Spiro- and substituted tetrahydrobenzo[b]thiophene-triazaphospholes and phosphoramidates as potent antineoplastic agents: Synthesis, biological evaluation, and SAR studies

Article abstract of DOI:10.1007/s00706-015-1542-4

New and efficient conjugate addition reaction of trimethyl, triethyl, and triisopropyl phosphites with 2-azido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile was developed and furnished spiro-triazaphosphole-oxide derivatives in ≈70 % yield. Contrary to these results, linear substituted phosphoramidates were obtained from the reaction of the azide with dimethyl, diethyl, and diisopropyl phosphites. N-Alkylamino- or 2-aminotetrahydrobenzo[b]thiophene-3-carbonitrile was also isolated in ～10 % yield from the previous two reactions. In the context of this work, hexaalkylphosphorus triamides were caused to react with the same substrate afforded the corresponding phosphoric triamides only in the presence of a protonating agent (dil. alcohol). The three reactions proceeded smoothly, cleanly, and were completed within ～6 h at r.t. Pharmacological evaluation results of antibreast, anticolon, and antiprostate carcinoma cell lines properties for the products were discussed in terms of structure-activity relationship to define a chromophore for lead compounds.

Full text of DOI:10.1007/s00706-015-1542-4

Monatsh Chem
DOI 10.1007/s00706-015-1542-4
ORIGINAL PAPER
Spiro- and substituted tetrahydrobenzo[b]thiophene-
triazaphospholes and phosphoramidates as potent antineoplastic
agents: synthesis, biological evaluation, and SAR studies
Wafaa M. Abdou1 Neven A. Ganoub1 Eman Sabry1
•
•
Received: 27 April 2015 / Accepted: 22 July 2015
Ó Springer-Verlag Wien 2015
Abstract New and efficient conjugate addition reaction of
trimethyl, triethyl, and triisopropyl phosphites with 2-azido-
Keywords Trialkyl phosphites Á Dialkyl phosphites Á
Azides Á Triazaphospholes Á Phosphoramidates Á
Anticancer agents
4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile
was
developed and furnished spiro-triazaphosphole-oxide
derivatives in &70 % yield. Contrary to these results, linear
substituted phosphoramidates were obtained from the reac-
tion of the azide with dimethyl, diethyl, and diisopropyl
Introduction
phosphites.
N-Alkylamino-
or
2-aminotetrahy-
The search for biologically promising new chemical enti-
ties against the deadly cancer disease remains an important
theme in drug discovery [1]. The preliminary cancer
treatment options are usually a combination of surgery,
radio-, and chemotherapy. The latter one is considered one
of the effective approaches in suppressing tumor growth
and eradication of tumors. However, many patients, who
undergoing chemotherapy, suffer from associated serious
side effects emphasizing the need to new anticancer agents
with improved efficacy and reduced side effects are still
needed. Generally, cancers of the breast, lung, colon, and
prostate are the most frequent types of cancer worldwide
[1–3]. This paper along with our recent efforts focuses on
developing, identifying, and optimizing new entities of
anticancer properties [4–10].
drobenzo[b]thiophene-3-carbonitrile was also isolated
in *10 % yield from the previous two reactions. In the
context of this work, hexaalkylphosphorus triamides were
caused to react with the same substrate afforded the corre-
sponding phosphoric triamides only in the presence of a
protonating agent (dil. alcohol). The three reactions pro-
ceeded smoothly, cleanly, and were completed within *6 h
at r.t. Pharmacological evaluation results of antibreast,
anticolon, and antiprostate carcinoma cell lines properties
for the products were discussed in terms of structure–activity
relationship to define a chromophore for lead compounds.
Graphical abstract
Organophosphonates, over the last three decades, have
become notably recognized for their pharmacological
antiinflammatory [11, 12] and anticancer activity [13, 14],
especially when associated with various heterocycles [15].
One of the most important of these bioactive heterocycles
is the triazoles, which have not been widely reported in the
literature [16, 17]. Also, phosphoramidates in particular
constitute an important class of compounds that attracts
medicinal chemists because of their wide use in drug
development [18–20].
& Wafaa M. Abdou
wabdou@link.net
Based on these considerations and with the aim of
synthesizing new phosphor esters containing one, two or
1
Chemical Industries Division, National Research Centre,
Elbohouth St., Dokki, Cairo 12622, Egypt
123

W. M. Abdou et al.
three amino functions directly linked to the phosphorus
atom, we describe herein the reactions between 2-azido-
4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (1) and
the phosphorus reagents trialkyl phosphites (TAPs), dialkyl
phosphites (DAPs), and hexaalkylphosphorus triamides.
Pharmacological evaluation results of antibreast, anticolon,
and antiprostate carcinoma cell lines properties for the
products were also discussed in terms of structure–activity
relationship (SAR) in an attempt to define a chromophore
for lead compounds.
stable P=O bond [23]. Similar triazaphosphole ring was
previously obtained from the reaction of trialkyl phosphites
with 3-x-azidoacetyl coumarin [24]. Furthermore, 4H-
isooxazol-4-ylphosphonate was formed via [1 ? 4]
cycloaddition mechanism from the reaction of triketoin-
dan-2-oxime with phosphorus reagents [25]. In support of
the formation of the intermediates 3, the 31P NMR spec-
trum was recorded in an early stage of the reaction for the
crude mixture of trimethyl phosphite and azide 1. The
spectrum (after 2 h) revealed two big peaks at
d = 141.3 ppm P(OMe)3, and at 11.7 ppm that corre-
sponds to the phosphazole product 4a whereas only a
minor peak was displayed at -9.6 ppm assigned to the
intermediate 3, which indicated the short life of this
structure. Unfortunately, all trials to obtain any of 4a–4c in
proper crystallized form for running single crystal crys-
tallography failed. On the other hand, the amines 5a–5c
arose from slight collapse of the intermediates 3 (due to
unfortuitous moisture), resulting in its denitrogenation and
the loss of phosphorus moiety (Scheme 1). 31P NMR
spectra of the crude mixtures of the reaction of (MeO)3P
and azide 1 after 2h (a), and at the end of the reaction
(b) was displayed in Fig. 1.
