Characterization of hydrazine derivative: Proposed decomposition mechanism and structure elucidation of decomposition compounds

Article abstract of DOI:10.1248/cpb.51.346

Decomposition of protected hydrazine diol (1) hemi-oxalate, a key intermediate of the potent indolocarbazole-based DNA topoisomerase I inhibitor (2), was investigated. Spectroscopic analysis revealed that the main decomposition compounds of the hydrazine

Full text of DOI:10.1248/cpb.51.346

346
Notes
Chem. Pharm. Bull. 51(3) 346—350 (2003)
Vol. 51, No. 3
Characterization of Hydrazine Derivative: Proposed Decomposition
Mechanism and Structure Elucidation of Decomposition Compounds
Takayuki NEMOTO,* Eliada LAZOURA, and Takashi NOMOTO
Process R & D, Laboratories for Technology Development, Banyu Pharmaceutical Co., Ltd.; 3–9–1 Kamimutsuna,
Okazaki, Aichi 444–0858, Japan.
Received November 8, 2002; accepted December 16, 2002; published online December 16, 2002
Decomposition of protected hydrazine diol (1) hemi-oxalate, a key intermediate of the potent indolocar-
bazole-based DNA topoisomerase I inhibitor (2), was investigated. Spectroscopic analysis revealed that the main
decomposition compounds of the hydrazine derivative were a peroxide (3) and an alcohol derivative (4). The per-
oxide derivative (3) was proposed to form in the presence of oxygen- and/or H2O-generated radicals, which was
subsequently reduced to the more stable alcohol derivative (4). A plausible decomposition mechanism was pro-
posed and our findings were substantiated by chemical conversion.
Key words hydrazine; radical; decomposition; peroxide; DNA topoisomerase I
CH3CN/0.1% H3PO4 (10/90) to CH3CN/0.1% H3PO4 (50/50) over 40 min,
then to CH3CN/0.1% H3PO4 (90/10) over 20 min and maintained at these
conditions for a further 5 min, flow rate 1.0 ml/min. Absorbance was de-
tected at 220 nm.
Preparative HPLC The HPLC equipment was as follows : an octadecyl
silica (ODS) column [Shim-pack PREP-ODS(H) KIT, 250ϫ20 mm i.d.] and
Shimadzu VP Series HPLC. Gradient elution was performed from
CH3CN/H2O (10/90) to CH3CN/H2O (50/50) over 40 min, then to
CH3CN/H2O (90/10) over 20 min and maintained at these conditions for a
further 5 min, flow rate 10.0 ml/min. Absorbance was detected at 220 nm.
Pure 3 (tRϭ56.7 min), and 4 (tRϭ54.0 min) were collected for further analy-
sis. While 4 was freeze dried, 3 was extracted from H2O using benzene to
avoid decomposition.
DNA topoisomerase I (topo I), presently represents an at-
tractive target for the development of inhibitors for use as
anti-bacterial, anti-fungal, and anti-cancer chemotherapeutic
agents. Recently our group reported the practical synthesis of
a potent indolocarbazole-based topo I inhibitor (2), produced
by coupling the glucoside (5) and protected hydrazine diol
(1) hemi-oxalate (Chart 1).1) Often, however, unexpected ob-
stacles are encountered in a scale-up synthesis. In this case,
the decomposition of a key intermediate, protected hydrazine
diol (1) hemi-oxalate, posed problems.
Hydrazine derivatives are known to easily decompose in
the presence of radicals.2,3) Commonly used as rocket fuels,
most of them are endothermic compounds.3) Upon storage,
protected hydrazine diol (1) hemi-oxalate was found to de-
LC/MS Spectrometry (MS) MS spectra were recorded on a JEOL
JMS-LC mate and/or a Finnigan Thermoquest Model LC-Q mass spectrom-
eter coupled to an Agilent 1100 Series HPLC. An ODS column (YMC AM-
303, 250ϫ4.6 mm i.d.) was used, with gradient condition from CH3CN/
compose, even when kept in the dark at 5 °C and under nitro- 0.04% HCOOH (10/90) to CH3CN/0.04% HCOOH (50/50) over 30 min and
subsequently to CH3CN/0.04% HCOOH (90/10) for a further 30 min, flow
rate 1.0 ml/min (0.2 ml/min split to LC-MS). Absorbance was detected at
220 nm. Mass spectra were recorded in positive electrospray ionization
(ESIϩ) mode.
gen.
Here we reports the decomposition of protected hydrazine
diol (1) hemi-oxalate, structure elucidation of the decompo-
sition compounds and propose a plausible decomposition
mechanism.
NMR Spectrometry (NMR) NMR spectra were recorded on a Bruker
DRX500 (500 MHz). NMR chemical shifts are reported as d values in ppm
relative to TMS. For LC-NMR of the peroxide derivative 3, an Agilent 1100
Series HPLC was coupled to the NMR spectrometer. An ODS column
(YMC AM-303, 250ϫ4.6 mm i.d.) was used, with gradient condition from
CD3CN/D2O (10/90) to CD3CN/D2O (50/50) in 5 min, then to CD3CN/D2O
(90/10) for 30 min, and maintained at these conditions for a further 5 min,
Experimental
Materials All compounds were prepared by Banyu Pharmaceutical Co.
Ltd. (Okazaki, Japan). The purity of protected hydrazine diol (1) hemi-ox-
alate was above 99.9% at the time of storage, in the dark and under nitrogen
at 5 °C for 6 months in a polyethylene bag stored in a steel drum. All chemi-
cals and solvent were of analytical reagent grade.
flow rate 1.0 ml/min. Absorbance was detected at 220 nm. Pure
3
(tRϭ17.7 min) was collected for further online analysis.
Analytical HPLC The HPLC equipment was as follows : an octyl silica
(C8) column (Waters Symmetry C8, 250ϫ4.6 mm i.d.), Waters 2695 separa-
tion module coupled to a Waters 2487 dual l absorbance detector and a Wa-
ters 2996 photodiode array detector. Gradient elution was performed from
Results and Discussion
Structure Elucidation of Decomposition Compounds
The HPLC profile of decomposed protected hydrazine diol
(1) hemi-oxalate is shown in Fig. 1. Initial purity was over
99.9%, however, after storage at 5 °C for 6 months under ni-
trogen in a polyethylene bag and inside a steel drum, the pu-
rity decreased to 95.9 % with impurity levels ranging be-
tween 0.01—0.96% (Fig. 2). The following decomposition
compounds were detected (Fig. 3): a secondary amine (6)
[m/z 272 (MϩH)ϩ; 0.11%], formed by homolytical cleavage
of the N–N bond by HO ; 1-benzyloxy-3-benzoyloxy-2-
hydrazinopropane (7) [m/z 301 (MϩH)ϩ; 0.06%], formed by
oxidation of one of the benzyl oxy groups; and the dimer
compound (8) [m/z 539 (MϩH)ϩ; 0.27%], formed by cou-
pling 1 to 1,3-dibenzyloxyketone (9), formed by oxidation of
2,3)
·
Chart 1. Synthesis of Indolocarbazole Compound (2)
* To whom correspondence should be addressed. e-mail: nemototy@banyu.co.jp
© 2003 Pharmaceutical Society of Japan

