An efficient synthesis of O,O- di propyl (E)-2-[1-methyl 2-oxopropylidene]phosphorohydrazidothiolate (E) oxime and its analogues: A potential marine toxin

Article abstract of DOI:10.1080/10426500701796249

An efficient method for the synthesis of Ptychodiscus brevis toxin O,O- di n-propyl (E)-2-[1-methyl 2-oxopropylidene]phosphorohydrazidothiolate (E) oxime (TG-1) and its analogues has been developed using thermally stable and recyclable silica gel and Na2SO4 as a condensing agent and water scavenger, respectively. The compounds were evaluated against fish Rasbora daniconius by determining the LC50 and LC90 values. The results of biological evaluation showed that these compounds have high degree of toxicity. Copyright Taylor & Francis Group, LLC.

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An efficient synthesis of O,O-
Di Propyl (E)-2-[1-methyl 2-
oxopropylidene]phosphorohydrazid
(E) Oxime and Its Analogues: A
Potential Marine Toxin
Arvind K. Gupta a , D. K. Dubey a , B. D. Parashar a &
M. P. Kaushik a
a Process Technology Development Division ,
Defence R & D Establishment , Gwalior, India
Published online: 28 Jul 2008.
To cite this article: Arvind K. Gupta , D. K. Dubey , B. D. Parashar & M. P.
Kaushik (2008) An efficient synthesis of O,O- Di Propyl (E)-2-[1-methyl 2-
oxopropylidene]phosphorohydrazidothiolate (E) Oxime and Its Analogues: A Potential
Marine Toxin, Phosphorus, Sulfur, and Silicon and the Related Elements, 183:8,
1892-1910, DOI: 10.1080/10426500701796249
To link to this article: http://dx.doi.org/10.1080/10426500701796249
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Phosphorus, Sulfur, and Silicon, 183:1892–1910, 2008
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500701796249
An efficient synthesis of O,O- Di Propyl (E)-2-[1-methyl
2-oxopropylidene]phosphorohydrazidothiolate (E) Oxime
and Its Analogues: A Potential Marine Toxin
Arvind K. Gupta, D. K. Dubey, B. D. Parashar,
and M. P. Kaushik
Process Technology Development Division, Defence R & D
Establishment, Gwalior, India
An efficient method for the synthesis of Ptychodiscus brevis toxin O,O- di n-propyl
(E)-2-[1-methyl 2-oxopropylidene]phosphorohydrazidothiolate (E) oxime (TG-1)
and its analogues has been developed using thermally stable and recyclable silica
gel and Na2 SO4 as a condensing agent and water scavenger, respectively. The com-
pounds were evaluated against fish Rasbora daniconius by determining the LC50
and LC90 values. The results of biological evaluation showed that these compounds
have high degree of toxicity.
Keywords Fish toxin; phosphoric acid hydrazides; ptychodiscus brevis; thiophosphoric
acid hydrazide
INTRODUCTION
Ptychodiscus brevis (Gymnodinium breve) is a marine dinoflagellate,
which is the cause of massive fish kills, mollusk poisoning, and human
food poisoning along the Florida coast and in the Gulf of Mexico. This
class of marine toxins have attracted the attention of organic chemists
due to their involvement in human intoxication and socioeconomic im-
pact brought by those incidents.1 Several attempts have been made to
isolate the toxins from the cultured cells; however, discrepancies ex-
ist in the reported physical properties.1–5 Presumably, the main rea-
son is the difficulty associated with the separation and purification
of the toxin mixture. Elucidation of the chemical formula is impera-
tive not only for the understanding the molecular basis of mechanism
of action but for the design of proper defensive countermeasures such
Received 28 August 2007; accepted 18 September 2007.
Address correspondence to M. P. Kaushik, Process Technology Development Divi-
sion, Defence R & D Establishment, Jhansi Road, Gwalior 474002 (MP) India. E-mail:
mpkaushik@rediffmail.com
1892

A Potential Marine Toxin
1893
FIGURE 1 O,O-Dipropyl(E)-2-[1-methyl 2-oxopropylidene] phosphorohydra-
zidothiolate(E)oxime (TG-1) and O,O-Diphenyl N-cyclooctylphosphoramidate
(PB-1).
as detection, protection, and decontamination methods, as well.6 Over
the past decade, phosphorus-containing Gymnodinium breve toxins,
such as O,O- di n-propyl (E)-2-[1-methyl 2-oxopropylidene] phospho-
rohydrazidothiolate (E) oxime7 (TG-1) and O,O-diphenyl, N-cyclooctyl
phosphoramidate [Ptychodiscus brevis (PB-1)], were isolated from the
dinoflagellate8 and their formula’s (Figure 1) were established on the
basis of X-ray crystallography.
In order to study the structure–activity relationship, there was a
need to synthesize these toxins containing phosphorus moiety. More-
over, the synthesis of P=O analogues of TG-1 is also not reported in
the literature. Due to their unusual and unique formula, and their as-
sociation with high degrees of activity, much attention was focused to
study their biological aspects, only.9 The non availability of TG-1 from
the natural sources and biological activity shown by this compound
prompted us to develop new strategy for the synthesis of TG-1 and its
analogues. It is noteworthy to mention that P=O derivatives gener-
ally are more toxic than P=S derivatives, as has already been observed
in case of paraoxane and parathion.10 A literature search reveals that
there has been little attention towards synthetic studies of TG-1 and
its analogues. To the best of our knowledge, there is only one report
for the synthesis of TG-1 using azeotropic distillation.7b This method,
however, suffers from drawbacks such as use of carcinogenic solvent,
long reaction times, requires chromatographic technique for purifica-
tion, and gives rise to three minor unidentified impurities that reduced
the yield of the desired product.
RESULTS AND DISCUSSION
Retrosynthetic analysis revealed many synthetic routes, however, the
most convenient and logical synthetic method for the synthesis of target
compound (TG-1) is depicted in Scheme 1.
TG-1 was synthesised by condensing the phosphorahydrazide with
the corresponding diketone monoxime (Scheme 2).

