Inhibitory effect of novel S,N-bisphosphonates on some carcinoma cell lines, osteoarthritis, and chronic inflammation

Article abstract of DOI:10.1016/j.ejmech.2012.02.047

A new series of S,N-bisphosphonate derivatives was synthesized and evaluated as antitumor agents against breast-, cervix-, liver, and colon cancer diseases. Antiarthritic and antichronic inflammatory properties of the new bisphosphonates (BPs) were also investigated. The studies demonstrated an efficient site selective method for making condensation products of BP-derivatives in high yields from thiazinethiones and tetraethyl methylenebisphosphonate reagent. The bioscreening evaluation showed that one of the tested BPs exhibited remarkable antitumor activity against the four tested carcinoma cell lines; nevertheless, all tested S,N-BP-derivatives (11 compounds) showed significant to moderate anti-inflammatory activity and capable of inhibiting polyarthritis.

Full text of DOI:10.1016/j.ejmech.2012.02.047

European Journal of Medicinal Chemistry 51 (2012) 239e249
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Inhibitory effect of novel S,N-bisphosphonates on some carcinoma cell lines,
osteoarthritis, and chronic inflammation
,
Azza A. Kamel a, Athina Geronikaki b, Wafaa M. Abdou a
*
a Chemical Industries Division, National Research Elbohouth St., Dokki, Cairo 12662, Egypt
b Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle, University of Thessaloniki, Thessaloniki, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of S,N-bisphosphonate derivatives was synthesized and evaluated as antitumor agents
against breast-, cervix-, liver, and colon cancer diseases. Antiarthritic and antichronic inflammatory
properties of the new bisphosphonates (BPs) were also investigated. The studies demonstrated an effi-
cient site selective method for making condensation products of BP-derivatives in high yields from
thiazinethiones and tetraethyl methylenebisphosphonate reagent. The bioscreening evaluation showed
that one of the tested BPs exhibited remarkable antitumor activity against the four tested carcinoma cell
lines; nevertheless, all tested S,N-BP-derivatives (11 compounds) showed significant to moderate anti-
inflammatory activity and capable of inhibiting polyarthritis.
Received 3 November 2011
Received in revised form
17 February 2012
Accepted 23 February 2012
Available online 6 March 2012
Keywords:
S,N-bisphosphonates
Thiazinethiones
Ó 2012 Elsevier Masson SAS. All rights reserved.
Antitumor activity
Antiarthritic- and anti- inflammatory
activity
1. Introduction
proteins, including the small GT-Pases Ras, Rac, Rho and Cdc42. The
latter three, as well as numerous others, are signaling proteins that
Recently, bisphosphonates (BPs) have been proven to be an
important asset in the treatment and prevention of bone metastatic
breast cancer [1e5]. In addition, a number of in vitro studies indi-
cated growth inhibition and induction of apoptosis of multiple
myeloma cells by BP-drugs [1]. This result of antitumor activity-
based (N-BPs) and their relevant BP-acids has been confirmed
and reported by our group [6e8]. Our findings also showed that the
order of BP-potency on inflammatory is not, however, equivalent to
that of inhibiting cell viability in carcinoma cells [6].
Over the last two decades, there was a significant progress in
elucidating the mechanism of action of BPs, which in general can
shorten the life span of osteoclasts by induction of programmed cell
death (apoptosis) [9e11]. For all N-BPs, in particular, apoptosis seems
to be caused by the inhibition of important biosynthetic enzymes,
e.g. farnesyl diphosphate synthase, in the mevalonate pathway, on
which depends the synthesis of cholesterol and isoprenoid lipids.
Isoprenylation involves covalent linkage of the 15 or 20 carbon
of isoprene moiety of farnesyl diphosphate or geranylegeranyl
diphosphate, respectively, to the carbon-terminus of regulatory
regulate a variety of cellular processes. In this way, N-BPs can deprive
osteoclasts of important regulators of intramolecular dynamics,
leading to poor cell functioning and eventually programmed cell
death. This targeted osteoclast inhibition accounts for the potency of
the N-BPs and for their ability to elicit the desired therapeutic
response of suppressing bone turnover [1,5].
In the present article, it is intended to utilize the chemistry of the
easily available thiobenzothiazine scaffold for formation of a new
series of N-heterocyclic methylenethiobis-phosphonates in order to
compare their chemistry with their oxygen-counterparts previously
studied [7], and to evaluate their antitumor- as well as chronic
inflammation properties. The work is a pursuance of our research
activity directed toward construction of bioactive heterocycle gem-
diphosphor esters, especially those associated with antitumor
[6e8], anti-inflammatory [6,12e15], and antiosteoporosis potencies
[12,15e17].
In an earlier stage, we had adopted the computer-assisted
approach, PASS program [18,19] for designing in silico the struc-
tures of potentially active molecules for the future synthesis. An
innovative approach to the simultaneous computer-aided predic-
tion and structure-activity relationship analysis of many biological
activities has been developed and widely used in finding and
optimization of new leads [20,21].
* Corresponding author. Tel.: þ20 2 3371615; fax: þ20 2 33370931.
E-mail address: wabdou@link.net (W.M. Abdou).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.047

240
A.A. Kamel et al. / European Journal of Medicinal Chemistry 51 (2012) 239e249
2. Results and discussion
with subsequent ring cleavage leads to the formation of the sodium
salt 11, which is the key intermediate for subsequent transformations.
12a and 12b were formed from 11, aspreviously reported [22e24], via
carbon disulfide elimination whereas intramolecular cyclization of
the protonated intermediates 13 yielded 14a and 14b with concom-
itant loss of ethanol moiety (Scheme 1). Considering the previous
report [7], the results of the reaction of the oxygen analog 1 with 10
showed that we were able to isolate the products 12 parallel to those
oxygen-analogs, but with 14a and 14b we have isolated different
condensed products. The structures 12 and 14 were deduced on the
basis of IR, 1H, and 13C spectroscopy and elemental analyses.
In a recent study [7], we have prepared a number of methyl-
enediphosphonate compounds 7, 8 and 9 by conducting the
condensation reaction of oxazines 1, 3 and 5 with Horner reagent,
tetraethyl methylenebisphosphonate. Antitumor properties of the
bisphosphonate products of types 7e9 were discussed.
Tetraethyl
1-methyl-3-thioxoindoline-2,2-diyldiphosphonate,
12b was obtained in 41.4% yield. The IR spectrum of 12b showed
absorptions bands at 1329 (C]S), 1258, 1242 (2P]O), and at 1135,
1052 (2PeOeC groups). Its 1H NMR (CDCl3,
d ppm) spectrum
exhibited two doublet-triplets and two doublet-quartets recognized
as arising from the two P-ester moieties (1.11, 1.41 and 3.84, 3.97 Hz,
respectively). One doublet signal at 3.14 ppm and two doublet signals
at 7.42, 7.85 ppm are due to the H3CN and the HeAr, respectively. The
1H decoupled 13C NMR (CDCl3,
d ppm) spectrum of 12b showed
signals at 256.4 (d, C]S), 82.9 (t, 1JPeC ¼ 136.4 Hz, CeP2), and 38.3
(d, CH3N). The 31P NMR spectrum of 12b displayed two separate
doublets with equal 2JPeP coupling constants ¼ 28 Hz.
Diethyl (2-ethoxy-4-mercapto-1-methyl-2-oxido-1,2-dihydro-
1,2-benzazaphosphinin-3-yl)-phosphonate, 14b was obtained in
37.2% that showed stretching bands at
n
3429, 1424 cmꢀ1 that were
attributed to the bonded SH group. In its 1H NMR (CDCl3,
d
ppm) the
thiol and N-methyl protons were given as two doublet at 1.91 and
3.23, respectively. The two phosphor ester moieties were displayed
at 0.99, 1.12 (2dt) and at 3.88e4.01 [m (2dq)], respectively.
Furthermore, 13C NMR spectrum of 14b revealed, among other
signals, a doublet at 162.7 (CeSH), one triplet at 102.6 (CeP2), and
a doublet at 31.1 (CH3N) ppm. Its 31P NMR spectrum showed two
doublets at dP 19.8 and 25.6 ppm (2JPeP ¼ 22 Hz, CeP2).
In the context of this work, the synthesis of the required sulfurand
nitrogen containing-BP derivatives was accomplished by applying
the same methodology on the analogously thiothiazines 2, 4 and 6.
When 2H-3,1-benzothiazine-2,4(1H)-dithiones 2a and 2b were
allowed to react with tetraethyl methylenebisphosphonate reagent
10 in boiling ethanol solution containing sodium ethoxide, BPs of
types 12 and 14 in almost equal yields (z39%) were formed. The
spectroscopic data showed 14a and 14b to be present in the thiol
form. Formation of the reaction products 12 and 14 was attained
according to the transformations outlined in Scheme 1. The initial
nucleophilic attack of the carbanion center in 10 on the C(1)]S group
Application of the phosphorus reagent 10 to 3,1-benzothiazine-
4-thiones 4ae4d was next investigated. Treatment of 4ae4d with
a slight excess of 10 under the same reaction conditions, afforded
the corresponding bisphosphonate products 18ae18d in high
yields (z70%) as a sole reaction product (Scheme 2).
