Synthesis of novel phosphorylated guanidine derivatives from cyanamide and their anti-inflammatory activity

Article abstract of DOI:10.1248/cpb.c12-00582

A series of novel guanidine derivatives were synthesized in three steps and their anti-inflammatory activities in vitro and in vivo evaluated. 2-Aminopyridin-3-ol (1) was reacted with thiophosphoryl chloride (2) to give a monochloride (3). It was further reacted with cyanamide to afford the corresponding cyanamine (4), which was subsequently reacted with different heterocyclic amines to form the title compounds (5a-l). The substituent in the guanidine function affected the potency of anti-inflammatory activity. The compounds having benzothiazole, fluorophenyl, and piperazinyl moieties enhanced the anti-inflammatory activity.

Full text of DOI:10.1248/cpb.c12-00582

Regular Article
Chem. Pharm. Bull. 61(1) 25–32 (2013)
January 2013
25
Synthesis of Novel Phosphorylated Guanidine Derivatives from
Cyanamide and Their Anti-inflammatory Activity
Venkata Ramana Katla,a Rasheed Syed,a Chandra Sekhar Kuruva,a
,a
Hema Kumar Kuntrapakam,b and Naga Raju Chamarthi*
b
a Department of Chemistry, Sri Venkateswara University; Tirupati 517–502, India: and Aptus BioSciences Private
Limited; Mahabub Nagar 509–002, India. Received June 30, 2012; accepted September 24, 2012
A series of novel guanidine derivatives were synthesized in three steps and their anti-inflammatory
activities in vitro and in vivo evaluated. 2-Aminopyridin-3-ol (1) was reacted with thiophosphoryl chloride
(2) to give a monochloride (3). It was further reacted with cyanamide to afford the corresponding cyanamine
(4), which was subsequently reacted with different heterocyclic amines to form the title compounds (5a–l).
The substituent in the guanidine function affected the potency of anti-inflammatory activity. The compounds
having benzothiazole, fluorophenyl, and piperazinyl moieties enhanced the anti-inflammatory activity.
Key words phosphorylated guanidine; 2-aminopyridin-3-ol; anti-inflammatory activity
Compounds containing a guanidine functional group ex- 2-aminopyridin-3-ol (1) with thiophosphoryl chloride (2) in
hibit many biological, chemical and medicinal applications.1) tetrahydrofuran (THF) using triethylamine (TEA) as mild
Guanidine is a ubiquitous group in natural products and plays base by stirring at room temperature for 3h. The formation of
a key role in many biological activities.2) In peptides, gua- monochloride 3 was ascertained by TLC eluting with a 3:2
nidine, a residue of l-arginine, exists in the protonated form mixture of ethyl acetate and hexane. After completion of the
as guanidinium ions functioning as an efficient recognition reaction, the resulting triethylamine hydrochloride (Et3N–HCl)
moiety of anionic substrates such as carboxylate, phosphate was removed from the reaction mixture by filtration. The
and intonate functionalities.3) A wide range of biological filtrate containing the monochoride 3 was used for the next
activities and chemical applications have been found for gua- step of the reaction without further purification. Second, cy-
nidine derivatives. These have motivated the development anamide was added in the presence of TEA to the filtrate and
of many reagents for their synthesis and various methods to stirred at 30–40°C for 2h. After completion of the reaction,
achieve guanylation by nucleophilic addition of an amine to the mixture was concentrated under vacuum and the residue
a cyanamide.4) The guanidine derivatives exhibited diverse purified by column chromatography on silica gel (100–200
biological and pharmacological activities such as neuronal mesh) using petroleum ether–ethyl acetate (3:7) as elutent to
Na+ and Ca2+ channel blockers,5) glutamate release inhibi- obtain the P-cyanamine intermediate 4. Finally, the title com-
tors, anti-ischemic agents,6) anti-seizure agents,7) aderenergic pounds (5a–l) were prepared by treatment of 4 with various
neuron-blocking agents,8) human immunodeficiency virus-1 heterocyclic/aryl amines in the presence of TEA with stirring
(HIV-1) protease inhibitors,9) K+/ATP channel openers, nitric at 50–60°C for 2h. All the title compounds were purified by
oxide (NO) synthase inhibitors, influenza neuraminidase in- column chromatography on silica gel (100–200 mesh) using
hibitors,10) cardiotonic agents,11) and histamine H3 receptor petroleum ether–ethyl acetate (2:3) as eluent; the title com-
antagonists.12) Therefore, the guanidine functional group is an pounds were obtained in excellent yields (71–88%).