Results and discussion
Chemistry
The azide 1 readily reacted with trimethyl, triethyl, and
triisopropyl phosphites (2a–2c) in absolute tetrahydrofuran
(THF) under ambient conditions in 4–6 h to give spiro-
triazaphosphole-oxides 4a–4c in good yields. Alkylated
2-aminotetrahydrobenzo[b]thiophene-3-carbonitriles 5a–
5c were also isolated (&10 % yield) from the reactions.
Compounds 4a-4c could be formed in a one-step
mechanism [1 ? 4] cycloaddition to give initially the
intermediates 3 [21]. Further extrusion of the appropriate
alcohol moiety, and an alkyl group shift led to the forma-
tion of the triazaphosphole-oxide products 4a–4c [21, 22].
The latter step was provoked by the formation of the highly
Compounds 4a–4c showed 31P NMR chemical shifts
around 12 ppm confirming the presence of N–P–C linkage
in an azaphosphole nucleus [23, 24]. In their EI mass
spectra, 4a–4c showed the molecular ion peaks in addition
to the expected fragmentations of the corresponding
Fig. 1 31P NMR spectra of the crude mixtures of the reaction of (MeO)3P and azide 1 after 2h (a), and at the end of the reaction (b)
123

Spiro- and substituted tetrahydrobenzo[b]thiophene-triazaphospholes…
Staudinger reaction of azides with phosphines [27, 28].
Phosphorimidates 7 underwent rearrangement to their
corresponding phosphoramidates 8 via a 1,2-proton shift by
Lewis acid activation, whereas the amine 9 was formed
through the Staudinger reduction [29, 30] (Scheme 2).
Structural reasons for 8 are: (a) elementary analyses and
molecular weight determination (MS) confirmed the
assigned structure; (b) the IR spectra (KBr) of 8a–8c
revealed the presence of NH and CN absorption bands
around 3322 and 2198 cm-1; (c) The 1H NMR spectrum of
8a (CDCl3) showed a signal at 11.35 ppm (NH) and a
doublet (3JPH = 11.5 Hz) at 3.79 ppm due to the two
methoxyl groups attached to the phosphorus atom; and
(d) 31P NMR signals of 8a–8c were found at &18 ppm.
Next, the azide 1 was allowed to react also with hex-
aalkyl-phosphorus triamides 10a and 10b in THF at 20 °C.
The resulting crystalline 1:1 adducts (&90 % yield) were
formulated as phosphoranylidene-triazenes 11a, 11b [31,
32]. The 31P NMR signals of 11a, 11b were observed
at &40 ppm. Correct elemental analyses and expected 1H,
13C NMR data also confirmed the assigned structure 11
(Scheme 3). Compounds 11a and 11b were water sensitive,
quite stable only for few days in a desiccator. When a
protonating agent (2 cm3 dil. EtOH) was present in the
reaction medium, the reaction markedly accelerated lead-
ing to the formation of the phosphoric triamides 13a and
13b (&82 % yield). Furthermore, when compounds 11
were warmed in dil. alcohol, 13a and 13b were again
obtained. According to the Staudinger mechanism, the first
step is the nucleophile attack of the trivalent phosphorus
reagent 10 on the terminal nitrogen of the azide 1 that
results in the formation of the phosphazide 11 [29, 30].
Compound 11 is stable under drastic conditions due to the
presence of the strong donor, hexaalkylphosphorane tri-
amides moiety [33]. Quenching 11 with a molecule of H2O
afforded 13 via the intermediate 12 with concomitant loss
of dialkylamine molecule (Scheme 3). The 31P NMR
spectrum of these compounds showed signals around
Scheme 1
compound. The IR spectrum of 4b showed the presence of
absorption bands at 2211 (CN), 1243 (P=O, free), and at
1029 (P–O–C) cm-1. The strong presence of azide fre-
quency in the IR spectrum of 1 at 2090 cm-1 was absent in
the IR spectrum of 4a–4c. In the 1H NMR spectrum of 4b,
the two ethyl–methyl groups were observed as a multiplet
within the range 1.16–1.25 ppm (6H, Me.C–N and
MeC.OP), while the 1-H proton of the trihydrobenzene
nucleus was displayed as a triplet (3JHH = 6.6 Hz) at
3
6.93 ppm. A doublet of quartet (2H, JHH = 6.7 Hz,
3JPH = 8.3 Hz) was revealed at 3.79 ppm due to CH2
protons linked to 3-N in the triazaphosphole ring. A quintet
3
3
(2 H, JHH = 5.6, JPH = 8.9 Hz) at 4.02 ppm due to
OCH2 protons attached to the phosphorus atom. Moreover,
the spiro-C was located as a doublet (1JPC = 133.6 Hz) at
d = 110.3 ppm in the 13C NMR spectrum of 4b.