March 2003
347
Fig. 1. HPLC Chromatographic Profiles from Hydrazine Derivative (1) (Initial)
* Oxalic acid peak.
Fig. 2. HPLC Chromatographic Profiles from Hydrazine Derivative (1) (Stored at 5 °C for 6 Months under Nitrogen in a Polyethylene Bag)
* Oxalic acid peak.
hydrazine to hydrazone, and subsequent hydrolysis to ketone. standard sample.
To confirm this dimer formation, 1 was mixed with 9 in an
The main decomposition compounds 3 (0.71%) and 4
CH3CN–H2O (1 : 1) solution, since 9 was only detectable (0.96%) could not be elucidated by LC/MS since m/z peak
under accelerated decomposition conditions (Fig. 5). Fur- intensities were too low and complex following chromato-
thermore, the benzyl alcohol (10) (0.33%), which could not graphic separation of the crude decomposed hydrazine mix-
be observed by LC-MS was determined by co-injecting a ture. Following purification by preparative HPLC, approxi-

348
Vol. 51, No. 3
mately 1 mg of sample was obtained, which was used for (CD3CN–D2O) of 3, which indicated two methylene groups
1
NMR studies (Fig. 4). H-NMR (C6D6) of 4 indicated two attached to oxygen [d 3.55 (dd, Jϭ11.3, 5.1 Hz, 2H), 3.58
methylene groups attached to oxygen [d 3.43 (d, Jϭ5.4 Hz, (dd, Jϭ11.3, 4.6 Hz, 2H)], one oxymethine proton [d 4.12 (tt,
4H)], one oxymethine proton [d 4.00 (tt, Jϭ5.4, 5.4 Hz, 1H, Jϭ5.1, 4.6 Hz)], and two benzyloxy groups [d 4.46 (s,
1H)], and two benzyloxy groups [d 4.26 (s, 4H), 7.0—7.2 4H), 7.1—7.3 (m, 10H)], which were similar to those of al-
(m, 10H)]. Compound 4 was determined to be the secondary cohol (4). Compound 3 was determined to be the peroxide
alcohol [m/z 273 (MϩH)ϩ], however, 3 decomposed after derivartive.4) Furthermore, the peroxide was extracted with
freeze drying. Therefore, LC-NMR was employed to eluci- deuterated benzene. This sample was subsequently used for
date the structure of 3 [m/z 289 (MϩH)ϩ]. 1H-NMR 1H-NMR experiments [d 3.61 (dd, Jϭ10.7, 4.7 Hz, 2H), 3.64
(dd, Jϭ10.7, 5.6 Hz, 2H), 4.40 (tt, Jϭ5.6, 4.7 Hz, 1H), 4.33
(s, 4H), 7.0—7.2 (m, 10H)].
Factors Promoting Decomposition The effect of oxy-
gen on the decomposition of the protected hydrazine diol (1)
hemi-oxalate, which was kept in the dark, in open air and
heated at 60 °C for 1 week was examined. As shown in Fig.
5, accelerated decomposition of 1 was observed under these
conditions, with increased levels of each decomposition
compound (0.01—0.96 % vs. 0.01—3.56%). To confirm
our findings, 1 was stirred vigorously for several days in
MeOH/H2O (1/1) in open air at 25 °C. As expected, acceler-
ated decomposition of 1 was observed (data not shown).
Chemical Conversion Chemical conversion of the hy-
Fig. 3. Structures of Decomposition Compounds
drazine derivative (1) to the peroxide (3) and the peroxide (3)
to the secondary alcohol (4) was performed (Chart 2). The
conversion of 1 to 3 (76%) was accomplished by reacting 1
with 30% H2O2 in MeOH/H2O (1/1) for 14 h at 25 °C. Unre-
Fig. 4. 500 MHz 1H-NMR Spectra of 3 and 4 in C6D6
The LC-NMR 1H-NMR spectrum of 3 in CD3CN–D2O is also shown.
Chart 2. Chemical Conversion of Hydrazine (1) and Peroxide (3)