P(O)(OEt)2
R1
N
O
R1
N
R1
N
-
S
S
P(O)(OEt)2
10
P(OEt)2
- CS2
-
S
S
3
P(OEt)2
O
NaOEt/EtOH
CH[P(O)(OEt)2]2
Na+
S
S
S
11a, 11b
2a, 2b
12a, 12b
b
l
2
C
1
H
y
l
.
n
c
o
n
o
c
O
Me
N
R1
N
NHR1
O
P
O
P(OH)2
OEt
OEt
-
ETOH
1
P
OEt
P(OEt)2
4
P(OH)2
O
P(OEt)2
O
S
S
HS
O
15
13a, 13b
14a, 14b
14a, R = H
14b
, R = Me
Scheme 1. Synthesis of BPs 12a, 14a, 12b, 14b, and BP-acid 15.

A.A. Kamel et al. / European Journal of Medicinal Chemistry 51 (2012) 239e249
241
O
O
P(OEt)
_
-
S
[(EtO) P] HC
S
2
2
X
2
NaOEt/EtOH
S
X
+
+
-
S
Na
P(OEt)
N
Z
2
N
Z
O
10
4a-4d
16a-16d
O
S
S
P(O)(OEt)
2
P(O Et)
2
X
X
-
H S
4
P(O)(OEt)
SH
2
2
P(OEt)
O
2
1
N
N
Z
Z
17a-17d
18a-18d
O
S
N
P(OH)
2
X
Conc. HCl
18c, 18d
P(OH)
O
2
Z
19a, 19b
4, 16-18: a, X = H; Z = Ph
4, 16-18: b
, X = H; Z = Me
19a
, X = H; Z = C H
6
11
4, 16-18: c, X = H; Z = C H
6 11
, X = Br; Z = Me
19b, X = Br; Z = Me
4, 16-18: d
Scheme 2. Synthesis of BPs 18ae18d, and BP-acids 19a, 19b.
A reasonable mechanism of the condensation of 4ae4d with 10
involved an initial attack [22,25e28] of the carbanion carbon in 10
on the C(1)]S group with subsequent ring opening to afford the
intermediated 17 via 16. Under thermal conditions and the basic
medium, 17 was formed. Further intramolecular cyclization led to
the final BPs 18ae18d accompanied with the loss of hydrogen
sulfide molecule (Scheme 2).
a promised cytotoxicity. Some common types of activities for new
class of BPs and the known BP-drugs (zoledronic acid and clodro-
nate) were observed. Thus, such activities as cytotoxicity and breast
cancer were predicted almost for all tested new compounds as well
as for zoledronic acid and clodronate. By default, in PASS Pa ¼ Pi
value is chosen as a threshold, therefore all compounds with Pa > Pi
are suggested to be active.
Finally, the inserted products 21ae21d were obtained in high
yields when 4-substituted-1H-2,3-benzothiazine-1-thiones 6ae6d
were treated with Horner reagent 10 under the alkaline condition.
The substituted isoquinoline-bisphosphonate esters were
produced via the intermediates 20 according to Scheme 3.
As recent advances in pharma laboratories have identified
impressively remarkable therapeutics from 1,1-bisphosphonic acid
to 1,1-bisphosphonate ester counterparts [29], hydrolysis of the
representatives 12b, 18c, 18d and 21d was undertaken to give the
corresponding BP-acids 15, 19a, 19b and 22 (Schemes 1e3).
The structures suggested for all new compounds are in good
agreement with their analytical and spectral data (see Experimental
section).
In sequel, antitumor activity screening of 12b, 18a, and 21a in
assays applying a human breast carcinoma (MCF7)-, a human
cervix (HeLa)-, a human liver (HEPG2)-, and a human colon
(HCT116) cell lines was investigated. The evaluation was considered
vc the known anticancer drugs: doxorubicin (DOX) or cisplatin
(CIS) by sulfo-rhodamine-B-Stain (SRB) using the method of Ske-
han et al. [30]. The obtained results (Table 1) represent concen-
trations (four different concentrations: 5, 12.5, 25, and 50 mg/mL) of
the used investigated compounds resulting in growth inhibition of
50% (IC50) for the tested human cell lines compared to DOX or CIS;
the highest concentration of each compound used was 50 mg/mL.
Each concentration is evaluated three times (each dose is incubated
with the cells in three different wells) thereby the data represent
the average of the total inhibition observed. The deviation in the
obtained data was ranged between: p < 0.001 and p < 0.05
(Table 1).
3. Pharmacological evaluation
3.1. Antitumor activity
The antitumor activity results displayed in Table 1, indicated that
the tested BP-derivatives 12b,18a, and 21a reflect good to moderate
activity against the used human tumor cells. The order of activity is
12b > 18a > 21a. It is also been noticed that 12b exhibited
remarkable antitumor activity against the four tested carcinoma
cell lines. The relation between surviving fraction and drug
concentration of the tested compounds 12b, 18a, and 21a vc DOX or
CIS is plotted in Fig.1, to get the survival curve of each tumor cell for
each compound. Further studies in experimental tumors in vivo for
evaluating the possible antineoplastic potential by these and the
other synthesized compounds are warranted.
Biological activity spectra were predicted for all synthesized BPs
and the known drugs: zoledronic acid and clodronate with the free
available internet version of PASS 2009.1: http://www.ibmc.msk.
ru/PASS. The potency spectrum for a substance is a list of the bio-
logical activity types for which the probability to be revealed (Pa)
and the probability not to be revealed (Pi) are calculated. Pa and Pi
values are independent and their values vary from 0 to 1. As it is
observed from the PASS results (Supplementary), 12b, 18a, and 21a
are the most three out of eight predicted compounds showed

242
A.A. Kamel et al. / European Journal of Medicinal Chemistry 51 (2012) 239e249
S
S
P(O)(OEt)2
S
N
NaOEt/EtOH
+
10
P(O)(OEt)2
NSH
Y
Y
20a-20d
6a-6d
O
O
S
S
P(OH)2
P(OH)2
O
P(OEt)2
P(OEt)2
- H2S
only 21d
conc. HCl
N
N
O
4-Et2NC6H4
Y
21a-21d
22
6, 20, 21: a, Y = 4-H3C.C6H4
6, 20, 21: b
, Y = 4-H3CO.C6H4
6, 20, 21: c
, Y = C6H5
6, 20, 21: d, Y = 4-Et2NC6H4
Scheme 3. Synthesis of BPs 21ae21d and BP-acid 22.
3.2. Antiarthritis bioassay
According to the histopathology of the human arthritic joint, the
adjuvant-induced polyarthritis (AIP) model expresses synovitis,
infiltration of the subsynovial tissue with numerous inflammatory
cell types, and pannus formation leading to erosion of articular
cartilage and bone. Perhaps the most distinctive symptom of the
joint pathology in AIP is the marked resorption of bone that is
caused by a granulomatous reaction and periosteal bone formation.
The rapid onset (the first appearance of the signs or symptoms of an
illness) (24 h) of arthritis in the injected paw is considered to be
largely a nonimmune inflammatory response to complete Freund‘s
adjuvant. In contrast, the subsequent arthritic reaction in the
noninjected hindpaw and forepaws is delayed in onset and is
mediated by immunological components. The suppression of bone
destruction and periarticular inflammation in the noninjected paw
in AIP, therefore, is considered to be an indication of potential
antiarthritic activity in human rheumatoid arthritis.
The antiarthritic effect of seven new BPs 12a,b, 14a,b, 18a,b and
21d as well as four BP-acids 15, 19a,b and 22, in the rat adjuvant-
induced polyarthritis (AIP) over 28 days, was examined. Clodro-
nate 23, the BP drug was used as the relative control in our
experiments as it demonstrated activity in this model [33]. The
changes in paw volume over time (APV), which occurred in the
injected and noninjected hindpaws of treated and control rats were
quantitated by mercury displacement plethysmography. Clodro-
nate, when administered at 10e50 mg/kg, exerted significant
inhibitory effects (p < 0.001) on the noninjected hindpaw arthritis,
whereas it was less effective (23e38% inhibition) against the
swelling in the injected hindpaw. None of the tested BPs signifi-
cantly altered the arthritis in the 28-day injected paw; however,
12b and 14b (10e50 mg/kg) caused a marginal suppression
(18e37%) of this component of AIP (injected hindpaw) and matched
that observed with the standard control 23. In contrast, all tested
BPs significantly inhibited (p < 0.05) noninjected paw arthritis
when given at the same doses. The suppressive effects of tested BPs
as well as the standard control 23 on AIP were not dose-related. BPs
14a and 14b, which contain free thiol group, exerted the highest
inhibitory effect (56e76%) on the noninjected hindpaw arthritis.