The reaction of dialkylphosphonates, dimethyl, diethyl,
and diisopropyl phosphites (6a–6c) with the azide 1,
however, did not proceed in the same way, but gave the
respective phosphoramidates 8a–8c (*72 % yield) along
with the known [26] 2-amino-4,5,6,7-tetrahydrobenzo-
[b]thiophene-3-carbonitrile (9, 10 % yield; Scheme 2). The
reaction sequence firstly included the reaction of the azide
with phosphites to give phosphorimidates 7a–7c, which are
analogous to the iminophosphoranes formed during the
1
d = 34 ppm. The H NMR spectrum of 13a revealed a
Scheme 3
Scheme 2
123

W. M. Abdou et al.
doublet centered at 2.45 ppm (3JPH = 9.4 Hz) due to 12 H
against all tested cell lines. On the other hand, phosphorami-
dates 8a and 8b showed excellent response to the three
parameters GI50, TGI, and LC50 of the 5 colon cell lines. As
such, 8b produced GI50 between 6.84 and 21.36 9 10-6
M
of the two magnetically equivalent dimethylamino groups.
Pharmacology
concentration, TGI between 3.542 and 5.942 9 10-5 M, and
LC50 value between 5.084 and 6.306 9 10-5 M. For com-
parison, the standard drug, 5-fluorouracil showed GI50
between 11.36 and 41.63 9 10-6 M concentration, TGI
between 3.368 and 6.651 9 10-5 M, and LC50 value between
3.894 and 8.894 9 10-5 M concentrations.
Evaluation of anticancer activity on the synthesized stable
phosphorus compounds 4a–4c, 8a–8c, 13a, and 13b was
performed at the Cancer Research Institute (CRI). First, all
these compounds were evaluated in primary anticancer assay
at 10-4 M concentration against MCV7 (breast), COLO205
(colon), and PC-3 (prostate cancer) cell lines (Table 1).
For CRI criteria, compounds that reduce the growth of any
one of the cell lines to approximately 32 % or less are passed
on for evaluation in a full panel of cell lines over a 3-long
dose range. Triazaphospholes 4a, 4b and phosphoramidates
8a, 8b that met these criteria were evaluated for their anti-
cancer activity following the known in vitro disease-oriented
antitumor screening program, which is based on the use of a
multiple panels of 12 human tumor cell lines [34]. Each
compound is tested as a minimum of three concentrations at
10-fold dilution against every cell line in the panel. A 48 h
continuous drug exposure protocol is used and sulforho-
damine B (SRB) protein assay is used to estimate cell
viability or growth [35, 36]. The response parameters GI50,
TGI, and LC50 (Table 2) refer to the drug concentration that
produce 50 % inhibition, total growth inhibition, and 50 %
cytotoxicity, and are expressed in 10-5 molar concentration.
In the table, we report only the activity of those compounds,
which exhibited GI50, TGI, and LC50 less than 10 9 10-5 M.
The GI50 reported data in Table 2 indicated that nominated
compounds 4a, 4b, 8a, and 8b showed a good level of cyto-
Considering the structure–activity relationship (SAR), as
shown in Tables 1 and 2, the highest protection was observed
for 4a, followed by 4b while 8a and 8b displayed less
activity. On the other hand, compounds 4c, 8c, 13a, and 13b
revealed only a weak antitumor activity toward all tested cell
lines. It seems that the phosphonate group is essential for the
biological activity. As far as cyclic substituted pyrazol-based
phosphonate is concerned, it is obvious that the moieties
attached to phosphorus determine the potency. As such,
cyclic triazaphospholes (4a, 4b) are more favorable for
antitumor activity compared to open-chain phosphorami-
dates (8a and 8b). Furthermore, the introduction of a long
chain (e.g., isopropyl) or replacing the alkoxy group (Me or
Et) with the amino group at the phosphor atom was totally
unfavorable and dramatically decreased the antitumor
potencies of compounds (4c, 8c; 13a, 13b).
Conclusion
In conclusion, we have developed a benign convenient and
rapid procedure for the synthesis of two types of phosphor
families: spiro-triazaphosphole-oxides and phosphoramidate
derivatives in significant yields. The products were evaluated
for their in vitro antitumor activities against 12 carcinoma cell
lines represent (breast, colon, and prostate cancer diseases).
Four compounds out of 8 phosphor ester derivatives exhibited
micromolar remarkable inhibition against several cancer cell
lines in comparison to 5-flourouracil.
static activity at 10-5 M and in some cases at 10-6
M
concentration against all subpanel cell lines. Compound 4a
displayedasignificantgrowthinhibitoryactivityonthe12cell
lines showing GI50 values between 6.35and 28.56 9 10-6 M,
and total growth inhibitory activity at 2.789–8.888 9 10-5
M. 4a also showed cytotoxic activity against all cell lines
(4.411–8.564 9 10-6 M concentrations) with particular
selectivity against breast and prostate cancer cell lines.