attractive pharmacophore for the development of novel drugs
The intermediate 4 and all the title compounds were
1
against tumors, hypertension, glaucoma, and inflammation.13) characterized by IR, H-, 13C- and 31P-NMR, mass spectra,
We previously reported that phosphorylation of various drugs and elemental analysis. The disappearance of band at 3425
1
improves their biological activity.14) This finding led us to in- (–O–H) in IR, 10.2ppm (–OH) in H-NMR of 2-aminopyri-
vestigate the anti-inflammatory activities of a novel series of din-3-ol (1) and appearance of bands at 2323 (–CN), 764
1
phosphorylated guanidine derivatives. Their anti-inflammatory (–P=S) in IR, 3.58ppm (–NH–CN) in H-NMR, 31P-NMR
activities in vitro and in vivo were compared versus those of and molecular ions confirm the formation of key intermedi-
conventional anti-inflammatory drugs such as aspirin, diclo- ate compound 4. Finally, IR bands appeared in the regions
fenac sodium, phenyl butazone, and flufenamic acid.
of 3472–3436, 3358–3322, and 1670–1624 for N–H, C=N–H,
1
and C=N, respectively. H-NMR spectra exhibited peaks at
δ 3.93–5.62 (br), 6.89–8.00, and 8.31–10.43 due to guanidine
Results and Discussion
Chemistry The synthesis of new biologically active –NH, aromatic protons, and –C=N–H protons, respectively.
substituted N′-(2-thioxo-2,3-dihydro-2λ5-pyrido[2,3-d][1,3,2] 31P-NMR chemical shifts were observed in the region −11.8 to
oxazaphosphol-2-yl)guanidine derivatives was accomplished 15.2 ppm.15) In addition, 13C-NMR, mass spectra, and elemen-
in straight forward manner as shown in Chart 1. To syn- tal analysis provided further evidence for confirmation of the
thesize target title derivatives, the key intermediate com- title compounds (5a–l).
pound 4 was achieved in two steps. First, the monochloride
intermediate,
Biological Evaluation. In Vitro Anti-inflammatory
2-chloro-2,3-dihydro-2λ5-pyrido[2,3-d][1,3,2] Activity (a) Inhibition of Albumin Denaturation: The
oxazaphosphole-2-thione (3) was synthesized by reaction of newly synthesized compounds 5a–l were assayed for their
anti-inflammatory activity with respect to inhibition of al-
bumin denaturation.16) In this method, the reaction mixture
The authors declare no conflict of interest.
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: rajuchamarthi10@gmail.com

26
Vol. 61, No. 1
Chart 1
comprised test compounds at different concentrations and 1% mL. Flufenamic acid, a standard anti-inflammatory drug,
aqueous solution of bovine albumin fraction. pH of the reac- showed maximum inhibition of, 85.36 0.56% at a concentra-
tion mixture was adjusted to 7 using small amount of 1n HCl. tion of 0.2µg/mL. In this bioassay 5a, 5b, 5g, and 5j com-
The samples were incubated at 37°C for 20min then heated at pounds exhibited promising percent inhibition compared with
57°C for 20min. After cooling the samples, the turbidity was that of standard.
measured spectrophotometrically at 660nm. The experiment
Membrane Stabilization Test (a) Preparation of Red
was performed in triplicate. Percent inhibition of protein dena- Blood Cells (RBCs) Suspension: Fresh whole human blood
turation was calculated as follows:
(10mL) was collected and transferred to heparinized centri-
fuge tubes. The tubes were centrifuged at 3000rpm for 10min
then washed three times with equal volume of normal saline.