1894
A. K. Gupta et al.
SCHEME 1
The intermediates 1–4 were synthesized by the reported
methods.10–13However Step 4 of Scheme 2 appears simple, ap-
parently, but it requires a chemoselective condensation of butane
2,3-dionemono (E) oxime and thiophosphoric acid hydrazide; Although
a variety of reagents are available to transform carbon-nitrogen
double bond.14–18 However, the chemistry of condensation of carbonyl
group with NH2-Y (Y=OH, NH, NHCONH2, etc.) reveals that an
additional elimination reaction in which more basic nitrogen atom
of amine adds to the carbonyl moiety and results in the formation of
SCHEME 2 Synthesis of Tg1 (5) via use of thiophosphorichydrazides and
butane 2,3 dione monoxime. Reaction Conditions: 1, 5:1 mole ratio; benzene,
90◦C, refluxed till P2S5 dissolved, under N2 atmosphere,3–4 h 2, solvent-CCl4,
temp.0◦C, under N2 atmosphere; 2–4 h 3, mole ratio1:2; solvent-ethanol; temp.
0◦C followed by r.t., 12 h; 4, mole ratio 1:1: 1; acetonitrile, 2–3 h.

A Potential Marine Toxin
1895
tetrahedral intermediate which gave carbon-nitrogen compounds after
the elimination of water.13 Furthermore, the removal of water is a
reversible process and needs to be removed by azeotropic distillation.19
Molecular sieves, dehydrating solvents, or the use of acid catalysts
have been reported to facilitate such kind of reactions, as well.20 The
transformation of C=N double bond is challenging in TG-1 type of
molecules essentially because of sensitive functionalities present in the
molecule. These compounds are prone to Beckmann rearrangement
due to the presence of free oximino functionality and/or can also
undergo cyclization reaction through hydrazimino group. This is
likely the reason that limited attempts have been made so far for the
synthesis of TG-1 and its analogues. Recently, the use of insoluble
inorganic materials as a condensing and water scavenger has received
significant interest in organic chemistry. These heterogeneous reac-
tions have advantages over the homogenous reactions due to ease of
set-up and work-up, reaction time, high yields, and greater selectivity.
Na2SO4–silica has been reported as condensing agent and as water
scavengers for dehydration of alcohols.21 In spite of the availability of
various synthetic methods for transformation of C=N double bond, no
attempt has been made to synthesize O, O-dialkyl, (E)-2-[1-methyl 2-
oxopropylidene]phosphorohydrazidothiolate (E) oxime, and analogues
by using Na2SO4-silica as a condensing agent and as water scavengers.
In continuation of our recent work,22 we report a new method for the
synthesis of (TG-1) and its analogues via chemoselective condensation
of thiophosphoric acid hydrazide and alkane dionemono (E) oxime
using Na2SO4-silica resulting high yields of (TG-1) Scheme 2. This
method efficiently afforded the TG-1 and its derivatives and allowed
us to obtain excellent yields of the products in reduced reaction
time. The condensing agent was prepared by mixing sodium sulfate
solution with chromatographic silica gel and evaporating the water
under reduced pressure by heating at 150◦C .The condensing agent
was dried at 200◦C in a vacuum oven for 2 h (see Experimental
section).
Initially, the reaction conditions were optimized by carrying out
the reactions of diethyl thiophosphoric acid hydrazide with acetone as
model reaction in the presence of different condensing agents such as
silica, sodium sulfate, CuSO4, ZnSO4. SiO2- Na2SO4, symctone clay,
montmorillonite KSF clay, kiesel gel, SiO2, Celite 521, kiesal gel, and
activated carbon under various reaction conditions using different
solvents. The reaction was monitored by TLC and GC to find out the
consumption of diethyl thiophosphoric acid hydrazide and formation
of the corresponding hydrazone (O,O-diethyl isopropylidenehydrazino
thiophosphate). The result of this analysis showed that combination

1896
A. K. Gupta et al.
TABLE I Optimization of Conditions for the Synthesis of O,
O–Diethyl Isopropylidenehydrazino Thiophosphate
Entry Condensing agent
Solvent
Mole (%)
Yielda(%)
1.
2.
3.
Nil
SiO2
Na2SO4
Symctone clay
benzene
nil
100
100
10
50b
57
58
acetonitrile
acetonitrile
acetonitrile
4.
14
5.
6.
7.
8.
Montmorillonite KSF clay acetonitrile
10
10
10
10
10
50
75
100
120
100
100
100
23
SiO2- Na2SO4
Kiesel gel
Celite521
Activated carbon
SiO2- Na2SO4
SiO2- Na2SO4
SiO2- Na2SO4
SiO2- Na2SO4
ZnSO4
acetonitrile
acetonitrile
acetonitrile
DCM
acetonitrile
acetonitrile
acetonitrile
acetonitrile
acetonitrile
acetonitrile
acetonitrile
86c
52
16
NIL
26
68
86
9.
14.
15.
16.
17.
18.
19.
20.
89
34d
27d
36e
CuSO4
SiO2- Na2SO4
aIsolated yield; bDean- Stark receiver was used and reaction was found incomplete in
2 h; cin 31PNMR, a signal of diethyl thiophosphoric acid hydrazide (δ 73.20)
disappeared, and a new signal at δ 68.43 appeared in entry 6; da metal complex formed
within 30–45 min; and ethe reaction mixture was refluxed up to 10 h.
of SiO2- Na2SO4 gave best results. However, the mole ratio of
SiO2-Na2SO4 plays a crucial role in increasing the yield of products.
Equimolar ratio of anhydrous SiO2-Na2SO4 was found an ideal choice
as compared to others in terms of conversion and reaction time (Table
I). It was also observed that all the reactions reached completion within
3–4 h when anhydrous SiO2-Na2SO4 was used under reflux conditions.
Extended reaction time did not have any significant change in the yield
of products (Table I). The effects of solvent were also studied by using
various solvents like THF, dioxane, acetonitrile, ethanol, diethyl ether,
DCM, chloroform, and CCl4; we observed that acetonitrile afforded
the best results. Reactions were monitored by TLC, GC, and 31P NMR
at 162 MHz using CDCl3. To explore the generality and scope of
this condensing agent, various aldehydes and ketones with diverse
structure were condensed with diethyl phosphoric hydrazides and the
results are summarized in Table II.