Compound 4b indicated more or less close activity to 4a
Table 1 Antiproliferative activity of 4a–4c, 8a–8c, 13a, and 13b at
10-4 M concentration expressed in growth percentage
Experimental section
Compounds
MCF7
COLO205
PC-3
Melting points were determined with open capillary tube
on an Electrothermal (variable heater) melting point
apparatus and were corrected. IR spectra were recorded
on a JASCO FT-IR 6100 using KBr disc (JASCO,
Japan). NMR spectra were measured with a JEOL
4a
10
35
78
22
56
87
72
73
18
42
4
24
84
14
32
73
67
77
4b
4c
89
8a
33
1
E.C.A-500 MHz (13C: 125.4 MHz, H: 500.7 MHz, 31P:
8b
8c
64
200.7 MHz) spectrometer (JEOL, Japan). 31P NMR
101
62
spectra were recorded with H3PO4 (85 %) as external
1
13a
13b
reference. H and 13C NMR spectra were recorded with
101
trimethylsilane as internal standard in CDCl3. Chemical
123

Spiro- and substituted tetrahydrobenzo[b]thiophene-triazaphospholes…
Table 2 GI50, TGI, and LC50 values of compounds 4a, 4b and 8a, 8b
Panel/cell lines
Type of test
4a
4b
8a
8b
5-Fluorouracil
GI50 TGI
LC50 GI50 TGI
LC50 GI50 TGI
LC50 GI50 TGI
LC50 GI50 TGI
LC50
Breast cancer
MCF7
0.635 4.662 5.084 0.503 4.732 3.190 0.947 8.852 6.143 0.764 6.864
–
2.284 4.831 7.521
MDA-MB-231/ATCC 1.445 3.025 5.284 1.457 3.186 4.780 2.205 4.946 6.985 1.843 7.893 6.874 2.635 6.572 3.894
MDA-MB-435
NCl/ADR-RES
HS578T
0.828 2.789 8.076 0.954 3.433 8.063 1.766 5.780 8.864 0.366 7.075
1.409 4.062 6.053 1.273 4.689 6.423 1.684 6.250 8.974 2.065 8.443 8.531 4.163 6.651 8.894
1.924 5.864 4.762 1.746 6.786 4.628 1.336 6.876 6.740 0.541 8.468 2.065 4.101 6.184
–
3.022 4.073 5.776
–
Colon cancer
COLO205
HCC-2998
HCT-15
1.843 8.748 7.962 1.780 8.042 8.053 0.865 6.947 5.234 0.945 3.542 6.306 1.168 3.479 6.032
1.278 8.432 6.895 2.942 8.353 8.756 0.761 4.037 5.846 0.684 4.314 6.143 1.434 4.673 5.074
2.633 8.888 8.564 3.653 6.406 8.684 1.776 3.621 6.346 2.136 5.942 5.084 1.776 4.055 8.063
HCT-116
2.062 5.562 7.053 2.064 4.985
2.856 5.883 6.941 2.868 8.043
–
–
0.764 6.875 5.647 0.857 4.634 5.173 2.148 3.435 4.079
0.982 4.043 7.023 0.906 4.959 5.430 2.982 3.368 7.653
SW-620
Prostate cancer
PC-3
1.243 3.356 4.411 0.997 4.636 4.650 1.053 6.620 5.878 0.788 2.865
1.053 4.325 4.664 2.843 3.513 5.844 1.448 5.974 5.036 0.816 3.906
–
–
1.439 4.658 6.008
1.136 4.502 6.016
DU-145
(–) means values [10 9 10-5
[38], but no physical data have been published. m.p.: 73 °C
(from pentane); IR (KBr): v = 3334 (NH), 2222 (CN)
shifts (d) are given in ppm. The mass spectra were
performed at 70 eV on an MS-50 spectrometer provided
with a data system spectrometer (Kratos, UK). Elemental
analyses (C/H/N/S) were carried out at the Microanalysis
Laboratory, Cairo University, Cairo, Egypt, using ele-
mentary Analysen-systeme GmbH-Vario EL III Element
Analyzer, Germany. The values obtained are favorably
agreed with the calculated ones ( 1). The appropriate
precautions in handling moisture-sensitive compounds
were observed and solvents were dried by standard
techniques. TLC: Merck 0.2 mm silica gel 60 F154 anal
aluminum plates. Column chromatography (CC): silica
gel (Kieselgel 60 mesh, particle size 0.2–0.5 mm; E.
Merck, Darmstadt). The substrate 2-azido-4,5,6,7-te-
1
cm-1; H NMR (500.7 MHz, CDCl3): d = 2.68 (s, 3H,
MeN), 1.82, 2.79 (2 m, 2 9 4H, 4 H2C-hexyl), 6.98 (br,
1H, HN) ppm; 13C NMR (125.4 MHz, CDCl3): d = 163.2
(C–NH), 130.4, 128.3 (C=C-hexyl), 118.7 (CN), 87.3 (C–
CN), 34.7 (MeN), 26.4, 26.2, 23.5, 21.3 (CH2-hexyl) ppm;
MS (EI, 70 eV): m/z (%) = 192 (56) [M?], 191 (100)
[M?-1].
2-(Ethylamino)-4,5,6,7-tetrahydrobenzo[b]-thiophene-3-
carbonitrile (5b)
Obtained as orange material; yield: 735 mg (9.6 %).
Compound 5b is commercially available, but no physical
data have been published. m.p.: 49 °C (from pentane); IR
trahydrobenzo[b]thiophene-3-carbonitrile (1)
prepared according to the reported method [37].
was
(KBr): v = 3333 (NH), 2217 (CN) cm-1
;
1H NMR
3
(500.7 MHz, CDCl3): d = 1.24 (t, JHH = 7.1 Hz, 3H,
MeH2C), 1.82, 2.95 (2 m, 2 9 4H, 4 H2C-hexyl), 3.54 (q,
3JHH = 6.8 Hz, 2H, H2CMe), 6.88 (br, 1H, HN) ppm; 13C
NMR (125.4 MHz, CDCl3): d = 164.3 (C–NH), 130.5,
128.6 (C=C-hexyl), 118.8 (CN), 87.7 (C–CN), 43.6
(CH2.Me), 26.4, 26.2, 23.5, 21.3 (CH2-hexyl), 15.3 (HC2-
Me) ppm; MS (EI, 70 eV): m/z (%) = 206 (64) [M?], 205
(100) [M?-1].