The volume of the blood was measured and reconstituted as
10% v/v suspension with normal saline.
percentage inhibition
(absorbance control  absorbance sample)
=
×100
absorbance of control
Denaturation of protein is a well-documented cause of in-
(b) Heat-Induced Hemoloysis: The reaction mixture (2mL)
flammation. Phenylbutazone, flufenamic acid (anti-inflamma- consisting of 1mL of test drug solution and 1mL of 10%
tory drugs), etc., have shown dose-dependent ability to reduce RBCs suspension, was added to the test tube.17) Aspirin was
thermally induced protein denaturation. As part of the inves- taken as a standard drug. All the centrifuge tubes containing
tigation on the mechanism of the anti-inflammatory activity, reaction mixture were incubated in a water bath at 56°C for
ability of extract to inhibit protein denaturation was studied.
30min. At the end of the incubation, the tubes were cooled
It was effective in inhibiting heat-induced albumin denatur- under tap water. The reaction mixture was centrifuged at
ation at different concentrations as shown in (Table 1, Fig. 1). 2500rpm for 5min and the absorbance of the supernatants
Maximum inhibition, 85.92 1.48%, was observed at 0.8µg/ taken at 560nm. The experiments were performed in triplicate

January 2013
27
Table 1. In Vitro Anti-inflammatory Activity of Title Compounds (5a–l)
S. No.
Compounds
Dose (µg/mL)
Mean OD
S.D.
% Inhibition
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
NC
PC1
PC1
5a
5a
5b
5b
5c
5c
5d
5d
5e
5e
5f
RO Water
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
2.509
1.004
0.367
1.008
0.570
1.002
0.708
1.260
0.863
1.139
0.842
1.292
1.021
1.294
0.993
0.972
0.605
1.101
0.767
1.066
0.766
0.861
0.594
1.102
0.881
1.314
0.984
0.143
0.017
0.028
0.015
0.016
0.005
0.014
0.038
0.044
0.018
0.035
0.049
0.037
0.035
0.025
0.015
0.006
0.045
0.031
0.038
0.010
0.018
0.030
0.051
0.046
0.038
0.034
—
59.97
85.36
59.81
77.27
60.05
71.79
49.79
65.62
54.59
66.43
48.52
59.31
48.44
60.44
61.27
75.90
56.12
69.43
57.53
69.47
65.70
76.31
53.28
68.41
54.64
67.85
5f
5g
5g
5h
5h
5i
5i
5j
5j
5k
5k
5l
5l
NC=phenyl butazone; PC1=flufenamic acid. PC1=0.1µg/mL value should be given 59.97 and 0.2µg/mL value should be given 85.36.
Fig. 1. In Vitro Anti-inflammatory Activity of Title Compounds (5a–l)
and presented as (Table 2, Fig. 2). Percent membrane stabiliza- during 24h observation. The common clinical signs observed
tion activity was calculated by the following formula:
in the treated animals were dullness, piloerection and recum-
bency with 5a to 5l compounds 50mg/kg bw treated animals
during first 4h the of observation. Abnormalities were not
detected during the observation of gross pathology for all the
animals.
percentage inhibition
(absorbance control  absorbance sample)
=
×100
absorbance of control
In Vivo Anti-inflammatory Activity (a) Acute Toxicol-
(b) Anti-inflammatory Studies: In vivo anti-inflammatory
ogy Studies: Acute toxicology study was performed on the hy- activity was performed by carrageenan-induced paw edema
brid compounds according to the Organisation for Economic and cotton pellet-induced granuloma models. Doses were se-
Cooperation and Development (OECD) guidelines for Testing lected based on the acute toxicity studies.
of Chemicals, No. 423 “Acute Oral Toxicity—Acute Toxic
(c) Carrageenan Induced Paw Oedema: The test was con-
Class Method,” adopted December 17, 2001. For compounds ducted to determine the anti-inflammatory activity of the
5a to 5i mortalities were found at 5mg/kg body weight (bw) hybrid compounds.18) The animals were pretreated with hybrid

28
Vol. 61, No. 1
Table 2. In Vitro Anti-inflammatory Activity of Title Compounds (5a–l) by the Membrane Stabilization Method
S. No.
Compounds
Dose (µg/mL)
Mean OD S.D.