A Potential Marine Toxin
1897
Before applying the optimized reaction conditions for synthesis of
TG-1 and its analogues, chemo-selectivity of the method was exam-
ined by performing two sets of control experiments under identical
reaction conditions. In the first experiment, an equimolar mixture of
benzophenone oxime and acetone were allowed to react with diethyl
phosphoric acid hydrazide under similar reaction conditions and
monitored by TLC and GC-Ms. The results showed that acetone
reacted chemo-selectively and gave corresponding hydrazone in 83%
yield, while there was no change in the mass spectrum of benzophe-
none oxime. Further, no Beckmann rearrangement product was also
observed in IR and GC-Ms. In the second experiment, an equimolor
mixture of benzophenone oxime and butane-2,3-dionemono-(E)-oxime
were allowed to react with diethyl phosphoric acidhydrazide, the
butane-2,3-dionemono-(E)-oxime reacted chemo-selectively and gave
corresponding derivatives of TG-1. It indicated competitive condensa-
tion of butane-2,3-dionemono-(E)-oxime with diethyl phosphoric acid
hydrazide. By following the above method, a variety of thiophosphoric
acid hydrazides were reacted either with butane-2,3-dionemono-
(E)-oxime or with 1-phenyl – 2-oxopropylidene-2-(E) oxime for the
synthesis of TG-1 and its analogues, and the results are enumerated in
Table III.
Since, it is known that compounds bearing P=O groups are more
potent /active than their P=S derivatives. Therefore, to validate
the reported fact, P=O analogues were also synthesized as per
Scheme 3. The results of all the P=O derivatives are summarized in
Table IV.
It is evident from Tables III and 4 that change in alkyl chain length
in phosphorohydrazides influences the reaction time (entry 5a–5h and
8a–8l) to some extent. However, it was also observed that to butane-2, 3-
dionemono (E) oxime reacted faster than 1-phenyl – 2-oxopropylidene-
2-(E)-oxime. This is probably due to resonance of phenyl ring, which
might reduce electrophillic nature of carbonyl carbon. The reactivity
of phosphorohydrazides and thiophosphorohydrazides were also com-
pared, the results of this study indicated that phosphoric acid hy-
drazides reacted faster compared to later one. The reaction of dianilino
phosphoric acid hydrazide was observed slowest one as it took longer
time (Table III, 8l). One of the unique features of this method is conve-
nient work-up.
Bioassay
Any assay or estimation of any compounds for their biological activity in
a particular organism or fish is defined as bioassay. All the compounds

1898

A Potential Marine Toxin
1899
TABLE III Synthesis of O,O-dialkyl-2-(E)(1-alkyl-2-oxopropylidine)
phosphorohydrazido thioate-(E)-oximea
Entry
R
Rꢀ
Time (h)
m.p. (◦ C)
31PNMR (CDCl3)
Yield (%)
5a.
5b.
5c.
5d.
5e.
5f
CH3
C2H5
CH3
CH3
CH3
CH3
CH3
C6H5
C6H5
CH3
2.00
2.15
2.30
2.30
2.75
2.80
3.00
2.75
61–62
67–69
80–82
79–80
89–91
104–105
100–101
97–99
67.62
65.32
64.74
61.50
63.90
64.35
61.19
60.25
86
80
86
87
82
87
84
89
n-C3H7
i-C3H7
n-C4H9
n-C3H7
i-C3H7
i-C4H9
5g
5h
aIsolated yield; and ball the above reactions were performed by using 100 mole %
of SiO2-Na2SO4 in acetonitrile at reflux temperature. The reactions were monitored
by silica TLC and 31 P NMR.
were subjected for biological evaluation against freshwater fish R. dan-
iconius and their LC50 and LC90 were determined (see experimental).
Table V showed the LC50 and LC90 values of TG-1 and its analogues. The
results of fish toxicity are summarized in increasing order of LC50value
in Table V.
The LC50value of parent synthetic toxin TG-1 was found to be 3.25
ppm. It indicates that at given concentration 50% mortality is observed
However, out of 20 compounds, the toxicity of the first three compounds
were found to be more or less similar (i.e., 3.0–3.32 ppm). The toxicity
of seven analogues of synthetic toxin TG-1 have been observed to vary
from 6.49–9.95 ppm, while rest of the compounds showed LC50 values
ranging from 13.12–97.27 ppm (see Experimental section). The toxicity
SCHEME 3

1900
A. K. Gupta et al.
TABLE IV Synthesis of O,O-dialkyl-2-(E) (1-alkyl-2-oxopropylidine)
phosphorohydrazido–(E)-oximea
Entry
R
Rꢀ
Time (h)
m.p. (◦ C)
31PNMR (CDCl3)
Yield (%)
8a.
8b.
8c.
8d.
8e.
8f.
8g.
8h.
8i.
CH3
C2H5
CH3
CH3
CH3
CH3
CH3
C6H5
C6H5
CH3
C6H5
C6H5
C6H5
CH3
1.75
2.00
2.20
2.30
2.50
2.60
2.75
2.75
3.25
2.50
4.50
2.75
52–53
57–59
62–64
76–77
82–83
110–111
107–108
90–91
156–157
87–88
3.20
2.89
2.37
1.51
1.31
2.07
1.49
1.65
88
85
82
85
82
87
87
84
87
83
79
85
n-C3H7
i-C3H7
n-C4H9
n-C3H7
i-C3H7
i-C4H9
C6H5
C2H5
C6H5NH
C6H5
−0.65
8j.
8k.
8l.
2.24
235–237
148–150
−3.71
−0.34
aIsolated yield, All the above reactions were performed by using 10 mole % of
SiO2-Na2SO4 in acetonitrile. The reactions were monitored by silica TLC and 31
NMR.
P
of oxo analogues are in some instances slightly higher than their thio
analogues. Probably, this may be due to higher binding characteristics
of the former with the target enzyme.
CONCLUSION
In conclusion, we report a general and highly efficient method for the
synthesis of biologically active TG-1 and its P=O analogues by con-
densation of thiophosphoric acid hydrazides as well as and phosphoric
acid hydrazides in excellent yields using thermally stable condensing
agent (SiO2- Na2SO4). This method offers several advantages over ear-
lier reported method in terms of versatility, clean reaction, easy work
up, recyclability of condensing agent, avoids the use of column chro-
matography for purification and shorter reaction times.
EXPERIMENTAL
All the solvents were dried according to published methods and dis-
tilled before use. The reactions were carried out in oven-dried glass-
ware. Analytical thin layer chromatography (TLC) was performed on
aluminum plates with Merck Kieselgel 60F254 and visualized by UV
irradiation (254 nm) or with iodine. The purity of compounds was fur-
ther checked on a country make Chemito GC model 1000 instrument.
It was used with flame ionization detector (FID). The capillary column