Preparation of compounds 4a–4c and 5a–5c by reaction of
azide 1 with trialkyl phosphites 2a–2c
A mixture of 0.8 g of the azide 1 (3.9 mmol) and 4.1 mmol
trimethyl, triethyl, or triisopropyl phosphite (2a–2c) in
15 cm3 THF was stirred at r.t. for &4–6 h (TLC). Excess
of the volatile materials was removed under vacuum, and
the resulting residue was chromatographed on silica gel.
Elution with n-hexane/CHCl3 (9:1 v/v) produced alkylated
amines 5a–5c. Elution with n-hexane/CHCl3 (7:3 v/v)
afforded triazaphospholes 4a–4c.
2-(Isopropylamino)-4,5,6,7-tetrahydrobenzo[b]thiophene-
3-carbonitrile (5c, C12H16N2S)
Yellow material; yield: 850 mg (10.3 %); m.p.: 62 °C
(from CH2Cl2/diethylether 1:1); IR (KBr): v = 3325 (NH),
2218 (CN) cm-1; 1H NMR (500.7 MHz, CDCl3): d = 1.24
2-(Methylamino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
carbonitrile (5a)
3
(d, JHH = 7.1 Hz, 2 9 3H, 2 Me.C), 1.82, 2.93 (2 m,
Obtained as orange substance; yield: 766 mg (10.4 %).
Compound 5a is commercially available and described in
2 9 4H, 4 H2C-hexyl), 3.01 (m, 1H, HC.(Me)2), 7.81 (br,
123

W. M. Abdou et al.
1H, HN) ppm; 13C NMR (125.4 MHz, CDCl3): d = 162.2
(C–NH), 130.1, 128.6 (C=C-hexyl), 118.6 (CN), 86.7 (C–
CN), 45.5 (CH.Me2), 26.4, 26.2, 23.5, 21.3 (CH2-hexyl),
23.7 (HCMe2) ppm; MS (EI, 70 eV): m/z (%) = 219 (47)
[M?-1].
4JPH = 4.1 Hz,
3JHH = 6.5 Hz,
2 9 3H,
Me2CHO),
2.69
3.68
(t,
2H, H2C-hexyl),
(dsept,
3JPH = 7.9 Hz, 1H, HC.N), 4.17 (dsept, JPH = 8.2 Hz,
3
3
1H, HC.O), 6.49 (t, JHH = 6.4 Hz, 1H, HC-hexyl) ppm;
13C NMR (125.4 MHz, CDCl3): d = 161.2, 149.6, 120.6,
26.6, 25.6, 22.6 (C-hexyl), 122.8 (CN), 109.8 (d,
1JPC = 139.5 Hz, C–P), 93.8 (C–CN), 75.5 (d,
40-Methoxy-30-methyl-30,40,5,6-tetrahydro-4H-spiro[1-ben-
zothiophene-2,50-[1,2,3,4]triazaphosphole]-3-carbonitrile
40-oxide (4a, C11H13N4O2PS)
2JPC = 12.9 Hz, CH.O), 49.6 (d, JPC = 11.9 Hz, HC.N),
2
24.1 (d, 3JPC = 7.9 Hz, Me2CHO), 21.3 (d, 3JPC = 8.5 Hz,
Me2CHN) ppm; 31P NMR (200.7 MHz, CDCl3):
d = 11.9 ppm; MS (EI, 70 eV): m/z (%) = 352 (47)
[M?], 324 (36) [M?-28 (N2)], 298 (25) [M?-54
(N2 ? CN)], 255 (46) [M?-97 (N2 ? CN ? C3H7)], 149
(100) [M?-203 (N2 ? CN ? C3H7 ? P(O)(OC3H7))].
Strew yellow needles; yield: 0.77 g (66.4 %); m.p.: 146 °C
(CH2Cl2); IR (KBr): v = 2214 (CN), 1237 (P=O, free), 1034
1
(P–O–C) cm-1; H NMR (500.7 MHz, CDCl3): d = 1.21,
3
2.15 (2 m, 2 9 2H, 2 H2C-hexyl), 2.78 (t, JHH = 6.8 Hz,
2H, H2C-hexyl), 3.51 (d, 3JPH = 7.1 Hz, 3H, Me.N), 3.78 (d,
3JPH = 6.7 Hz, 3H, Me.O), 6.87 (t, 3JHH = 6.8 Hz, 1H, HC-
hexyl) ppm; 13C NMR (125.4 MHz, CDCl3): d = 158.5,
149.7, 115.8, 27.1, 26.2, 21.9 (C-hexyl), 123.1 (CN), 110.6
Preparation of phosphoramidates 8a–8c by reaction of
azide 1 with dialkyl phosphites 6a–6c
1
(d, JPC = 136.4 Hz, C–P), 95.1 (C–CN), 55.3 (d,
A mixture of 0.8 g azide 1 (3.9 mmol) and 4.1 mmol of
dimethyl, diethyl, or diisopropyl phosphite (6a–6c) in
15 cm3 THF was stirred at r.t. for 6–10 h (TLC) and
volatile materials were removed under vacuum. The
resulting residue was chromatographed on silica gel with
n-hexane/CHCl3 (8:2, v/v) to give 2-amino-4,5,6,7-tetrahy-
drobenzo-[b]thiophene-3-carbonitrile (9) as buff substance
in 10.3 % yield; m.p. 147 °C (Ref. [26] 145 °C). Elution
with n-hexane/CHCl3 (1:1, v/v) afforded 8a–8c.