Also, the results displayed in Table 2 indicated that N-alkylated
All bisphosphonates (BPs) are similar in terms to their inhibitory
effects on bone resorption, but seem to have different effects on
hypocalcemia resulted from diseases such as tumor-induced-
osteolytic bone diseases, and human arthritis. However, it is
recently reported that sulfur-containing BPs [31e33], e,g., mer-
captomethyl-1,1-bisphosphonic acid (HSEDPÒ) [31], have demon-
strated remarkable activity in the rat adjuvant arthritis model. In
sequel, it is encouraging to evaluate the new S,N-BPs in animal
models of arthritis: rat adjuvant-induced polyarthritis (AIP) and
delayed type hypersensitivity granuloma assay as a model of
chronic inflammation.
O
Cl
C
OH
P
OH
P
P(OH)2
HS
HO
O
O
P
-
O
OH Cl
OH
O
23
HSEDP
Table 1
Anti-tumor properties of the tested compounds DOX, CIS, 12b, 18a, and 21a.
Compd. IC50 g/mL ( M)
MCF7
,
m
m
HeLa
HEPG2
HCT116
(colon cancer)
(breast cancer) (cervical cancer) (liver cancer)
DOX
CIS
12b
18a
21a
4.4 (8.09)* 3.10 (5.70)***
e
3.73 (6.86)*
e
2.8 (9.30)**
7.20 (16.54)*** 6.60 (15.16)*
7.95 (15.60)* 13.80 (27.08) *** 10.40 (20.40)* 11.60 (22.77)***
15.90 (30.37)** 14.00 (26.74)* 13.20 (25.20)** 9.60 (18.34)*
e
e
6.40 (14.70)** 3.60 (8.27)*
DOX ¼ doxorubicin; CIS ¼ cisplatin.
Deviation error: (***) p < 0.001, (**) p < 0.01, (*) p < 0.05; p is the percentage of
inhibition.

A.A. Kamel et al. / European Journal of Medicinal Chemistry 51 (2012) 239e249
243
Fig. 1. Dose response curves of compounds 12b, 18a, 21a vc DOX or CIS against HCT116, HEPG2, HeLa, and MCF7 cell lines.
derivatives 12b and 14b caused better inhibition than their coun-
terparts 12a and 14a at the same doses whereas conversion of BPs
12b, 18c, 18d, and 21d to the corresponding BP-acids, 15, 19a, 19b,
and 22, resulted in loss of activity even at higher dose (100 mg/kg)
in noninjected hindpaw-associated arthritis.
Next, the same standard 23, BPs 12a,b, 14a,b, 18a,b and 21d as
well as BP-acids 15, 19a,b and 22 were profiled in a delayed-type
hypersensitivity granuloma model using the reported procedure
by Nugent et al. [33]. The compounds were administered subcu-
taneously in mice, which were previously sensitized to methylated
bovine serum albumin (mBSA) and surgically implanted with
hydroxyapatite disks (two per mouse), soaked in mBSA, in order to
generate granulomas. This model is unaffected by traditional
nonsteroidal anti-inflammatory drugs, such as aspirin, indometh-
acin, or ibuprofen [34]. Clodronate reproducibly inhibited granu-
loma wet and dry weights and served as a positive control in our
experiments; the results are shown in Table 2.
Both 14a and 14b significantly inhibited the granuloma in
a dose-dependent manner at the doses examined (25e100 mg/kg).
12a, 12b, 18a, 18b, and 21d all displayed inhibitory activities, which
were equivalent matched to that of clodronate at 100 mg/kg. BP-
acids 15, 19a, 19b, and 22, on the other hand, showed only
marginal activity against the dry and the wet weight granuloma.
This result is not surprising as it has been reported that, in some
cases, conversion of BPs to the corresponding acid, resulted in the
loss of activity [33]. Also data in Table 2 showed that replacement of
the acidic N-1 hydrogen in 12a, 14a with a methyl group, resulted in
enhancing the activity.
stage in order to decide if it is worthy to be in vivo evaluated, and to
compare the results of the prediction with the experimental one.
The prediction result is presented as a list of activities (Table 2) with
appropriate Pa and P/E, which reflects the accuracy of the predic-
tion with the experiment results thereby it can be deduces the
average accuracy of prediction (AAP) for anti-inflammatory activity
to 54.5%. This observation suggests that these compounds differ
significantly from the classic ant-inflammatory compounds and
that they may be new chemical entities (NCEs).
3.3. Toxicity of the most promised products
Toxicological studies of the most promising synthesized anti-
inflammatory active compounds 12b, 14b and 21d were performed
using LD50 standard method in mice in 500, 750 and 1000 mg/kg
(body weight), i.e. 10e20 folds of the used anti-inflammatory
effective dose. However, no toxic symptoms or mortality rates
were observed after 24 h post-administrations explaining the safe
behavior of the used doses.
4. Conclusion
The studied reactions in the previous [7] and the present
investigations are offered as an easy route for the transformations of
easily available starting materials to the title BPs and the related BP-
acids in satisfactory yields. In addition, our protocol demonstrates
an efficient site selective method for making condensation products
in high yields from thiazinethiones and methylene-bisphosphonate
reagent under mild conditions. We have also attempted in this
research work to utilize the high bone (joint) specificity of sulfur
Prediction of anti-inflammatory was made by using the
computer-assisted approach, PASS program [18,19] at the earlier

244
A.A. Kamel et al. / European Journal of Medicinal Chemistry 51 (2012) 239e249
Table 2
Antiarthritic activity and delayed-type hypersensitivity granuloma resultsa of BPs 23, 12a,b, 14a,b, 18a,b, 21d, and BP-acids 15, 19a,b, 22.
No
AIP (% inhibn) (APV, 28 days)
Dose (mg/kg)
sc
% inhibn of granuloma
Prediction
(AIA) Pa
Coincidence
P/E
Dose
(mg/
kg)
Injected
paw
Noninjected
paw
Dry
wt
Wet
wt
23
50
30
20
10
50
30
20
10
50
30
20
10
50
30
20
10
50
30
20
10
100
50
50
30
20
10
50
30
20
10
100
50
100
50
50
30
20
10
100
50
35
34
28
23
15
11
7
68***
62***
70**
56***
64***
58***
60**
48***
68*
70***
55**
45***
75**
68**
56***
66***
76***
73***
60***
66***
27
100
60
25
e
48***
44
33
44***
40
30
0.786
e
þ/þ
ꢀ/þ
ꢀ/þ
þ/þ
þ/þ
e
e
12a
12b
14a
14b
100
60
25
e
46***
42**
30
44***
31*
24
5
e
e
30
32
23
15
25
14
10
<5
37
35
27
18
13
<5
28
30
12
<5
20
18
23
<5
16
<5
21
<5
24
16
20
<5
18
<5
100
60
25
e
48*
45
36
38*
45
33
e
e
e
100
60
25
e
50***
42
38
51***
44
33
0.741
0.641
e
e
100
60
25
e
55***
53
50
50**
48
33
e
e
15
100
50
100
60
25
e
32***
16
48***
32
22
e
25**
15
43***
35
13
e
0.213
0.442
þ/þ
þ/þ
18
18a
70***
68***
60***
40**
66***
72***
61***
38***
14
8
18
12
65**
52***
56***
44***
24
18b
100
60
25
e
44**
37
24
40**
30
20
0.311
þ/þ
e
e
19a
19b
21d
100
50
100
50
100
60
25
e
36
28
25
18
44***
40**
32
24*
19*
13
<10
34***
31*
22
0.466
0.284
0.533
þ/þ
ꢀ/þ
þ/ꢀ
e
e
22
100
50
30
26
23**
16**
e
ꢀ/ꢀ
10
sc: subcutaneously.
(P/E): P e prediction; E e experiment; P/E e accuracy of prediction.
þ/þ means that both prediction and experiment give positive results; ꢀ/ꢀ means that both prediction and experiment give negative results; þ/ꢀ means that prediction gives
positive results but experiment gives negative results; ꢀ/þ means that prediction gives negative results but experiment give positive results.
a
(***) p < 0.001, (**) p < 0.01, (*) p < 0.05; p is the percentage of inhibition.
containing bisphosphonic acids [31e33] with other chemical
moieties of potential anticatabolic pharmacology for testing as
a new series of compounds for the treatment of human rheumatoid
arthritis. Screening results indicated that 14b and 14b that have free
thiol group are the highest potent BP-product for inhibition chronic
arthritis, which may bind to a metal atom in the active site of the
matrix metalloproteinases (MMPs) [35]. In parallel, the bioassays
are in agreement with the previously reported [29] that small
changes in the structure of the N-heterocyclic moiety in the BP-
derivatives can lead to extensive alterations in their physicochem-
ical, biological and therapeutic characteristics.
spectrawere recorded with H3PO4 (85%) as external reference.1H and
13C NMR spectra were recorded with trimethylsilane as internal
standard in CDCl3 or DMSO-d6. Chemical shifts (
d) are given in ppm.
The mass spectrawere performed at 70 eV on an MS-50 Kratos (A.E.I.)
spectrometer provided with a data system. The appropriate precau-
tions in handling moisture-sensitive compounds were observed. The
purity of all new samples was verified by microchemical analysis (H/
C/N) and spectroscopy. All international principles and local regula-
tions concerning the care and use of laboratory animals were
considered during the pharmacological screening. The known start-
ing substrates 2a, 2b [36], 4a [37], 4b [38], 4c [39], and 4d [40] were
prepared according to the reported methods.