Fig. 5. HPLC Chromatographic Profiles from Hydrazine Derivative (1) (Stored at 60 °C for 1 Week under Open Air)
* Oxalic acid peak.

March 2003
349
Fig. 6. Proposed Decomposition Mechanism of the Hydrazine Derivative (1)
acted hydrazine derivative (1) (10%) was recovered and a by- compound (8) as the minor decomposition compounds, is
product (10%), whose structure was not elucidated, were shown in Fig. 6. The removal of all traces of oxygen and/or
present. The conversion of 3 to 4 (100%) was accomplished H2O from powdered materials is very difficult. Therefore, re-
by reacting 3 with saturated Na2S2O3 in MeOH/H2O (1/1) for maining amounts of oxygen and/or H2O can be reduced to
1 h at 25 °C. The reactions were monitored by LC-MS and/or form numerous radicals [e.g. hydrogen peroxide radicals
1H-NMR.
(HOO ) and/or hydroxy radicals (HO )] and initiate a decom-
· ·
Radical Formation and Stability of Hydrazines Light, position cascade, i.e. conversion of 1 to 3 and subsequently
heat and trace amounts of iron, copper, aluminum, platinum to the stable secondary alcohol (4), and 1 to 9 and subse-
and other transition metals reduce molecular oxygen to su- quently to the coupling compound (8).
Ϫ
·
peroxide (O2 ) and promote hydrogen peroxide radical
·
(HOO ) formation and water homolysis, to yield hydroxy Conclusions
7)
·
radicals (HO ). The reaction of hydrazines, which are
Hydrazine, which is known to be an unstable compound
known to be reactive compounds, with hydroxyl radical was found to decompose when stored at 5 °C for 6 months
2—6)
·
(HO ), hydrogen peroxide (H2O2), O3 and O2 gas
results under nitrogen in a polyethylene bag and inside a steel drum.
in the formation of hydrazones, diazenes, NH3, N2, alkyl rad- Our studies revealed that the hydrazine derivative (1) easily
icals, and/or amines.2,3,5) In this study, the presence of a small oxidized in the presence of oxygen and/or H2O generated
amount of molecular oxygen and/or H2O initiated a chain re- radicals to give the peroxide (3) and secondary alcohol (4)
action with the formation of radical species, although it is not derivatives. A degradation pathway was proposed, which was
clear which radical species were generated. However, we can supported by chemical conversion experiments. As a result
assume that the hydrazine derivative (1) was decomposed to of these studies, the protected hydrazine diol (1) hemi-ox-
Ϫ
·
the peroxide derivative (3) by superoxide (O2 ), and/or hy- alate is presently stored at temperatures below Ϫ20 °C but
·
·
drogen peroxide (HOO ) and/or hydroxyl radicals (HO ), not under N2 gas. To date, after 9 months storage, no decom-
which also catalyze homolytic cleavage of the N–N bond to position has been observed.
give the secondary amine (6).2)
Acknowledgments The authors wish to thank Drs Takashi Araki and
Decomposition Mechanism The proposed decomposi-
tion mechanism of 1, to give the peroxide (3) and secondary
Asayuki Kamatani, and Miss Makiko Kanamaru for analytical support.
alcohol (4) derivatives as the main decomposition com-
pounds, and a ketone derivative (9), hydrazine, and a dimer
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