% Inhibition
1
1
2
3
4
5
6
7
8
NC
PC1
PC1
5a
5a
5b
5b
5c
5c
5d
5d
5e
5e
5f
RO Water
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.2
2.297 0.117*
0.866 0.056*
0.724 0.034*
0.971 0.049*
0.678 0.048*
0.961 0.028*
0.664 0.018*
1.192 0.016*
0.875 0.022*
1.108 0.009*
0.929 0.034*
1.455 0.045*
1.152 0.025*
1.856 0.008*
1.641 0.034*
0.955 0.039*
0.547 0.041*
1.017 0.005*
0.844 0.019*
0.962 0.036*
0.768 0.012*
0.863 0.018*
0.401 0.013*
0.822 0.013*
0.754 0.011*
0.822 0.013*
0.724 0.012*
—
60.28
79.18
57.73
72.50
58.15
73.09
48.12
61.89
51.78
59.56
36.66
49.83
29.18
48.57
58.41
76.19
55.74
73.27
58.12
66.58
56.44
68.53
64.24
68.12
65.28
68.12
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
5f
5g
5g
5h
5h
5i
5i
5j
5j
5k
5k
5l
5l
PC1, salicylic acid; *p<0.05.
Fig. 2. In Vitro Anti-inflammatory Activity of Title Compounds (5a–l) by the Membrane Stabilization Method
compounds or diclofenac sodium 1h before carrageenan injec- (100 1mg) inserted with blunted forceps in that area. All the
tion. The animals were injected with 100µL of 1% carrageen- animals received either hybrid compounds, diclofenac sodium
an (0.5% carboxymethyl cellulose (CMC)) solution into the or vehicle (0.5% CMC) orally depending on their respective
sub-plantar region of right hind paw. Paw volume was mea- grouping for 7d consecutively from the day of cotton pellet
sured by dislocation of the water column in a plethysmometer insertion. On day 8, animals were anesthetized again and
after carrageenan application at 0, 1, 3, 6 and 12h after test cotton pellets removed, dried, and weighed. The results are
compound stimulus. Reduction in the paw volume compared shown in (Table 4, Fig. 4).
with the vehicle-treated (0.5% CMC) control animals was con-
sidered as anti-inflammatory response; the results are shown Graph Pad InStat version 3.0 for Windows 95, Graph Pad
in (Table 3, Fig. 3). Software, San Diego, CA, U.S.A. The anti-inflammatory ac-
Statistical Analysis Statistical analysis was performed
(d) Cotton Pellet-Induced Granuloma: The test was per- tivity by membrane stabilization (in vitro), paw edema, cotton
formed on Wistar rats using cotton pellet-induced granuloma pellet (in vivo) and percent inhibition of albumin denaturation,
method.19) The rats were anesthetized under light ether and an was compared versus negative control group by one-way anal-
incision was made on the lumbar region by curved scissors; a ysis of variance (ANOVA) with Dunnett’s post-test; a value of
subcutaneous tunnel was made and a sterilized cotton pellet p<0.05 was considered significant.

January 2013
29
Table 3. In Vivo Anti-inflammatory Activity of Title Compounds (5a–l) by the Carrageenan Induced Paw Oedema Method
Paw volume
3h
Dose
(mg/kg)
Group
0h
1h
6h
12h
Mean
S.D.
Mean
1.95
S.D.
Mean
2.00
S.D.
Mean
S.D.
Mean
S.D.