5. Experimental section
5.1. General procedure of bisphosphonates 12a, 12b, 14a,
14b, and 18ae18d
Melting points (uncorrected) were determined with open capil-
lary tube on an electrothermal (variable heater) melting point
apparatus. IR spectra were recorded on a JASCO FT-IR 6100 using KBr
disc. NMR spectra were measured with a JEOL E.C.A-500 MHz (13C:
125.8 MHz, 1H: 500.6 MHz, 31P: 200.7 MHz) spectrometer. 31P NMR
To a stirred solution of 9.12 mmol of sodium (Na) in 10 mL EtOH-
was added drop wise 4.2 mmol of tetraethyl methylenebi-
sphosphonate 10 followed by a solution of 3.8 mmol of 2H-3,1-

A.A. Kamel et al. / European Journal of Medicinal Chemistry 51 (2012) 239e249
245
benzothiazine-2,4(1H)dithione 2a, 1-methyl-2H-3,1-benzothiazine-
5.3.1. Tetraethyl 1-methyl-3-thioxoindoline-2,2-
2,4(1H)dithione 2b, 2-phenyl-4H-3,1-benzothiazine-4-thione 4a,
2-methyl-4H-3,1-benzothiazine-4-thione 4b, 2-cyclohexyl-4H-
3,1-benzothiazine-4-thione 4c, or 6-bromo-2-methyl-4H-3,1-
benzothiazine-4-thione 4d in 20 mL EtOH at 0 ꢁC. The resulting
mixture was heated under reflux for 15e20 h (thin layer chroma-
tography, TLC). The reaction mixture was poured into 100 mL of
distilled water and HCl (1 N) was added at ꢀ5 ꢁC until the pH of the
reaction mixture became acidic, followed by extraction with AcOEt
(3ꢂ50mL).ThecombinedorganicphasewasdriedoveranhyNa2SO4.
After removal of the solvent, under reduced pressure, the resulting
residue was washed several times with light petroleum (40e60 ꢁC),
and recrystallized from the solvent indicated after the m.p., to give
therespectivebisphosphonates (BPs) 12a,12b,14a,14b, and18ae18d,
respectively.
diyldiphosphonate, 12b
Yield 40.4%. Orange material, mp 119e121 ꢁC (from cyclohexane).
IR (cmꢀ1, KBr): nmax 1329 (C]S), 1258, 1242 (2P]O), 1135, 1052
(PeOeC). 1H NMR [CDCl3] ppm:
d
1.11, 1.41 (2dt, JHeH ¼ 6.6,
4
4JPeH ¼ 3.4, 2 ꢂ 6H, 2(H3CCO)2), 3.14 (d, JPeH ¼ 3.3, 3H, H3CN),
3.84, 3.97 (2dq, JHeH ¼ 6.6, 3JPeH ¼ 4.8, 2 ꢂ 4H, 2(H2CO)2), 7.42, 7.85
(2d, JHeH ¼ 8.1, 2 ꢂ 2H, HeAr). 13C NMR [CDCl3] ppm:
d 256.4 (d,
2JPeC ¼ 13.4, C]S), 155.2, 133.3, 131.2, 127.3, 124.5, 114.2 (CeAr), 82.9
(t,1JPeC ¼ 136.4, CeP2), 60.9 (d, 2JPeC ¼ 13.6, CH2O), 38.3 (d, 3JPeC ¼ 8.2,
3
CH3N), 16.4 (d, JPeC ¼ 3.9, CH3CO). 31P NMR [CDCl3] ppm:
d 23.6,
2
27.5 (2d, JPeP ¼ 28, CP2). EI-MS: in m/z (%): 435 (17) [Mþ], 420
(42) [Mþ ꢀ 15 (Me)], 388 (24) [Mþ ꢀ (15 þ 32) (Me þ S)], 283
(38) [Mþ
ꢀ
(15
þ
137) (Me
þ
C4H10O3P)], 146 (55)
[Mþ ꢀ (15 þ 274) (Me þ C4H10O3P)2],114 (100) [Mþ ꢀ (15 þ 32 þ 274)
{Me þ S þ (C4H10O3P)2}], 102 (36), 77 (98). Anal. Calcd for
C17H27NO6P2S (435.4): C, 46.89; H, 6.25; N, 3.22; P, 14.23; S, 7.36.
Found: C, 46.94; H, 6.33; N, 3.15; P, 14.16; S, 7.28.
5.2. Reaction of 2a with 10
Reagents: NaOEt (0.2 g of Na, 9.1 mmol in 30 mL EtOH), BP-
reagent 10 (1.2 mL, 4.2 mmol), and the substrate 2a (0.8 g,
3.8 mmol). The product mixture was washed several times with
light petroleum (40e60 ꢁC), and recrystallized from the solvent
indicated after the mp, to give BPs 12a and 14a.
5.3.2. Diethyl (2-ethoxy-4-mercapto-1-methyl-2-oxido-1,2-
dihydro-1,2-benzazaphosphinin-3-yl)phosphonate, 14b
Yield 37.2%. Yellow crystals, mp 138e140 ꢁC (from CH2Cl2). IR
(cmꢀ1, KBr): nmax 3429,1424 (br, SH), 1245,1222 (2P]O),1155, 1096
(2PeOeC). 1H NMR [CDCl3] ppm:
d
4
0.99 (dt, JHeH ¼ 6.6, 4JPeH ¼ 3.5,
5.2.1. Tetraethyl 3-thioxoindoline-2,2-diyldiphosphonate, 12a
Yield 41.2%. Yellow crystals, mp 198e200 ꢁC (from MeCN). IR
(cmꢀ1, KBr): nmax 3356w (NH),1333 (C]S),1256,1246 (2P]O),1153,
3H, H3CCOP), 1.12 (dt, JHeH ¼ 6.6, JPeH ¼ 3.6, 2 ꢂ 3H, (H3CC.O)2P),
4
3
1.91 (d, JPeH ¼ 3.8, 1H, HS), 3.23 (d, JPeH ¼ 4.7 Hz, 3H, H3CN),
3.88e4.01 (m, 6H, CH2OP & (CH2O)2 P), 7.46, 7.84 (2d, JHeH ¼ 8.4 Hz,
1061 (PeOeC). 1H NMR [CDCl3] ppm:
d
0.99, 1.13 (2dt, JHeH ¼ 6.6,
2 ꢂ 2H, HeAr). 13C NMR [CDCl3] ppm:
d
162.7 (d, 2JPeC ¼ 13.7, CSH),
4JPeH ¼ 3.4, 2 ꢂ 6H, 2(H3CCO)2), 3.58, 3.78 (2dq, JHeH ¼ 6.6,
143.8, 131.3, 126.4, 125.5, 121.3 (CeAr), 102.6 (t, 1JPeC ¼ 137.4, CeP),
61.9, 59.7 (2d, 2JPeC ¼ 11.7, CH2O), 31.1 (d, 2JPeC ¼ 15.2, CH3N), 17.9,
3JPeH ¼ 4.7, 2 ꢂ 4H, 2(H2CO)2), 7.38, 8.22 (2d, JHeH ¼ 8.1, 2 ꢂ 2H,
HeAr), 8.79 (d, 3JPeH ¼ 4.6,1H, HN).13C NMR [CDCl3] ppm:
d
254.4 (d,
17.3 (2d, 3JPeC ¼ 8.8, CH3CO). 31P NMR [CDCl3] ppm:
d 19.8, 25.6 (2d,
2JPeC ¼ 12.4 Hz, C]S),153.6,134.3,128.3,126.5,112.3 (CeAr), 83.5 (t,
2JPeP ¼ 22, CP2). EI-MS: in m/z (%): 391 (15) [Mþ], 376 (28)
[Mþ ꢀ 15], 343 (14) [Mþ ꢀ (15 þ 33)(Me þ SH)], 251 (46)
1JPeC ¼ 137.4, CeP2), 61.7 (d, 2JPeC ¼ 13.7, CH2OP),16.4 (d, 3JPeC ¼ 8.8,
H3CCOP). 31P NMR [CDCl3] ppm:
d
22.4, 25.7 (2d, 2JPeP ¼ 28, CP2). EI-
[Mþ
ꢀ (15 þ 33 þ 92)(Me þ SH þ C2H5O2P)], 114 (100)
MS: in m/z (%): 420 (11) [Mþ ꢀ 1], 388 (16) [Mþ ꢀ 32(S)], 282 (32)
[Mþ ꢀ 137 (C4H10O3P)], 146 (48) [Mþ ꢀ 274 (C4H10O3P)2], 114 (100)
[Mþ ꢀ (32 þ 274)S þ (C4H10O3P)2}], 102 (38), 77 (96). Anal. Calcd for
C16H25NO6P2S (421.4): C, 45.60; H, 5.98; N, 3.32; P, 14.70; S, 7.61.
Found: C, 45.67; H, 6.03; N, 3.27; P, 14.64; S, 7.52.
[Mþ ꢀ (15 þ 33 þ 229)(Me þ SH þ C6H15O5P2)], 102 (42), 77 (88).
