arises. This spectral feature indicates the forma-

76
G. Bejcar et al. / Journal of Molecular Structure 830 (2007) 72–77
Table 4
˚
Interatomic distances (A) and angles (ꢁ) of the hydrogen bonds within N-imine–N-imine tautomeric form of GHTO.
˚
Compound
Hydrogen bond length (A) [angles (ꢁ)]
O7AHꢀ ꢀ ꢀN16
O1AHꢀ ꢀ ꢀO23-24
N17AHꢀ ꢀ ꢀO19-20
O6AHꢀ ꢀ ꢀO7
C11AHꢀ ꢀ ꢀO1
GHTO
2.45 [153.6]
2.68 [141.4]
2.86 [103.2]
2.82 [115.7]
2.59 [129.8]
Scheme 3. The most probable structure of (S)-GHTO in the N-imine–N-imine tautomeric form calculated by PM5 method (WinMopac 2003).
[8] W.M. Brown, L.E. Metzger, J.P. Barlow, L.A. Hunsaker, L.M. Deck,
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Agents Chemother. 46 (2002) 3133.
Research Project No R05 016 01. Dr Przybylski thanks to
Foundation of Polish Science for fellowship.
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