2JPC = 8.7 Hz, MeO), 28.6 (d, 2JPC = 8.4 Hz, MeN) ppm;
31P NMR (200.7 MHz, CDCl3): d = 11.7 ppm; MS (EI,
70 eV): m/z (%) = 296 (25) [M?], 268 (37) [M?-28 (N2)],
253 (25) [M?-54 (N2 ? CN)], 227 (23) [M?-69 (N2 -
? CN ? Me)],
149
(100)
[M?-147
(N2 ? CN ? Me ? P(O)(OMe))].
40-Ethoxy-30-ethyl-30,40,5,6-tetrahydro-4H-spiro[1-ben-
zothiophene-2,50-[1,2,3,4]triazaphosphole]-3-carbonitrile
(4b, C13H17N4O2PS)
Dimethyl 3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
ylphosphoramidate (8a, C11H15N2O3PS)
Strew yellow needles; yield: 0.94 g (74.2 %); m.p.: 132 °C
(cyclohexane); IR (KBr): v = 2211 (CN), 1243 (P=O, free),
;
1029 (P–O–C) cm-1 1H NMR (500.7 MHz, CDCl3):
Colorless crystals; yield: 0.76 g (68.2 %); m.p.: 174 °C
(EtOH); IR (KBr): v = 3322 (NH), 2198 (CN), 1234 (P=O,
;
bonded), 1029 (P–O–C) cm-1 1H NMR (500.7 MHz,
d = 1.16–1.25 (m, 6H, Me.C–N, MeC.OP), 1.11, 2.45
(2 m, 2 9 2H, 2 H2C-hexyl), 2.78 (t, JHH = 6.8 Hz, 2H,
3
CDCl3): d = 1.91, 2.45 (2 m, 4 9 2H, 4 H2C-hexyl), 3.79
(d, JPH = 11.5 Hz, 2 9 3H, 2 Me.OP), 11.35 (br, 1H,
3
3
H2C-hexyl), 3.79 (dq, JHH = 6.7, JPH = 8.3 Hz, 2H,
3
3
H2CN), 4.02 (qt, JHH = 5.6, JPH = 8.9 Hz, 2H, H2CO),
3
HN) ppm; 13C NMR (125.4 MHz, CDCl3): d = 151.6 (d,
3
6.93 (t, JHH = 6.6 Hz, 1H, HC-hexyl) ppm; 13C NMR
2JPC = 31.5 Hz, C-NH), 134.4, 130.2 (C = C-hexyl),
2
(125.4 MHz, CDCl3): d = 159.7, 149.4, 118.6, 26.7, 25.3,
22.4 (C-hexyl), 122.5 (CN), 110.3 (d, 1JPC = 133.6 Hz, C–
P), 94.7 (C–CN), 63.6 (d, 2JPC = 12.7 Hz, CH2O), 43.7 (d,
113.8 (CN), 74.3 (C–CN), 52.4 (d, JPC = 12.9 Hz, 2
MeOP), 26.4, 26.1, 23.6, 22.3 (CH2-hexyl) ppm; 31P NMR
(200.7 MHz, CDCl3): d = 17.6 ppm; MS (EI, 70 eV): m/z
(%) = 285 (53) [M?-1], 259 (34) [M?-27 (H ? CN)], 150
(100) [M?-136 (H ? CN ? P(O)(OMe)2)].
3
2JPC = 11.9 Hz, CH2N), 19.7 (d, JPC = 8.3 Hz, MeCH2-
3
O), 14.6 (d, JPC = 7.9 Hz, MeCH2N) ppm; 31P NMR
(200.7 MHz, CDCl3): d = 12.4 ppm; MS (EI, 70 eV): m/z
(%) = 324 (43) [M?], 296 (37) [M?-28 (N2)], 270 (29)
[M?-54 (N2 ? CN)], 241 (56) [M?-83 (N2 ? CN ? Et)],
149 (100) [M?-175 (N2 ? CN ? Et ? P(O)(OEt))].
Diethyl
3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
ylphosphoramidate (8b, C13H19N2O3PS)
Colorless crystals; yield: 0.86 g (70.6 %); m.p.: 162 °C
(EtOH); IR (KBr): v = 3325 (NH), 2194 (CN), 1229 (P=O,
bonded), 1032 (P–O–C) cm-1
40-Isopropoxy-30-isopropyl-30,40,5,6-tetrahydro-4H-
spiro[1-benzothiophene-2,50-[1,2,3,4]triazaphosphole]-3-
carbonitrile 40-oxide (4c, C15H21N4O2PS)
;
1H NMR (500.7 MHz,
3
4
CDCl3): d = 1.32 (dt, JHH = 6.7, JPH = 4.4 Hz,
2 9 3H, 2 MeCOP), 1.93, 2.46 (2 m, 4 9 2H, 4 H2C-
3
hexyl), 4.17 (dq, JHH = 6.7, JPH = 10.6 Hz, 2 9 2H, 2
3
Strew yellow crystals; yield: 0.98 g (71.2 %); m.p.: 152 °C
(MeCN); IR (KBr): v = 2213 (CN), 1240 (P=O, free), 1031
H2COP), 11.29 (br, 1H, HN) ppm; 13C NMR (125.4 MHz,
1
2
(P–O–C) cm-1; H NMR (500.7 MHz, CDCl3): d = 0.96,
2.12 (2 m, 2 9 2H, 2 H2C-hexyl), 1.26 (dd, JHH = 4.6,
CDCl3): d = 151.3 (d, JPC = 29.6 Hz, C–NH), 134.2,
129.7 (C=C-hexyl), 113.4 (CN), 74.7 (C–CN), 62.4 (d,
2JPC = 13.5 Hz, 2CH2.OP), 26.3, 26.1, 23.4, 21.9 (CH2-
3
3
4JPH = 3.8 Hz, 2 9 3H, Me2CHN), 1.33 (dd, JHH = 6.7,
123

Spiro- and substituted tetrahydrobenzo[b]thiophene-triazaphospholes…
3
hexyl), 16.3 (d, JPC = 8.2 Hz, 2Me.COP) ppm; 31P NMR
898 (P(NEt2)3) cm-1 1H NMR (500.7 MHz, CDCl3):
;
3
4
(200.7 MHz, CDCl3): d = 19.3 ppm; MS (EI, 70 eV): m/z
(%) = 313 (48) [M?-1], 287 (34) [M?-27 (H ? CN)], 150
(100) [M?-164 (H ? CN ? P(O)(OEt)2)].