Anal. Calcd for C15H23NO5P2S (391.4): C, 46.03; H, 5.92; N, 3.58; P,
15.83; S, 8.19. Found: C, 46.11; H, 5.98; N, 3.54; P, 15.76; S, 8.13.
5.4. Reactions of 4ae4d with 10
5.2.2. Diethyl (2-ethoxy-4-mercapto-2-oxido-1,2-dihydro-1,2-
benzazaphosphinin-3-yl)phosphonate, 14a
Reagents: NaOEt (0.2 g of Na, 9.12 mmol in 30 mL EtOH), BP-
reagent 10 (1.2 mL, 4.2 mmol), and the substrates 4ae4d
(3.8 mmol). The product mixture was washed several times with
light petroleum (40e60 ꢁC), and recrystallized from the solvent
indicated after the mp, to give BPs 18aed.
Yield 38.3%. Yellow needles, mp 219e221 ꢁC (from CHCl3). IR
(cmꢀ1, KBr): nmax 3429e3354 (br, NH, SH), 1421 (SH), 1248, 1226
(2P]O, bonded), 1156, 1086 (PeOeC). 1H NMR [CDCl3] ppm:
d
1.02
4
(dt, JHeH ¼ 6.6, JPeH ¼ 3.5, 3H, H3CCO), 1.26 (dt, JHeH ¼ 6.6,
4JPeH ¼ 3.6, 2 ꢂ 3H, (H3CCO)2P), 1.93 (br, 1H, HS), 3.85e4.03 [m,
6H, CH2 OP & (CH2O)2 P], 7.41, 7.88 (2d, JHH ¼ 8.4, 2 ꢂ 2 H, HeAr),
5.4.1. Tetraethyl 2-phenyl-4-thioxo-3,4-dihydroquinoline-3,3-
diyldiphosphonate, 18a
2
8.80 (br, 1H, HN). 13C NMR [CDCl3] ppm:
d
164.7 (d, JPeC ¼ 13.5,
CSH), 141.8, 131.3, 126.4, 123.5, 121.3 (CeAr), 106.6 (t,
1JPeC ¼ 148.4, CeP), 62.9, 60.1 (2d, 2JPeC ¼ 11.7, 2 ꢂ CH2O), 17.8, 17.6
Yield 72.4%. Yellow crystals, mp 191e193 ꢁC (from MeCN). IR
(cmꢀ1, KBr): nmax 1556 (C]N), 1330 (C]S), 1262, 1250 (2P]O),
(2d, 3JPeC ¼ 4.8, 2 ꢂ CH3C.O). 31P NMR [CDCl3] ppm:
d
20.4, 21.7 (2d,
1168, 1110 (2PeOeC). 1H NMR [CDCl3] ppm:
d
1.15, 1.43 (2dt,
2JPeP ¼ 29, CP2). EI-MS: in m/z (%): 376 (22) [Mþ ꢀ 1], 343 (17)
[Mþ ꢀ 33 (SH)], 251 (41) [Mþ ꢀ (33 þ 92) (SH þ C2H5O2P)], 114
(100) [Mþ ꢀ (33 þ 229) (SH þ C6H15O5P2)], 102 (46), 77 (92). Anal.
Calcd for C14H21NO5P2S (377.3): C, 44.56; H, 5.61; N, 3.71; P,16.42; S,
8.50. Found: C, 44.61; H, 5.69; N, 3.67; P, 16.50; S, 8.57.
JHeH ¼ 6.6, JPeH ¼ 3.6, 2 ꢂ 6H, 2(H3CCO)2), 4.00, 4.34 (2dq,
JHeH ¼ 6.6, 3JPeH ¼ 4.5, 2 ꢂ 4H, 2(H2CO)2), 7.38, 7.56, 7.68 (3m, 7H,
HeAr), 8.39, 8.42 (2d, JHeH ¼ 7.8, 2H, HeAr). 13C NMR [CDCl3]
4
2
2
ppm:
d
265.8 (d, JPeC ¼ 18.6, C]S), 171.6 (d, JPeC ¼ 22.7,
C(2) ¼ N), 151.1, 138.6, 135.7, 133.1, 130.7, 129.1, 128.2, 127.3
2
1
(CeAr), 61.8 (d, JPeC ¼ 9.7, CH2O), 58.8 (t, JPeC ¼ 148.6, CeP2),
3
5.3. Reaction of 2b with 10
16.1 (d, JPeC ¼ 8.2, CH3CO). 31P NMR [CDCl3] ppm:
d 22.4, 25.3
(2d, JPeP ¼ 28, CP2). EI-MS: in m/z (%): 510 (17) [Mþ þ 1], 509
2
Reagents: NaOEt (0.2 g of Na, 9.1 mmol in 30 mL EtOH), BP-
reagent 10 (1.2 mL, 4.2 mmol), and the substrate 2b (0.86 g,
3.8 mmol). The product mixture was washed several times with
light petroleum (40e60 ꢁC), and recrystallized from the solvent
indicated after the mp, to give BPs 12b, and 14b.
(13) [Mþ], 465 (20) [Mþ
ꢀ
44 (CS)], 235 (100)
[Mþ
ꢀ
274(C4H10O3P)2], 191 (88) [Mþ
ꢀ
(44
þ 274){CSþ
(C4H10O3P)2}],105 (67), 77 (78). Anal. Calcd for C23H29NO6P2S
(509.5): C, 54.22; H, 5.74; N, 2.75; P, 12.16; S, 6.29. Found: C,
54.28; H, 5.82; N, 2.69; P, 12.09; S, 6.23.

246
A.A. Kamel et al. / European Journal of Medicinal Chemistry 51 (2012) 239e249
5.4.2. Tetraethyl 2-methyl-4-thioxo-3,4-dihydroquinoline-3,3-
diyldiphosphonate, 18b
was added 20 mmol of P2S5 in 100 mL of dry xylene in one portion.
The mixture was boiled under reflux for 6e8 h (TLC), filtered upon
hot and then concentrated to its half. The product that separated on
cooling was recrystallized from the solvent indicated after the mp,
to give the corresponding thiones 6ae6d.
Yield 71.8%. Yellow crystals, mp 171e173 ꢁC (from CHCl3). IR
(cmꢀ1, KBr): nmax 1556 (C]N),1329 (C]S),1251,1244 (2P]O),1135,
1048 (2PeOeC). 1H NMR [CDCl3] ppm:
d
1.16, 1.33 (2dt, JHeH ¼ 6.3,
4JPeH ¼ 3.5, 2 ꢂ 6H, 2(H3CCO)2), 2.41 (d, 4JPeH ¼ 3.4, 3H, H3C), 4.04,
4.26 (2dq, JHeH ¼ 6.6, 3JPeH ¼ 4.6, 2 ꢂ 4H, 2(H2CO)2), 7.25, 7.78 (2d,
5.5.1. 4-(4-Methylphenyl)-1H-2,3-benzothiazine-1-thione, 6a
JHeH ¼ 8.2, 2 ꢂ 2 H, HeAr). 13C NMR [CDCl3]:
d
264.5 (d, 2JPeC ¼ 14.6,
Yield 53.7%. Red crystals, mp 178e180 ꢁC (from EtOH). IR (cmꢀ1
,
2
C]S), 173.6 (d, JPeC ¼ 16.8, C]N), 151.7, 150.8, 137.3, 130.4, 128.3,
128.03 (CeAr), 61.2 (d, 2JPeC ¼ 9.7, CH2O), 57.8 (t,1JPeC ¼ 148.4, CeP2),
24.3 [d, 3JPeC ¼ 7.8, CH3eC(2)], 16.9 (d, 3JPeC ¼ 6.8, CH3CO). 31P NMR
KBr): nmax 1562 (C]N), 1369 (C]S). 1H NMR [CDCl3] ppm:
(s, 3H, H3Cetolyl), 7.32e8.37 (m, 8H, HeAr). 13C NMR [CDCl3] ppm:
213.4 (C]S),163.4 (C(4)]N),139.1,137.3,130.9,129.8,129.2,127.5,
d 2.51
d
[CDCl3] ppm:
d
23.4, 28.7 (2d, 2JPeP ¼ 34, CP2). EI-MS: in m/z (%): 448
125.9, 125.1 (CeAr), 21.6 (CH3etolyl). EI-MS: in m/z (%): 269 (33)
[Mþ], 254 (100) [Mþ ꢀ 15 (Me)], 193 (68) [Mþ ꢀ 76 (CS2)], 178 (21)
[Mþ ꢀ (15 þ 76) (Me þ CS2)],102 (68), 77 (98). Anal. Calcd for C15H11
NS2 (269.4): C, 66.88; H, 4.12; N, 5.20; S, 23.81. Found: C, 66.91; H,
4.17; N, 5.13; S, 23.76.