A Potential Marine Toxin
1901
TABLE V Toxicity of TG-1(5 and 8) and Its Analogues
against Fresh Water Fish Rasbora Daniconius
S. no.
R
Rꢀ
X
LC50(ppm)
LC90(ppm)
1.
2.
C3H7
C3H7
CH3
CH3
O
S
3.00
3.25
4.50
5.43
3.
4.
C2H5
C2H5
CH3
CH3
O
S
3.32
6.49
5.69
9.90
5.
6.
7.
8.
CH3
CH3
C3H7
C6H5
CH3
CH3
C6H5
CH3
C6H5
CH3
O
S
O
O
S
O
S
O
S
7.06
8.13
9.60
9.68
9.71
9.95
13.12
19.98
25.48
25.53
27.74
29.01
33.28
43.62
95.99
97.27a
12.45
12.94
12.64
13.51
14.22
16.96
18.15
26.76
33.46
35.39
38.13
39.19
68.00
51.27
110.35
130.67
9.
C3H7
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20
i-C3H7
i-C3H7
i-C3H7
i-C3H7
n-C4H9
i-C4H9
C6H5
i-C4H9
C2H5
n-C4H9
C6H5NH
CH3
C6H5
C6H5
CH3
CH3
C6H5
CH3
C6H5
C6H5
C6H5NH
S
O
O
S
O
O
O
aThe compound was dissolved in DMSO.
(30 m × 0.25 mm I.D-BP5) packed with 5% phenyl and 95% dimethyl
polysiloxane (SGE) coated on fused silica was employed. The injection
port and detector block were maintained at 280◦C and 260◦C respec-
tively and the column oven was at programmed temperature profile
started at 50◦C, ramped up to 280◦C at 25◦C/min. Nitrogen was used
as a carrier gas (at a flow rate of 30 ml/min). Air for FID was supplied
at 300 ml/min and hydrogen at 30 ml/min. In all analyses, 1 µl sam-
ple was injected and peaks recorded on computerized data acquisition
station. Melting points were determined on a hot stage microscope and
are uncorrected. FT-IR spectra were recorded on Bruker FT-IR spec-
1
trometer model TensorTM 27 on KBr disk. H and 31P NMR spectra
were recorded on Bruker DPX Avance 400 MHz FT- NMR in CDCl3 or
(CD3)2SO at ambient temperature using tetramethylsilane as an inter-
1
nal standard for H and 85% H3 PO4 as an external standard for 31P

1902
A. K. Gupta et al.
NMR. Chemical shifts (δ) are given in parts per million (ppm) and cou-
pling constants (J) are given in Hertz (Hz). GC- MS data were recorded
on Varian 3400 GC coupled to a TSQ 7000 mass spectrometer (Finnigan
Mat). In order to operate GC the injector temperature 250◦C, Transfer
line temperature 280◦C, Column temperature programming 50◦C (2
min) @ 10◦C/min to 280◦C (5 min), carrier gas helium at pressure of 10
psi conditions were used. To obtain EI mass spectra Ion source pressure
1.5 × 10−6 torr, source temperature 150◦C, electron energy 70 eV and
emission current 400 µA were used as the operating conditions. To per-
form chemical ionization (CI) technique, the ion source pressure with
methane as the reagent gas 1.5×10−3 torr, source temperature 150◦C,
electron energy 100 eV and emission current 300 µA were maintained
to operate the mass spectrometer. Elemental analysis was performed
on elemental analyzer Carlo Erba Instrumantazione Model NOD1106
by using benzanilide as a reference compound.
Biological Evaluation against Freshwater Fish, R. Daniconious
To study the biological activity/toxicity of synthetic toxin TG-1, PB-1
and their various analogues as fish toxin freshwater fish Rasboradani-
conius were collected, from fresh water ponds present around Gwalior.
For each biological evaluation, 20 fish were released in enamel bowl
containing 3 L of water with different concentration of compounds
used under this study. Before performing actual experiments fish were
conditioned with non-chlorinated water under laboratory conditions
(28◦C 2◦C). For each experiment, three replicates with controls (each
of 20 fish) were used. Fish were subjected to various concentrations
of compounds continuously for 96 h. After this exposure period,
data were recorded on mortality of fish under various toxic stresses.
Data on various concentrations and corresponding mortality of fish
were subjected to probit analysis.23 For the determination of lethal
concentration for 50% and 90% mortality, data have been subjected to
determine fiducial limit for LC50 and LC90, and chi square value was
calculated to ascertain the heterogeneity among the fish population
taken for experiments. To conduct the experiment, compounds were
dissolved in ethanol and same amount of ethanol was used for control
experiments, which were also conducted simultaneously.
Preparation and Characterization of Silica- Na2SO4
Silica- Na2SO4 was prepared by combination of silica (column chro-
matography 10.0 g, 166.0 mmol) and anhydrous Na2SO4 (20.0 g,
140.0 mmol) in a mortar and pestle by grinding together until a fine,