Control
PC
5a
CMC
100
5
1.92
0.12
0.08
0.08
0.10
0.08
0.08
0.08
0.05
0.05
0.04
0.08
0.08
0.07
0.06
0.08
0.08
0.13
0.11
0.05
0.05
0.08
0.04
0.09
0.08
0.08
0.05
0.08
0.09
0.13
0.10
0.14
0.11
0.05
0.09
0.08
0.05
0.10
0.08
0.05
0.08
0.08
0.07
1.83**
1.17**
1.38**
1.38**
1.42**
1.43**
1.43**
1.48**
1.43**
1.42**
1.40**
1.33**
1.32**
1.31
0.14
0.12
0.13
0.10
0.08
0.08
0.08
0.04
0.05
0.08
0.06
0.08
0.08
0.07
1.67**
1.08**
0.98**
0.93**
1.15**
1.17**
1.17**
1.23**
1.13**
1.17**
1.17**
0.92**
0.91**
0.90
0.10
0.08
0.15
0.16
0.08
0.05
0.10
0.08
0.10
0.10
0.05
0.12
0.09
0.08
1.83ns
1.82ns
1.83ns
1.87ns
1.85ns
1.82ns
1.87ns
1.85ns
1.88ns
1.85ns
1.83ns
1.81ns
1.80
1.57**
1.70**
1.70**
1.75**
1.77**
1.72**
1.82**
1.70**
1.72**
1.68**
1.65**
1.64**
1.63
1.35**
1.57**
1.50**
1.57**
1.60**
1.57**
1.63**
1.58**
1.57**
1.53**
1.52**
1.51**
1.50
5b
5c
5
5
5d
5e
5
5
5f
5
5g
5
5h
5i
5
5
5j
5
5k
5l
5
5
One-way ANOVA with Dunnett’s post-test was performed, p<0.05 was considered statistically significant. **p<0.01, ns p>0.05.
Fig. 3. In Vivo Anti-inflammatory Activity of Title Compounds (5a–l) by the Carrageenan Induced Paw Oedema Method
Table 4. In Vivo Anti-inflammatory Activity of Title Compounds (5a–l) by the Cotton Pellet-Induced Granuloma Method
Granuloma dry weight (mg)
Group
Dose (mg/kg bw)
% inhibition
Mean
63.67
S.D.
Control
PC
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
0.5% CMC
100
5
1.37
1.21
2.56
1.87
1.47
5.04
3.54
1.64
1.60
1.87
1.21
2.23
2.24
2.25
—
29.67**
31.17**
30.50**
38.17**
37.83**
32.83**
39.50**
36.17**
37.50**
34.67**
27.17**
27.16**
27.15**
58.40
51.05
52.09
40.05
40.58
48.43
37.96
43.19
41.10
45.55
54.33
36.46
35.18
5
5
5
5
5
5
5
5
5
5
5
Control, 0.5% CMC (carboxymethyl cellulose); PC, positive control (dichlofenac), 5a–5l, title compounds. One-way ANOVA with Dunnett’s post-test was performed,
p<0.05 was considered statistically significant. **p<0.01.

30
Vol. 61, No. 1
Fig. 4. In Vivo Anti-inflammatory Activity of Title Compounds (5a–l) by the Cotton Pellet-Induced Granuloma Method
136.8 (C-2), 117.5 (C-3), 126.2 (C-4), 140.2 (C-5), 151.4 (C-6).
Conclusion
31
+
·
Synthesis of novel phosphorylated guanidine derivatives
P-NMR (DMSO-d6) δ: −6.4. m/z (%): 207 (100) [M +1],
containing various bioactive amines as substitutents in the 209 (50), 187 (25), 169 (21). Anal. Calcd for C5H4ClN2OSP:
guanidine function was accomplished in high yields. Based on C, 29.07; H, 1.95; N, 13.56. Found: C, 29.15, H, 1.91; N, 13.45.