d = 0.95, 1.39 (2 dt, JHH = 8.5, JPH = 6.4 Hz, 18H,
{(Me.C)2N}3P), 1.84, 2.63 (2 m, 4 9 2H, 4 H2C-hexyl),
3
3
2.91, 3.26 (2 dq, JHH = 8.5, JPH = 9.1 Hz, 12H,
[(H2C)2N]3P) ppm; 13C NMR (125.4 MHz, CDCl3):
d = 160.4 (C–N=N), 131.3, 128.6 (C=C-hexyl), 117.4
Diisopropyl 3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-
2-ylphosphoramidate (8c, C15H23N2O3PS)
2
(CN), 69.3 (C–CN), 39.5 (d, JPC = 13.7 Hz, [(CH2)2-
Colorless crystals; yield: 1.02 g (76.4 %); m.p.: 170 °C
(EtOH); IR (KBr): v = 3329 (NH), 2196 (CN), 1231 (P=O,
;
bonded), 1031 (P–O–C) cm-1 1H NMR (500.7 MHz,
CDCl3): d = 1.34, 1.56 (2 dd, 3JHH = 6.7, 4JPH = 4.7 Hz,
4 9 3H, (Me2CO)2P), 1.93, 2.51 (2 m, 4 9 2H, 4 H2C-
hexyl), 4.64-4.81 (2 m, 2H, (HCO)2P), 10.91 (br, 1H, HN)
ppm; 13C NMR (125.4 MHz, CDCl3): d = 151.5 (d,
2JPC = 31.2 Hz, C–NH), 133.9, 129.9 (C = C-hexyl),
N]3P), 26.5, 26.2, 23.6, 21.2 (CH2-hexyl), 14.7 (d,
31P
3JPC = 8.4 Hz,
[(Me.C)2N]3P)
ppm;
NMR
(200.7 MHz, CDCl3): d = 40.4 ppm; MS (EI, 70 eV):
m/z (%) = 451 (34) [M?], 423 (26) [M?-28 (N2)], 397 (19)
[M?-54 (N2 ? CN], 150 (100) [M?-301 (N2 ? CN ?
C12H30N3P)].
Reaction of 1 with hexaalkylphosphorus triamides 10a and
10b in the presence of a protonating agent. Preparation of
compounds 13a and 13b
2
113.5 (CN), 75.6 (C–CN), 71.2 (d, JPC = 12.7 Hz, 2
CH.OP), 26.4, 26.2, 23.5, 21.6 (CH2-hexyl), 24.2 (d,
3JPC = 8.5 Hz, 4 Me.COP) ppm; 31P NMR (200.7 MHz,
CDCl3): d = 18.4 ppm; MS (EI, 70 eV): m/z (%) = 341
(56) [M?-1], 315 (43) [M?-27 (H ? CN)], 150 (100) [M?-
192 (H ? CN ? P(O)(OC3H7)2)].
Phosphorus triamides 10a, 10b (5.2 mmol) in 5 cm3 of dry
THF were added in one portion to a mixture of 0.8 g azide
1 (3.9 mmol) and 2 cm3 dil. EtOH (1:1) in 10 cm3 THF.
The reaction mixture was stirred at r.t. for &6 h (TLC).
The solvent was evaporated to dryness; the residue was
washed several times with light petroleum (40–60 °C), and
crystallized from the proper solvent to give 13a and 13b,
respectively.
Preparation of compounds 11a and 11b by reaction of
azide 1 with 10a, 10b
Hexamethyl- or hexaethylphosphorus triamide (10a, 10b,
5.2 mmol) in 5 cm3 THF was added dropwise to 0.8 g of
azide 1 (3.9 mmol) in 10 cm3 THF. The reaction mixture
was stirred at r.t. for &4 h (TLC). The precipitate was
collected and washed several times with light petroleum
(40–60 °C) to give 11a and 11b. Compounds 11a and 11b
are pure enough for carrying out the spectroscopic
analyses, and stable for a week at -10 °C under argon.
N00-(3-Cyano-4,5,6,7-tetrahydro-1-benzothien-2-yl)-
N,N,N’,N’-tetramethylphosphoric triamide
(13a, C13H21N4OPS)
Pale yellow crystals; yield: 0.97 g (79.6 %); m.p.: 165 °C
(MeCN); IR (KBr): v = 3325 (NH), 2205 (CN), 1232 (P=O,
1
bonded) cm-1; H NMR (500.7 MHz, CDCl3): d = 1.91,
2.32 (2 m, 4 9 2H, 4 H2C-hexyl), 2.45 (d, 3JPH = 9.4 Hz,
2 9 6H, (Me2N)2P), 11.21 (br, 1H, HN) ppm; 13C NMR
2-[3-[Tris(dimethylamino)phosphoranylidene]triaz-1-en-1-
yl]-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile
(11a, C15H26N7PS)
2
(125.4 MHz, CDCl3): d = 150.6 (d, JPC = 28.3 Hz, C–
NH), 136.1, 130.2 (C=C-hexyl), 113.7 (CN), 87.3 (C–CN),
35.5 (d, 2JPC = 13.5 Hz, (Me2N)2P), 26.4, 26.2, 23.5, 21.8
(CH2-hexyl) ppm; 31P NMR (200.7 MHz, CDCl3):
d = 34.7 ppm; MS (EI, 70 eV): m/z (%) = 311 (45)
[M?-1], 285 (34) [M?-27 (H ? CN)], 150 (100) [M?-
162 (H ? CN ? C4H12N2OP)].