(15) [Mþ þ 1], 447 (12) [Mþ], 432 (36) [Mþ ꢀ 15], 388 (18)
[Mþ ꢀ (15 þ 44)], 158 (100) [Mþ ꢀ (15 þ 274){Me þ (C4H10O3P)2}],
114 (80), [Mþ ꢀ (15 þ 44 þ 274){Me þ CS þ (C4H10O3P)2}],105 (64),
77 (84). Anal. Calcd for C18H27NO6P2S (447.4): C, 48.32; H, 6.08; N,
3.13; P,13.85; S, 7.17. Found: C, 48.38; H, 6.12; N, 3.07; P,13.76; S, 7.19.
5.5.2. 4-(4-Methoxyphenyl)-1H-2,3-benzothiazine-1-thione, 6b
Yield 57.4%. Yellow crystals, mp 190e193 ꢁC (from CHCl3). IR
(cmꢀ1, KBr): nmax 1552 (C]N), 1370 (C]S). 1H NMR [CDCl3] ppm:
5.4.3. Tetraethyl 2-cyclohexyl-4-thioxo-3,4-dihydroquinoline-3,3-
diyldiphosphonate, 18c
Yield 73.6%. Red crystals, mp 118e120 ꢁC (from benzene); IR
d
3.53 (s, 3H, H3CO), 7.12e8.37 (m, 8H, HeAr). 13C NMR [CDCl3]
(cmꢀ1, KBr): nmax 1548 (C]N),1325 (C]S),1259,1242 (2P]O),1135,
ppm: d 213.4 (C]S), 163.4 (C(4)]N), 159.7, 137.2, 130.9, 129.2, 127.5,
1066 (2PeOeC). 1H NMR [CDCl3] ppm:
d
1.24, 1.38 (2dt, JHeH ¼ 7.6,
125.8, 125.1 (CeAr), 55.4 (CH3O). EI-MS: in m/z (%): 285 (42) [Mþ],
254 (100) [Mþ ꢀ 31(OMe)], 179 (27) [Mþ ꢀ (31 þ 76) (OMe þ CS2)],
102 (58), 77 (98). Anal. Calcd for C15H11NOS2 (285.4): C, 63.13; H,
3.89; N, 4.95; S, 22.47. Found: C, 63.17; H, 3.93; N, 4.89; S, 22.55.
4JPeH ¼ 3.7, 2 ꢂ 6H, 2(H3CCO)2),1.98 (m, 10H, H2C-hexyl), 3.43 (m,
3
1H, CHehexyl), 4.11, 4.44 (2dq, JHeH ¼ 7.6, JPeH ¼ 4.7, 2 ꢂ 4H,
2(H2CO)2), 7.55, 7.78 (2d, JHeH ¼ 8.2, 2 ꢂ 2H, HeAr). 13C NMR [CDCl3]
ppm:
d
266.6 (d, 2JPeC ¼ 13.6, C]S), 178.6 (d, 2JPeC ¼ 15.8, C(2) ¼ N),
2
151.8, 137.3, 136.3, 130.4, 128.3, 128.1(CeAr), 61.2 (d, JPeC ¼ 9.7,
5.5.3. 4-Phenyl-1H-2,3-benzothiazine-1-thione, 6c
1
3
CH2O), 58.8 (t, JPeC
¼
148.4, CeP2), 34.3 (d, JPeC
¼
9.2,
Yield 62.4%. Yellow crystals, mp 158e160 ꢁC (from MeCN). IR
(cmꢀ1, KBr): nmax 1554 (C]N), 1366 (C]S). 1H NMR [CDCl3]:
3
CHehexyl), 32.8, 26.6, 26.1 (CH2ehexyl), 16.3 (d, JPeC ¼ 9.2,
H3CC.O). 31P NMR [CDCl3] ppm:
d
24.1, 28.9 (2d, JPeP ¼ 18, CP2).
d d 213.4 (C]S),
7.33e8.37 (m, 9H, HeAr). 13C NMR [CDCl3] ppm:
2
EI-MS: in m/z (%): 516 (13) [Mþ þ 1], 515 (9) [Mþ], 513 (16) [Mþ ꢀ 2],
509 (18) [Mþ ꢀ 6], 465 (34) [Mþ ꢀ 6 þ 44 (6H þ CS)], 235 (100)
[Mþ ꢀ (6 þ 274) {6H þ (C4H10O3P)2}], 191 (88) [Mþ ꢀ (6 þ 44 þ 274)
{6H þ CS þ (C4H10O3P)2}], 105 (68), 77 (74). Anal. Calcd for
C23H35NO6P2S (515.5): C, 53.58; H, 6.84; N, 2.72; P, 12.02; S, 6.22.
Found: C, 53.64; H, 6.91; N, 2.66; P, 12.12; S, 6.29.
163.4 (C(4)]N), 137.2, 134.1, 133.9, 131.6, 129.6, 127.3, 125.9 (CeAr).
EI-MS: in m/z (%): 255 (100) [Mþ]. 179 (78) [Mþ ꢀ 76 (CS2)], 102
(76), 77 (90). Anal. Calcd for C14H9NS2 (255.4): C, 65.85; H, 3.55; N,
5.49; S, 25.11. Found: C, 65.89; H, 3.61; N, 5.41; S, 25.15.
5.5.4. 4-[4-(Diethylamino)phenyl]-1H-2,3-benzothiazine-1-
thione, 6d
5.4.4. Tetraethyl 6-bromo-2-methyl-4-thioxo-3,4-
Yield 66.3%. Yellow crystals, mp 111e113 ꢁC (from MeCN). IR
dihydroquinoline-3,3-diyldiphosphonate, 18d
(cmꢀ1, KBr): nmax 1558 (C]N), 1373 (C]S). 1H NMR [CDCl3] ppm:
Yield 70.6%. Yellowish brown substance, mp 177e179 ꢁC (from
d
0.94 (t, 6H, H3C.CN), 3.54 (q, 4H, H2CN), 6.48e8.37 (m, 8H, HeAr).
CH2Cl2). IR (cmꢀ1, KBr): nmax 1556 (C]N),1331 (C]S),1260,1247 (P]
13C NMR [CDCl3] ppm:
d
213.4 (C]S), 163.4 (C(4)]N), 149.1, 146.8,
O), 1154, 1078 (PeOeC). 1H NMR [CDCl3] ppm:
d
1.26, 1.30 (2dt,
139.2, 130.9, 129.4, 127.2, 126.9, 115.1 (CeAr), 44.8 (CH2N), 12.7
(CH3C. N). EI-MS: in m/z (%): 326 (32) [Mþ], 254 (100) [Mþ ꢀ 72
(NEt2)], 250 (38) [Mþ ꢀ 76 (CS2)], 178 (78) [Mþ ꢀ (72 þ 76)
{NEt2 þ CS2}], 102 (66), 77 (81). Anal. Calcd for C18H18N2S2 (326.5):
C, 66.22; H, 5.56; N, 8.58; S, 19.64. Found: C, 66.26; H, 5.61; N, 8.51;
S, 19.72.
JHeH ¼ 7.6, 4JPeH ¼ 4.3, 2 ꢂ 6H, 2(H3CCO)2), 2.49 (d, 4JPeH ¼ 3.6, 3H,
H3C), 4.04, 4.26 (2dq, JHeH ¼ 6.6, 3JPeH ¼ 12.8, 2 ꢂ 4H, 2(H2CO)2), 7.20,
7.53 (2d, JHeH ¼ 8.1, 2 ꢂ1H, HeAr), 8.58 (s,1H, HeAr).13C NMR [CDCl3]
2
ppm:
d
267.5 (d, JPeC ¼ 15.6, C]S), 173.6 (d, 2JPeC ¼ 11.8, C(2)]N),
152.7, 147.8, 131.3129.4, 128.4, 127.3 (CeAr), 121.83 (CeBr), 61.2 (d,
2JPeC ¼ 10.7, CH2O), 57.8 (t, 1JPeC ¼ 148.4, CeP2), 24.3 (d, 3JPeC ¼ 4.4,
CH3), 16.4 (d, JPeC ¼ 4.8, CH3CO). 31P NMR [CDCl3] ppm:
d
26.4,
5.6. General procedure of BPs 21ae21d
3
29.2 (2d, JPeP ¼ 28, CP2). EI-MS: in m/z (%): 526 (40) [Mþ], 527 (3)
[Mþ þ 1], 528 (2) [Mþ þ 2], 511 (28) [Mþ ꢀ 15 (Me)], 432 (46)
[Mþ ꢀ (15 þ 80)(Me þ Br)], 387 (36) [Mþ ꢀ (15 þ 80 þ 44)
2
Following the general procedure, a mixture of 9.2 mmol of Na,
4.2 mmol of 10, and 3.8 mmol of 4-(4-methylphenyl)-1H-2,3-benzo-
thiazine-1-thione 6a, 4-(4-methoxyphenyl)-1H-2,3-benzothiazine-
1-thione 6b, 4-phenyl-1H-2,3-benzothiazine-1-thione 6c or 4-[4-
(diethylamino)-phenyl]-1H-2,3-benzothiazine-1-thione 6d in 30 mL
EtOH was heated under reflux for 15e20 h (TLC). After the usual
workup, the resulting residue was collected, washed several times
with pentane and recrystallized from the solvent indicated after the
mp, to give the respective BPs 21ae21d.