A Potential Marine Toxin
1903
homogenous powder was obtained (10–15 min). It was mixed with 150
ml of distilled water and stirred for 1 h at room temperature and then
water was removed under vacuum using Heidolph rotary evaporator till
dryness. It was shaken with 100 ml acetonitrile, filtered, and washed
with 3 × 25 ml acetonitrile. It was further dried under vacuum at 150◦C
for 2 h and stored in a stoppard flask under desiccators. However, in or-
der to know the nature of Silica- Na2SO4, microstructural studies were
performed by scanning electron microscope (SEM). It was observed that
silica was finely and uniformly distributed on the Na2SO4.
General Procedure (5a–5h)—Typical Example
The solution of diacetylemonoxime (10.01 g, 100.0 mmol) in acetonitrile
(20 ml) was added to a suspended solution of O,O-dipropyl thiophos-
phorichydrazide (18.40 g, 100.0 mmol) and anhydrous silica-Na2SO4
(20.6 g, 100.0 mmol) in 100 ml acetonitrile. The reaction mixture was
refluxed with constant stirring and monitored periodically by TLC and
31P NMR. After complete consumption of O,O-dipropyl thiophospho-
richydrazide (4 h), the reaction mixture was cooled at room temperature
and was filtered off. The solvent was removed in rotary evaporator to
afford the desired crude product, which was re-crystallized from DCM-
ether mixture (6:4 vol/vol). Yield; (21.36g, 80%); m.p.67–69◦C. Spectral
data of newly synthesized compounds are given in the following.
5a O,O-Dimethyl,2-(1-methyl 2-Oxopropylidine)
Phosphorohydrazidothioate (E)-oxime
1H NMR (CDCl3); δ = 6.8 (S,1H,NOH, exchangeable), 5.62
(d,1H,NH,J
= 26.20Hz, exchangeable), 3.75(d, 6H, CH3JH-P =
P−H
12.21Hz), 2.31(S, 3H, CH3), 2.15(S,3H,CH3); IR(KBr disc); υ(cm−1) =
3450(-OH), 3220(NH), 2930(C-H),1660(-C=N-), 1590(NH, def), 1150,
1025, 970(P-O-C), 730(P=S); GC-MS(EI)% = 240(M+H+ 4.2), 239(M+
6.8), 222(22.0), 191(33), 160(16.5), 115(100), 98(65); Anal. calcd. for
C6H14N3O3PS: C, 30.13; H, 5.85; N, 17.57. Found: C, 30.35; H, 6.08;
N, 17.28.
5b O,O-Diethyl-2-(1-methyl-2-oxopropylidene)
phosphorohydrazidothioate-(E)-oxime
1H NMR (CDCl3); δ = 7.65 (S,1H,NOH, exchangeable), 5.69 (d, 1H,
NH,JP-H = 25.6Hz, exchangeable),4.1 (m, 4H, CH2O,JH-H = JH-P
=
=
7.05 Hz), 2.01(S,3H,CH3), 1.91(S,3H,CH3), 1.20(t, 6H, CH3,J
H-H
7.10Hz); IR KBr (disc) υ (cm−1); 3420(OH), 3245(NH), 2860(str C-H),
1625(>C=N-), 1590(N-H def), 1145, 1025(P-O-C), 735(P=S), GC-MS
(EI) m/z(%); 268(M+H+, 100), 267(M+, 7.4), 250(6.2), 184(4.2), 153

1904
A. K. Gupta et al.
(2.7), 125(5.8), 121(5.6), 114(5.3), 98 (2.8), 97(10.3); Anal. calcd. for
C8H18N3O3PS: C, 35.95; H, 6.74; N, 15.73. Found: C, 36.21; H, 6.98;
N, 15.95.
5c O,O-Dipropyl-2-(1-methyl-2-oxopropylidine)
phosphorohydrazidethioate-(E)-oxime
1H NMR (CDCl3); δ = 7.96(S,1H,NOH exchangeable), 6.75(d, 1H, NH,
J
= 26.16Hz, exchangeable), 4.09(m, 4H, CH2O, JH-P = 6.68Hz)
P-H
2.08(S,3H,CH3), 1.96 (S,3H,CH3), 1.74(m,4H,CH2, JH-H = 6.76Hz)
,
0.96(t,6H,CH3, JH-H = 6.72Hz); IR KBr disc υ (cm−1), 3410(-OH),
3225(-NH), 2850(C-H), 1630(C=N), 1595(-NH def.), 1135, 1020(P-O-C),
750(P=S); GC-MS(EI)m/z(%); 296(M+H+, 71.8), 295(M+,82.8),
278(16.2), 253(32.5), 236(12), 220(10), 211(58.9), 194(17.3), 178(24.3),
153(22.5), 139(34.7), 116(40.7), 115(78.1), 114(95.2), 98(100), 97(42.8)
Anal. calcd. for C10H22N3O3PS: C, 40.67; H, 7.45; N, 14.23. Found: C,
40.81; H, 7.75; N, 14.47.
5d O,O-Diisopropyl-2-(1-methyl-2-oxopropylidine)
phosphorohydrazido thioate (E)-oxime
1H NMR (CDCl3); δ = 7.8 (S, 1H, NOH, exchangeable), 6.02(d, 1H,
NH, JP-H = 25.96Hz, exchangeable),4.60(m, 2H, CH, JH-H = 6.68Hz),
2.08(S, 3H, CH3), 1.95(S, 3H, CH3), 1.30(d,d, 12H, CH3, J
=
H-H
6.28Hz; IR KBr disc υ (cm−1), 3400(-OH), 3215(-NH), 2860(C-H),
1635(C=N), 1590(NH def) 1125, 1030(P-O-C), 760(P=S), 296(M+H+,
3.8), 295(M+, 50.9), 278(46.9), 236(6.5), 220(54), 211(8.9), 194(27.3),
178(86.3), 153(12.5), 139(33.7), 116(10), 115(88.7), 114(85.2), 98(100),
97(28.8); Anal calcd. for C10H22N3O3PS: C, 40.67; H, 7.45; N, 142.3.
Found: C, 40.97; H, 7.98; N. 14.88.
5e O,O-Dibutyl 2-(1-methyl 2–oxopropylidene)
hydrazidothioate-E-oxime
1H NMR (CDCl3); δ = 6.94(S, 1H, NOH, exchangeable), 5.80(d, 1H,
NH, JP-H = 26.62Hz, exchangeable), 4.02(m, 4H, CH2, JH-H = JP-H
=
6.62Hz, 2.11(S,3H,CH3), 1.98(S,3H,CH3), 1.83(m, 8H,CH2) 0.97(t, 6H,
CH3, JH-H = 6.48Hz); FT-IR KBr(disc) υ (cm−1); 3440(-OH), 3250(NH),
2850(C-H), 1632(C=N), 1600(N-H def), 1025, 940(P-O-C), 750(P=S);
GC-MS(EI)m/z (%); 324(M+H+, 3.1), 323(M+, 5.8), 306(15.5), 267(18.5),
234(12), 250(10.8), 211(11)194(20.5), 178(6.5), 115(75), 98(100),97(8.5)
Anal. calcd. for C12H26N3O3PS: C, 44.58; H, 8.05; N, 13.00. Found: C,
44.25; H, 8.30; N, 13.28.