Yellow material; yield: 1.26 g (90 %); m.p.: 193 °C
(CHCl3); IR (KBr): v = 2206 (CN), 1333 (P=N), 1301,
;
923 (P(NMe2)3) cm-1 1H NMR (500.7 MHz, CDCl3):
d = 1.81, 2.61 (2 m, 2 9 4H, 4 H2C-hexyl), 2.75, 2.81 (2
d, JPH = 8.9 Hz, 18H, (Me2N)3P) ppm; 13C NMR
3
(125.4 MHz, CDCl3): d = 160.6 (C–N=N), 131.1, 128.7
(C=C-hexyl), 117.5 (CN), 69.4 (C–CN), 37.8 (d,
2JPC = 13.5 Hz, (Me2N)3P), 26.3, 26.1, 23.6, 21.4 (CH2-
hexyl) ppm; 31P NMR (200.7 MHz, CDCl3):
d = 40.2 ppm; MS (EI, 70 eV): m/z (%) = 367 (35)
[M?], 339 (24) [M?-28 (N2)], 313 (29) [M?-54
N00-(3-Cyano-4,5,6,7-tetrahydro-1-benzothien-2-yl)-
N,N,N’,N’-tetraethylphosphoric triamide
(13b, C17H29N4OPS)
Pale yellow crystals; yield: 1.1 g (82.4 %); m.p.: 153 °C
(CHCl3); IR (KBr): v = 3327 (NH), 2205 (CN), 1228 (P=O,
1
(N2 ? CN)],
150
(100)
[M?-217
(N2 ? CN ?
bonded) cm-1; H NMR (500.7 MHz, CDCl3): d = 1.32
(dt, JHH = 7.7, JPH = 5.4 Hz, 2 9 6H, [MeC.C)2N]2P),
3
4
C6H18N3P)].
1.92, 2.45 (2 m, 4 9 2H, 4 H2C-hexyl), 3.26 (dq,
3
2-[3-[Tris(diethylamino)phosphoranylidene]triaz-1-en-1-
yl]-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile
(11b, C21H38N7PS)
3JHH = 7.7, JPH = 8.6 Hz, 2 9 4H, [(H2C)2N]2P), 11.25
(br, 1H, HN) ppm; 13C NMR (125.4 MHz, CDCl3):
2
d = 150.4 (d, JPC = 28.6 Hz, C–NH), 135.8, 130.1
(C=C-hexyl), 113.6 (CN), 88.2 (C–CN), 40.8 (d,
Yellow material; yield: 1.49 g (92 %); m.p.: 184 °C
(EtOH); IR (KBr): v = 2210 (CN), 1330 (P=N), 1317,
123

W. M. Abdou et al.
2JPC = 13.5 Hz, [(MeCH2)2N]2P), 26.3, 25.9, 23.5, 21.2
7. Abdou WM, Barghash RF, Sediek AA (2012) Eur J Med Chem
57:362
8. Kamel AA, Geronikaki A, Abdou WM (2012) Eur J Med Chem
51:239
9. Abdou WM, Barghash RF, Bekhiet MS (2012) Arch Pharm
Chem Life Sci 345:884
3
(CH2-hexyl), 16.3 (d, JPC = 8.2 Hz, [(MeCH2)2N]2P)
ppm; 31P NMR (200.7 MHz, CDCl3): d = 33.3 ppm; MS
(EI, 70 eV): m/z (%) = 367 (42) [M?-1], 341 (31) [M?-27
(H ? CN)],
150
(100)
[M?-218
10. Abdou WM, Kamel AA, Khidre RE, Geronikaki A, Ekonomo-
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Determination of GI50, TGI, and LC50 values: A total of 12
human tumor cell lines, derived from three cancer types
(breast, colon, and prostate), formed the basis of this test
[35, 36]. The tumor cells were cultured in RPM11640
medium supplemented with 5 % fetal calf serum and
2 mM L-glutanine. The tumor cells are inoculated over a
series of standard 48-well microtiter plates in 100 cm3 of
medium. Density of inoculums depends on the type of
tumor cell and on its growth characteristics. These cells are
then preincubated on the microtiter plate for 24 h before
adding the compounds. They were tested in DMSO solu-
tion at three different concentrations (10-4, 10-5, and 10-6
M). After an incubation of the chemical agent for 48 h with
the tumor cell lines, a sulforhodamine B (SRB) protein
assay was used to estimate cell viability or growth. The
cytotoxic effects are evaluated, and the assay was used to
estimate cell viability or growth, and the assay results and
dose–response parameters were calculated as previously
described [34].
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48:1258
25. Abdou WM, Sediek AA, Khidre MD (2008) Monatsh Chem
139:617
Acknowledgments The authors gratefully acknowledge the finan-
cial support by the National Research Centre, Dokki, Cairo Egypt,
project# 10010101. They are also grateful to Cancer Research Insti-
tute, NY, USA for handling the pharmacological screening.
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Dalton 12
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