(Me
þ
Br
þ
CS)], 193 (100) [Mþ
ꢀ
(15
þ
44
þ
274)
{Me þ CS þ (C4H10O3P)2}], 114 (80), 105 (55), 77 (74). Anal. Calcd for
C18H26BrNO6P2S (526.3): C, 41.08; H, 4.98; Br,15.18; N, 2.66; P,11.77; S,
6.09. Found: C, 41.12; H, 5.06; Br, 15.12; N, 2.57; P, 11.71; S, 6.03.
5.5. General procedure of 4-aryl-1H-2,3-benzothiazine-1-
thiones, 6ae6d
To
a
solution of 10 mmol of 4-(4-methylphenyl)-2,3-
5.6.1. Tetraethyl 4-thioxo-1-p-tolyl-3,4-dihydroisoquinoline-3,3-
diyldiphosphonate, 21a
benzoxazin-1-one, 5a, 4-(4- methoxy-phenyl)-2,3-benzoxazin-1-
one, 5b, 4-(4-methylphenyl)-2,3-benzoxazin-1-one, 5c or 4-[4-
(diethylamino)phenyl]-2,3-benzoxazin-1-one, 5d in 10 mL xylene,
Yield 72.8%. Yellow crystals, mp 212e214 ꢁC (from MeOH). IR
(cmꢀ1, KBr): nmax 1562 (C]N), 1369 (C]S), 1252, 1239 (2P]O),

A.A. Kamel et al. / European Journal of Medicinal Chemistry 51 (2012) 239e249
247
1161, 1105 (2PeOeC). 1H NMR [CDCl3] ppm:
d
1.25, 1.31 (2dt,
{NEt2 þ CS þ (C4H10O3P)2}], 102 (76), 77 (88). Anal. Calcd for
C27H38N2O6P2S (580.6): C, 55.85; H, 6.60; N, 4.82; P, 10.67; S, 5.52.
Found: C, 55.92; H, 6.54; N, 4.77; P, 10.59; S, 5.45.
JHeH ¼ 6.6, 4JPeH ¼ 3.4, 2 ꢂ 6H, 2(H3CCO)2), 2.38 (s, 3H, H3Cetolyl),
4.02, 4.27 (2dq, JHeH ¼ 6.6, 3JPeH ¼ 4.6, 2 ꢂ 4H, 2(H2CO)2), 7.36, 7.58,
8.04, 8.74 (4m, 8H, HeAr). 13C NMR [CDCl3] ppm:
d 240.9 (d,
3
2JPeC ¼ 14.4, C]S), 178.6 (d, JPeC ¼ 7.4, C]N), 145.8, 137.7, 134.2,
5.7. General procedure of BP-acids 15, 19a, 19b, and 22
133.3, 130.9, 127.2, 126.8 (CeAr), 90.1 (t, 1JPeC ¼ 168.7, CeP2), 61.1 (d,
3
2JPeC¼ 8.3, CH2O), 21.5 (CH3etolyl), 16.3 (d, JPeC ¼ 5.3 Hz, H3CCO).
Bisphosphonate 12b, 18c, 18d, and 21d (0.5 g) was dissolved in
15 mL of conc HCl, and the mixture was heated under reflux
for z 10 h (TLC). After concentrating the product mixture to its half,
under reduced pressure, the crude material was diluted with AcOEt
and water and then stirred for 30 min. The layers were separated,
and the aqueous layer was evaporated to dryness. The precipitate
was collected and dried to give the corresponding BP-acid 15, 19a,
19b, and 22.
2
31P NMR [CDCl3] ppm:
d
20.7, 24.7 (2d, JPeP ¼ 28, CP2). EI-MS: in
m/z (%): 524 (14) [Mþ], 508 (22) [Mþ ꢀ 15, Me], 464 (36)
[Mþ ꢀ (15 þ 44) (Me þ CS)], 234 (100) [Mþ ꢀ (15 þ 274)
{Me
þ
(C4H10O3P)2}], 190 (85) [Mþ
ꢀ
(15
þ
44
þ
274)
{Me þ CS þ (C4H10O3P)2}], 102 (48), 77 (79). Anal. Calcd for
C24H31NO6P2S (523.5): C, 55.06; H, 5.97; N, 2.68; P, 11.83; S, 6.13.
Found: C, 55.11; H, 6.02; N, 2.74; P, 11.76; S, 6.05.
5.6.2. Tetraethyl 1-(4-methoxyphenyl)-4-thioxo-3,4-
5.7.1. 1-Methyl-3-thioxo-2,3-dihydro-1H-indole-2,2-
diyldiphosphonic acid, 15
dihydroisoquinoline-3,3-diyldiphosphonate, 21b
Yield 76.4%. Red crystals, mp 252e254 ꢁC (dec.) (from EtOH). IR
Yield 84.8%. White material, mp > 300 ꢁC (from MeOH). IR
(cmꢀ1, KBr): nmax 1572 (C]N),1370 (C]S),1265,1252 (2P]O),1161,
(cmꢀ1, KBr): nmax 3340e3325 (PeOH), 1329(C]S), 1232e1220 (P]
1110 (2PeOeC). 1H NMR [CDCl3] ppm:
d
1.29, 1.35 (2dt, JHeH ¼ 7.6,
O, bonded). 1H NMR [DMSO/D2O] ppm:
d
3.14 (d, JPeH ¼ 3.3, 3H,
4
4JPeH ¼ 3.3, 2 ꢂ 6H, 2(H3CCO)2), 3.48 (s, 3H, H3COAr), 4.19, 4.18
(2dq, JHeH ¼ 7.6, 3JPeH ¼ 4.8, 2 ꢂ 4H, 2(H2CO)2), 6.68, 6.70, 7.25, 8.41
H3CN), 7.42, 7.85 (2d, JHeH ¼ 8.1, 2 ꢂ 2 H, HeAr). 31P NMR [DMSO/
D2O] ppm:
d
21.5, 23.7 (CP2). EI-MS: in m/z (%): 319 (30) [Mþ ꢀ 4].
(4m, 8H, HeAr).13C NMR [CDCl3] ppm:
d
239.8 (d, 2JPeC ¼ 15.6, C]S),
Anal. Calcd for C9H11NO6P2S (323.2): C, 33.45; H, 3.43; N, 4.33; P,
19.17; S, 9.92. Found: C, 33.51; H, 3.51; N, 4.28; P, 19.25; S, 9.97.
3
178.6 (d, JPeC ¼ 8.8, C]N), 159.5, 145.4, 133.9, 131.7, 130.1, 126.3,
113.5 (CeAr), 88.3 (t, 1JPeC ¼ 166.7, CeP2), 61.5 (d, 2JPeC ¼ 133, CH2O),
55.5 (CH3O), 16.3 (d, 3JPeC ¼ 6.4 Hz, H3CCO). 31P NMR [CDCl3] ppm:
5.7.2. 2-Cyclohexyl-4-thioxo-3,4-dihydroquinoline-3,3-
diyldiphosphonic acid, 19a
d
24.7, 26.8 (2d, 2JPeP ¼ 29, CP2). EI-MS: in m/z (%): 539 (<8) [Mþ],
508 (33) [Mþ ꢀ 31, OMe], 464 (24) [Mþ ꢀ (31 þ44) (OMe þ CS)], 234
Yield 77.3%. White material, mp > 300 ꢁC (from MeOH). IR
(cmꢀ1, KBr): nmax 3338e3332 (PeOH), 1556 (C]N), 1322 (C]S),
(100) [Mþ
ꢀ
(31
þ
274) {OMe
þ
(C4H10O3P)2}], 190 (73)
[Mþ ꢀ (31 þ 44 þ 274) {Me þ CS þ (C4H10O3P)2}], 102 (62), 77 (88).
Anal. Calcd for C24H31NO7P2S (539.5): C, 53.43; H, 5.79; N, 2.60; P,
11.48; S, 5.94. Found: C, 53.51; H, 5.84; N, 2.53; P, 11.39; S, 5.86.
1233e1219 (P]O, bonded). 1H NMR [DMSO/D2O] ppm:
d 1.98 (m,
10H, CH2ehexyl), 3.43 (m, 1H, CHehexyl), 7.65, 7.88 (2d, JHeH ¼ 8.2,
2 ꢂ 2H, HeAr). 31P NMR [DMSO/D2O] ppm:
d 20.6e21.8 (broad,
CP2). EI-MS: in m/z (%): 399 (21) [Mþ ꢀ 4]. Anal. Calcd for
C15H19NO6P2S (403.3): C, 44.67; H, 4.75; N, 3.47; P, 15.36; S, 7.95.
Found: C, 44.73; H, 4.81; N, 3.41; P, 15.29; S, 7.88.