A Potential Marine Toxin
1905
5f O,O-isodibutyl,2-(1-methyloxopropylidene)
hydrazidothioate-E-oxime
1H NMR (CDCl3); δ = 7.58(S, 1H, NOH,exchangeable), 6.57(d, 1H,
NH, JP-H = 26.02Hz exchangeable), 4.03(m, 4H, CH2, JH-H = JP-H
=
6.50Hz), 2.13(S, 3H, CH3), 1.96(S, 3H, CH3), 1.88(m, 2H, CH), 1.32(d,
12H, CH3), 6.57(d, 1H, NH),; FT-IR KBr (disc) υ (cm−1); 3420(-OH),
3250(-NH), 2880(C-H), 1640(C=N), 1590(-NH def), 1140, 980(P-O-C),
760(P=S) GC-MS(EI) m/z(%); M+H+, 2.8),323(M+, 4.2), 306(12.7),
267(20.3), 234(14.5), 211(18), 194(22.6), 178(8.2), 115(71), 98(100),
97(9.7); Anal. calcd. for C12H26N3O3PS: C, 44.58; H, 8.05; N, 13.00.
Found: C, 44.88; H, 8.35; N, 13.23.
5g O,O-Dipropyl 2-(1-Phenyloxopropylidine) hydrazido
thioate (E) oxime
1H NMR (CDCl3); 7.78(S,1,NOH, exchangeable), 7.32(m,5H, C6H5),
6.52(S,1H,NH, JP-H = 26.40Hz, exchangeable), 4.12(m, 4H, CH2O,
JH-P = 6.60Hz), 1.94 (S,3H,CH3), 1.74(m,4H,CH2, JH-H = 6.76Hz)),
0.95(t,6H,CH3, JH-H = 6.70Hz); FT-IR KBr(disc) υ (cm−1);3425(-OH),
3230(-NH), 2875(C-H), 1625(C=N), 1585(-NH,def.)1125, 990(P-O-C),
745(P=S); GC-MS(EI)m/z(%) 357 (M+, 36.2), 340(3.55), 298(3.3),
273(3.5), 240 (3.5), 202 (5.8), 178 (13.8), 177(21.8), 160(34.6),104(8.2),
77(45.0); Anal. calcd. for C15H24N3O3PS: C, 50.42; H, 6.72; N, 11.76.
Found: C, 49.82; H, 6.93; N, 11.58.
5h O,O-Diisopropyl 2-(1-Phenyl-2-oxopropylidine)
hydrazidothioate-E-oxime
1H NMR (CDCl3); 7.73(S,1,NOH, exchangeable), 7.32(m,5H, C6H5),
6.67(S,1H,NH, JP-H = 26.79Hz,exchangeable), 4.25(m, 2H, CH, JH-H
=
6.88Hz),1.94(S, 3H, CH3), 1.32 (d,d,12H, CH3, JH-H = 6.28Hz);FT-
IR(KBr) disc υ (cm−1), 3400 (OH), 3210(NH), 2900 (C-H), 1640(C=N),
1590(NH def.)1120, 1050(P-O-C), 760(P=S), GC-MS (EI)m/z (%)
357(M+, 30.2), 340(5.0), 298(2.3), 273(4.5),177(18), 160(32.6),104(8.2),
77(42.7); Anal. calcd. for C15H24N3O3PS: C, 50.42; H, 6.72; N, 11.76.
Found: H, 6.98; N, 11.65.
General Procedure (8a–8l)—Typical Example
The solution of diacetylemonoxime (10.01 g, 100.0 mmol) in acetoni-
trile (20 ml) was added to a suspended solution of O,O-dialkyl phos-
phoric acid hydrazide (100.0 mmol) and anhydrous silica-Na2SO4(20.6
g, 100.0 mmol) in 100 ml acetonitrile. The reaction mixture was re-
fluxed with constant stirring and monitored periodically by TLC and
31P NMR. After complete consumption of corresponding phosphoric acid

1906
A. K. Gupta et al.
hydrazide(as per time given in Table III), the reaction mixture was
cooled at room temperature and was filtered off. The solid product was
washed with 2 × 15 ml acetonitrile. The filtrate and washings were
combined and the solvent was removed in rotary evaporator to afford
the desired crude product, which was triturated with ether to give solid
white product. Spectral data of newly synthesized compounds are given,
following.
8a O,O-Dimethyl,2-(1-methyl oxopropylidine)
Phosphorohydrazido(E)-oxime
1H NMR (CDCl3); δ = 8.41 (S,1H,NOH, exchangeable),
6.20(d,1H,NH, JP−H = 25.20Hz, exchangeable), 3.80(d, 6H, CH3
JH-P = 12.0Hz), 2.32 (S, 3H, CH3), 2.21(S,3H,CH3); IR(KBr disc);
υ(cm−1) = 3470 (-OH), 3270(NH), 2925(C-H),1640(-C=N-), 1570(NH,
def), 1255(P=O), 1045,960 (P-O-C),; GC-MS(EI)% = 224(M+H+ 6.2),
223(M+ 7.8), 206(27), 193(5.3), 163(18.5), 115(100), 109(31) 98(65);
Anal. calcd. for C6H14N3O4P: C, 32.28; H, 6.27; N, 18.83. Found: C,
32.05; H, 6.58; N, 18.58.
8b O,O-Diethyl-2-(1-methyl-2-oxopropylidene)
phosphorohydrazido-(E)-oxime
1H NMR (CDCl3); δ = 8.15 (S,1H,NOH, exchangeable), 5.75 (d, 1H,
NH,J
= 26.26Hz, exchangeable),4.21(m,4H,CH2O,JH-H = JH-P
=
=
P-H
7.0Hz), 2.10(S,3H,CH3), 2.01(S,3H,CH3), 1.23(t, 6H, CH3, JH-H
7.0Hz); IR KBr (disc) υ (cm−1); 3425(OH), 3255(NH), 2870(str C-
H),1635(>C=N-),1580(N-Hdef),1245(P=O),1035,950(P-O-C), GC-MS
(EI) m/z(%); 252(M+H+, 7.3), 251(M+, 3.4), 234(6.62), 206(24.2),
195(11.20, 178 (32.7), 115(85.3), 98 (42.8); Anal. calcd. for C8H18N3O4P:
C, 38.24; H, 7.17; N, 16.73. Found: C, 37.15; H, 6.98; N, 16.95.
8c O,O-Dipropyl-2-(1-methyl-2-oxopropylidine)
phosphorohydrazide-(E)-oxime
1H NMR (CDCl3); δ = 7.59(S,1H,NOH exchangeable), 6.15(d, 1H, NH,
JP-H = 26.40Hz, exchangeable), 4.20(m,4H,CH2O,JH-P = 7.08Hz), 2.18
(S,3H,CH3), 1.98(S,3H,CH3), 1.75 (m,4H,CH2,JH-H = 6.97Hz), 0.96
(t,6H,CH3,JH-H = 6.87Hz);IR KBr disc υ(cm−1), 3425(-OH), 3230(-NH),
2860(C-H), 1635(C=N), 1585(-NHdef.), 1235(P=O), 1020,940(P-O-C),
GC-MS(EI)m/z(%); 280(M+H+,12.28), 279(M+,8.8), 264(18.42),
262(9.32), 237(22),220(15.40), 195(16.73), 115(34.7),98(80); Anal.
calcd. for C10H22N3O4P: C, 43.07; H, 7.88; N, 15.05. Found: C, 43.40;
H, 8.02; N, 15.24.