5.6.3. Tetraethyl 1-phenyl-4-thioxo-3,4-dihydroisoquinoline-3,3-
diyldiphosphonate, 21c
Yield 74.7%. Yellow crystals, mp 176e178 ꢁC (from EtOH). IR
(cmꢀ1, KBr): nmax 1564 (C]N), 1374 (C]S), 1261, 1250 (2P]O),
5.7.3. 6-Bromo-2-methyl-4-thioxo-3,4-dihydroquinoline-3,3-
diyldiphosphonic acid, 19b
1162, 1113 (2PeOeC). 1H NMR [CDCl3] ppm:
d 1.25, 1.62 (2dt,
4
JHeH ¼ 7.6, JPeH ¼ 3.6, 12H, H3CC.OP), 4.42, 4.67 (2dq, JHeH ¼ 7.6,
Yield 80.7%. White material, mp > 300 ꢁC (from MeOH). IR
3JPeH ¼ 4.6, 2 ꢂ 4H, 2(H2CO)2), 7.08e8.23 (m, 9H, HeAr). 13C NMR
(cmꢀ1, KBr): nmax 3342e3325 (PeOH), 1559 (C]N), 1322 (C]S),
[CDCl3] ppm:
d
239.2 (d, 2JPeC ¼ 14.4, C]S), 178.6 (d, 3JPeC ¼ 6.8 Hz,
1229e1217 (P]O, bonded). 1H NMR [DMSO/D2O] ppm:
d
2.49 (s,
C]N), 145.8, 137.7, 133.7, 133.2, 128.9, 127.3, 126.1 (CeAr), 90.4 (t,
3H, H3C), 7.50, 7.75 (2d, JHeH ¼ 8.1, 2 ꢂ 1H, HeAr), 8.58 (s,1H, HeAr).
1JPeC ¼ 166.7, CeP2), 61.1 (d, JPeC¼ 8.3 Hz, CH2OP), 16.4 (d,
31P NMR [DMSO/D2O] ppm:
d 20.4, 23.7 (CP2). EI-MS: in m/z (%): 410
2
3JPeC ¼ 5.3, H3CCO). 31P NMR [CDCl3] ppm:
d
20.7, 24.7 (2d,
(52) [Mþ ꢀ 4]. Anal. Calcd for C10H10BrNO6P2S (414.1): C, 29.00; H,
2.43; Br, 19.30; N, 3.38; P, 14.96; S, 7.74. Found: C, 29.08; H, 2.51; Br,
19.22; N, 3.31; P, 15.08; S, 7.81.
2JPeP ¼ 32, CP2). EI-MS: in m/z (%): 509 (14) [Mþ], 465 (28) [Mþ ꢀ 44,
CS], 235 (100) [Mþ ꢀ 274 (C4H10O3P)2], 190 (92) [Mþ ꢀ (44 þ 274)
{CS þ (C4H10O3P)2}], 102 (65), 77 (96). Anal. Calcd for C24H31NO6P2S
(509.5): C, 54.22; H, 5.74; N, 2.75; P, 12.16; S, 6.29. Found: C, 54.30;
H, 5.81; N, 2.68; P, 12.09; S, 6.13.
5.7.4. 1-(4-(Diethylamino)phenyl)-4-thioxo-3,4-
dihydroisoquinoline-3,3-diyldiphosphonic acid, 22
Yield 72.4%. White material, mp > 300 ꢁC (from MeOH). IR
5.6.4. Tetraethyl 1-(4-(diethylamino)phenyl)-4-thioxo-3,4-
dihydroisoquinoline-3,3-diyldi-phosphonate, 21d
(cmꢀ1, KBr): nmax 3342e3325 (PeOH), 1550 (C]N), 1328 (C]S),
1228e1222 (P]O, bonded). 1H NMR [DMSO/D2O] ppm:
d
0.94 (t,
Yield 76.2%. Red crystals, mp 129e131 ꢁC (from cyclohexane). IR
6H, (H3CC)2N), 3.54 (q, 4H, (H2C)2N), 7.21, 7.57, 7.63, 8.96 (4m, 8H,
(cmꢀ1, KBr): nmax 1559 (C]N), 1367 (C]S), 1261, 1258 (2P]O), 1160,
HeAr). 31P NMR [DMSO/D2O] ppm:
d 19.5, 22.8 (broad, CP2). EI-MS:
1100 (2PeOeC).1H NMR [CDCl3] ppm:
d
0.93 (t, 6H, H3CCN),1.23,1.69
in m/z (%): 464 (18) [Mþ ꢀ 4]. Anal. Calcd for C19H22N2O6P2S
(468.4): C, 48.72; H, 4.73; N, 5.98; P, 13.23; S, 6.85. Found: C, 48.81;
H, 4.79; N, 5.89; P, 13.16; S, 6.77.
(2dt, JHeH ¼ 7.6, 4JPeH ¼ 3.6, 2 ꢂ 6H, 2(H3CCO)2), 3.55 (q, 4H, H2CN),
3.69, 3.73 (2dq, JHeH ¼ 7.6, 3JPeH ¼ 4.6, 2 ꢂ 4H, 2(H2CO)2), 7.21, 7.57,
7.63, 8.96 (4m, 8H, HeAr). 13C NMR [CDCl3] ppm:
d 239.2 (d,
2JPeC ¼ 14.4, C]S),179.1 (d, 3JPeC ¼ 6.8, C]N),149.8,147.7,133.7,131.2,
5.8. Pharmacology
1
128.9, 127.3, 126.7 (CeAr), 92.1 (t, JPeC ¼ 168.7, CeP2), 61.2 (d,
2JPeC ¼ 8.3, CH2O), 44.8 (CH2N), 16.4 (d, JPeC ¼ 5.3, H3CC.O), 15.8
5.8.1. Antitumor activity screening
Following the technique, previously reported by Skehan et al.
[30], antitumor activity screening of BPs 12b, 18a, and 21a was
3
(H3CC)2N. 31P NMR [CDCl3] ppm:
d
21.7, 25.7 (2d, 2JPeP ¼ 24, CP2). EI-
MS: in m/z (%): 581 (14) [Mþ þ 1], 580 (10) [Mþ], 508 (26) [Mþ ꢀ 72
(NEt2)], 464 (33) [Mþ ꢀ (72 þ 44) (NEt2 þ CS)], 234 (100)
[Mþ ꢀ (72 þ 274) (C4H10O3P)2)], 191 (87) [Mþ ꢀ (72 þ 44 þ 274)
evaluated at doses of 0, 5, 12.5, 25, and 50
mM/kg. Four different
human carcinoma cell lines, representing breast, cervix, liver, and

248
A.A. Kamel et al. / European Journal of Medicinal Chemistry 51 (2012) 239e249
colon were utilized based on comparison to the behavior of DOX or
CIS (Table 1).
vehicle solution in 500, 750, and 1000 mg/kg (body weight) were
given intra-peritoneally. The control groups were given in buffer
solution only. The toxic symptoms, mortality rates and postmortem
findings in each group were recorded 24 h post-administration.
LD50 of the tested compounds were calculated according to the
following formula:
5.8.2. Methods and materials
(i) Cells were plated in 96-multivated plate (104 cells/well) for
24 h before treatment with compounds to allow the attachment of
cells to the wall of the plate; (ii) different concentrations of each
X
LD50 ¼ Dm
ꢀ
ðz ꢂ dÞ=n
compound under test (5, 12.5, 25 and 50 mg/mL) were added to the
cell monolayer triplicate wells that prepared for each individual
dose. Each concentration is evaluated three times (each dose is
incubated with the cells in three different wells) and the monolayer
cells were incubated with the compounds for 48 h at 37 ꢁC and in
atmosphere of 5% CO2; (iii) after 48 h, cells were fixed, washed and
stained with sulfo-rhodamine-B-Stain; (iv) excess stain was
washed with acetic acid and attached stain was recovered with tris
EDTA buffer (pH 7.4); (v) color intensity was measured in an ELISA
plate reader; (vi) The relation between surviving fraction and drug
concentration is displayed in Table 1, and it is plotted to get the
survival curve of each tumor cell after the specified examined
compound (Fig. 1).
Where, Dm ¼ the largest dose which kill all animals, z ¼ mean of
dead animals between two successive groups, d ¼ the constant
factor between two successive doses, n ¼ number of animals in
each group, S ¼ the sum of (z ꢂ d).
Acknowledgment
We thank National Cancer Institute, Cairo University, Egypt, for
carrying out the anticancer screening.
Appendix A. Supplementary material
5.9. Adjuvant-induced polyarthritis
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2012.02.047.
Groups of 10 male rats (200 g) were challenged with an intra-
dermal injection of complete Freund’s adjuvant (Mycobacterium
tuberculosis in mineral oil) in the left hindpaw on day 0. Tested
compounds were dissolved or suspended in sterile saline and
sonicated where appropriate to homogeneous doses. All
compounds were then adjusted to pH 7.0 with 1 N NaOH and stored
frozen in aliquots. Fresh aliquots were used for each day of dosing.
Rats were dosed once daily (subcutaneous, sc) for 28 days on
a mg/kg of body weight basis. The normal rat control group
received vehicle po (prolecebo) and the arthritic rat control group
received vehicle sc. Changes in paw volume over time (APV) which
occurred in the arthritic control and treated rats (injected and
noninjected hindpaw) were quantitated on day 28 by mercury
displacement plethysmography. Results were analyzed by one way
analysis of variants, then Student’s unpaired t test. The results were
displayed in Table 2.
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