A Potential Marine Toxin
1907
8d O,O-Diisopropyl-2-(1-methyl-2-oxopropylidine)
phosphorohydrazido (E)-oxime
1H NMR (CDCl3);δ = 7.58(S,1H,NOH, exchangeable),6.10(d,1H,NH,
JP-H = 25.76Hz, exchangeable), 4.65(m,2H,CH,JH-H = 6.65Hz), 2.09
(S,3H, CH3),1.96 (S,3H,CH3), 1.32 (d,d,12H,CH3,JH-H = 6.45Hz;
IR KBr disc υ (cm−1),3415(-OH),3225(-NH),2860(C-H), 1625(C=N),
1575(NHdef),
1245(P=O),1030,950(P-O-C),GC-MS(EI)m/z(%);280
(M+H+,4.8), 279 (M+,24.4), 264(2.5), 237(16), 222(7.9), 195(37.3),
178(26.3), 137(12.85), 115(15.7), 114(12.2), 99(10.30), 98(100); Anal.
calcd. for C10H22N3O4P: C, 43.07; H, 7.88; N, 15.05. Found: C, 42.95;
H, 7.68; N, 14.91.
8e O,O-Dibutyl 2-(1-methyloxoprophlidine) hydrazido-E-oxime
1H NMR (CDCl3); δ = 7.54(S, 1H, NOH, exchangeable), 6.08(d, 1H,
NH, JP-H = 25.74Hz, exchangeable), 4.22(m, 4H, CH2, JH-H = JP-H
=
6.82Hz, 2.10(S,3H,CH3), 1.98(S,3H,CH3), 1.85(m, 8H,CH2) 0.97(t, 6H,
CH3, JH-H = 6.78Hz); FT-IR KBr(disc) υ (cm−1); 3440(-OH), 3260(NH),
2850(C-H), 1625(C=N), 1580(N-H def), 1250(P=O) 1035, 950(P-O-C),;
GC-MS(EI)m/z(%); 308(M+H+,6.7), 307(M+, 3.8), 290(13.5),251(15.8),
234(14), 195(22.25),115(45),98(87), Anal. calcd. for C12H26N3O4P: C,
46.90; H, 8.46; N, 13.68. Found: C, 46.75; H, 8.72; N, 13.80.
8f O,O-Dipropyl 2-(1-phenyl oxopropylidine)
hydrazido (E) oxime
1H NMR (CDCl3); 7.78(S,1,NOH, exchangeable), 7.43(m,5H, C6H5),
6.42(S,1H,NH, JP-H = 25.74Hz, exchangeable), 4.32(m, 4H, CH2O,
JH-P = 6.76Hz), 1.95 (S,3H,CH3), 1.75(m,4H,CH2, JH-H = 6.75Hz)),
0.97(t,6H,CH3, JH-H = 6.70Hz); FT-IR KBr(disc) υ (cm−1);3425(-OH),
3240(-NH), 2900(C-H), 1625(C=N),1575(-NH,def.)1245(P=O),1025,
950(P-O-C); GC-MS(EI)m/z(%) 342(M+H+, 15.94) 341(M+, 26.2),
299(13), 257(23.5), 256(23.5), 240(12.18), 178(3.8), 177(12.8), 176(20.6),
160(100), 130(25.0), 125(17), 105(15.5), 104(18.5), 103(27), 94(98),
77(29), Anal. calcd. for C15H24N3O4P: C, 52.79; H, 7.03; N, 12.32.
Found: C, 52.58; H, 6.90; N, 12.08.
8g O,O-Diisopropyl 2-(1-phenyl-2-oxopropylidine)
hydrazido-E-oxime
1H NMR (CDCl3); 7.87(S,1,NOH, exchangeable), 7.38(m,5H,C6H5),
6.37(S,1H,NH,JP-H = 26.12Hz, exchangeable), 4.24(m,2H,CH,JH-H
=
6.78Hz), 1.95(S,3H,CH3),1.33 (d,d,12H,CH3,JH-H = 6.20Hz); FT-IR
(KBr) discυ(cm−1),3420(OH),3230(NH),2900(C-H), 1628(C=N), 1580
(NHdef.), 1248(P=O), 1050, 930(P-O-C), GC-S(EI)m/z(%) 342(M+H+,
8.64) 341(M+,14.28), 299(9.24), 257(21.55), 256(28.5), 240(12.18),

1908
A. K. Gupta et al.
177(10.50), 160(86), 130(21.0), 125(11.75), 105(9.5), 104(12.5), 94(98),
77(11.50), Anal.calcd. for C15H24N3O4P: C, 52.79; H, 7.03; N, 12.32.
Found: C, 53.02; H, 7.23; N, 12.48.
8h O,O-isodibutyl,2-(1-methyloxopropylidene)
hydrazido-E-oxime
1H NMR (CDCl3); δ = 7.58(S, 1H, NOH, exchangeable), 6.69(d,
1H, NH, JP-H = 25.76Hz exchangeable), 6.57(d, 1H, NH), 4.23(m,
4H, CH2, JH-H = JP-H = 6.50Hz), 2.13(S, 3H, CH3), 1.99(S, 3H,
CH3), 1.81(m,2H,CH), 1.30(d, 12H, CH3),; FT-IR KBr (disc) υ (cm−1);
3420(-OH), 3250(-NH), 2880(C-H), 1640(C=N), 1590(-NH def), 1140,
980(P-O-C), GC-MS(EI)m/z (%); 308(M+H+, 15.14), 307(M+, 8.13),
290(34.25),251(19.25), 234(22.87), 195(17.22),115(39.62),98(77), Anal.
calcd. for C12H26N3O4P: C, 46.90; H, 8.46; N, 13.68. Found: C, 47.15; H,
8.32; N, 13.48.
8i O,O-Diphenyl 2-(1-phenyl-2-oxopropylidine)
hydrazido-E-oxime
1H NMR (CDCl3); δ = 7.86(s,1H,NOH, exchangeable)), 7.5(m,15H,
C6H5), 1.9(s,3H,CH3), 6.32(S,1H,NH, JP-H = 25.74Hz, exchangeable),);
FT-IR KBr (disc) υ (cm−1); 3420(-OH), 3250(-NH), 2880(C-H),
1640(C=N), 1590(-NH def), 1240(P=O), 1030,930(P-O-C),; GC-MS(EI)
m/z(%) 409(M+, 1.54),392(1.28), 249(3.54), 248(2.1), 235(1.75),
170(1.50), 160(26), 159(63), 130 (15.75), 118(22.5) 105(22.5), 104(19),
103(27), 94(100), 77(29.4), Anal. Calcd for C21H20N3O4P: C, 66.61; H,
4.88; N, 10.26. Found: C, 61.75; H, 5.00; N, 10.05.
8j O,O-Diethyl-2-(1-phenyl-2-oxopropylidene)
phosphorohydrazido-(E)-oxime
1H NMR (CDCl3); δ = 8.12 (S,1H,NOH, exchangeable), 7.38(m,5H,
C6H5), 5.75 (d, 1H, NH,J
= 26.18Hz, exchangeable),4.28 (m, 4H,
P-H
CH2O,JH-H = JH-P = 7.0 Hz), 1.99(S,3H,CH3), 1.24(t, 6H, CH3,J
=
H-H
7.0Hz); IR KBr (disc) υ (cm−1); 3440(OH), 3260(NH), 2880(str C-H),
1625(>C=N-), 1570(N-H def), 1255(P=O), 1035,940(P-O-C), GC-MS
(EI) m/z(%); 314(M+H+, 17.3), 313(M+, 8.4), 296(12.60), 285(20.2),
287(7.20), 257(22), 240(7.75), 177 (82.7), 178(25.13), 160 (12.8), 159(63),
130 (15.75), 118(22.5) 105(22.5), 104(19), 103(27), 94(100), 77(29.4);
Anal. calcd. for C13H20N3O4P: C, 49.84; H, 6.38; N, 13.42. Found: C,
50.12; H, 6.78; N, 13.29.
8k O,O-Dianilino-2-(1-phenyl2-oxopropylidene)
phosphorohydrazido-(E)-oxime
1H NMR (DMSO-d6); δ = 11.36(s,1H,NOH, exchangeable)),
8.51(d,2H, C6H5NH, J = 9Hz), 7.98(d,1H,NH, JP−H = 25.34Hz),
7.3–6.27(m, 15H, Ar), 5.78(S,1H,NH, J P−H = 25.34Hz, exchangeable),);

A Potential Marine Toxin
1909
FT-IR KBr (disc) υ (cm−1); 3420(-OH), 3360, 3150(-NH), 2870(C-
H), 1635(C=N), 1590(-NH def), 1235P=O), 1030,930(P-O-C),;
Mass(D.P.)(EI)m/z(%) 408(M+, 3.74),407(M+,2.1), 390(6.78), 330(2.54),
248(2.1), 315(14.75), 253(18.7),223(2.3), 177(12.50), 160(20.8),
94(90),93(48), 77(22.4), Anal. calcd. for C21H22N5O2P: C, 61.91;
H, 5.40; N, 17.19. Found: C, 61.78; H, 5.68; N, 17.40.
8l O,O-Diphenyl-2-(1-methyl-2-oxopropylidene)
phosphorohydrazido-(E)-oxime
1H NMR (CDCl3); δ = 7.83(s,1H,NOH, exchangeable)), 7.45(m,10H,
C6H5), 6.32(S,1H,NH, J
= 25.63Hz, exchangeable), 2.10(S, 3H,
P-H
CH3), 1.93(s,3H,CH3); FT-IR KBr (disc) υ (cm−1); 3420(-OH), 3250(-
NH), 2880(C-H), 1640(C=N), 1590(-NH def), 1240(P=O), 1030, 930(P-
O-C); GC-MS(EI)m/z(%)348(M+H+,24), 347(M+,16.5), 330(21.54),
254(13.54), 248(2.1), 233(8.75), 132 (15.75), 114(6.22) 98(30), 94(100),
77(12), Anal. calcd. for C16H18N3O4P: C, 55.33; H, 5.18; N, 12.10. Found:
C, 55.10; H, 5.00; N, 12.38.
REFERENCES
[1] (a) Y. Shimizu, Marine Natural Products, P.J. Scheuer, Ed. (Academic Press, New
York, 1978), vol. 1, chap. 1; (b) A. Abdelghani, W. R. Haetley, F. R. Esmundo, and T.
F. Harris, Biological and chemical contaminants in the Gulf of Mexico and the po-
tential impact on public health: a characterization report (Tulane University School
of Public Health and tropical medicine, New Orleans, LA, 1994), p. 77.
[2] (a) J. J. Spikes, S. M. Ray, D. V. Aldrich, and J. B.Nash, Toxicon., 5, 171 (1968).
[3] G. M. Padilla, Y. S. Kim, E. J. Rauckman, and G. M. Rosen, Toxic Dinoflagel-
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