the data obtained from the carrageenan-induced paw edema
Synthesis of [(2-Thioxo-2,3-dihydro-2λ5-pyrido[2,3-d]-
and cotton pellet-induced granuloma, all the title compounds [ 1 ,3 ,2 ] o x a z a p h o s p h o l - 2 - y l ) a m i n o ] m e t h a n e n i t r i l e 4 T h e fi -
showed good to excellent anti-inflammatory activity compared ltrate containing 3 was used for the next step of the reaction
with control (diclofenac). In particular, compounds 5a, 5b, 5g, without further purification. It was further reacted with cy-
and 5j showed potent anti-inflammatory activity in in vitro anamide (H2N–CN) in the presence of Et3N at 20–40°C for
and in vivo models. This enhanced activity may be due to the 2h to afford the corresponding cyanamine (P(S)–NH–CN) 4.
presence of benzothiazole, chlorobenzothiazole, fluoro-chloro- The reaction progress was monitored by TLC analysis using
phenyl, and fluorophenylpiperazinyl substituents in the guani- ethyl acetate and hexane (7:3). Yield: 85%; mp 126–128°C. IR
1
dine function. Selected compounds of the present series will (KBr) cm−1: 3362 (C=NH), 768 (P=S), 982 (P-N). H-NMR
be further optimized in vitro and in vivo and investigated to (DMSO-d6) δ: 7.83–6.97 (3H, m, Ar-H), 3.93 (1H, s, Ar-NH)
explore the pharmacological mechanism of anti-inflammatory 3.49 (1H, s, P-NH). 13C-NMR (DMSO-d6) δ: 136.8 (C-2),
activity.
117.5 (C-3), 126.2 (C-4), 140.2 (C-5), 151.4 (C-6), 156.2 (C-11).
31
+
·
P-NMR (DMSO-d6) δ: −11.8. m/z (%): 213 (100) [M ], 212
(51), 204 (16), 195 (28). Anal. Calcd for C6H4N4OSP: C, 33.97;
Experimental
Chemistry Chemicals were purchased from Sigma- H, 2.38; N, 26.41. Found: C, 33.85, H, 2.41; N, 26.31.
Aldrich, Merck, and Lancaster, and were used without further
General Procedure for Preparation of N-Substituted N′-
purification. All solvents used for spectroscopic and other (2-Thioxo-2,3-dihydro-2λ5-pyrido[2,3-d][1,3,2]oxazaphos-
physical studies were reagent grade and were further purified phol-2-yl)guanidine Derivatives (5a−l) The intermediate
by literature methods.20) IR spectra were recorded as KBr 4 was further treated with heterocyclic/aryl amines in THF
1
pellets on a Perkin Elmer 283 unit. H-, 13C-, and 31P-NMR in the presence of Et3N at 50–60°C for 2h to form title
spectra were recorded on a Bruker 400MHz NMR spec- compound 5a–l. Identification of the product and completion
1
trometer operating at 400MHz for H-, 100MHz for 13C-, of the reaction were monitored by TLC using ethyl acetate–
and 161.9MHz for 31P-NMR in DMSO-d6 and referenced to hexane (4:1) as eluent. After completion of the reaction, the
tetramethylsilane (TMS) (1H and 13C) and 85% H3PO4 (31P). mixture was concentrated in a rota-evaporator and the residue
LC-MS spectra were recorded on a Jeol SX 102 DA/600 purified by column chromatography on silica gel (100–200
mass spectrometer. Elemental analyses were performed on mesh) using petroleum ether–ethyl acetate (2:3) as eluent. The
a Thermo Finnigan Instrument at University of Hyderabad, structure of the title compounds (5a–l) were established by
Hyderabad, India.
spectral and elemental analysis.
Synthesis of 2-Chloro-2,3-dihydro-2λ5-pyrido[2,3-d]-
N-(1,3-Benzothiazol-2-yl)-N′-(2-thioxo-2,3-dihydro-2λ5-
[1,3,2]oxazaphosphole-2-thione 3 2-Aminopyridin-3-ol (1) pyrido[2,3-d][1,3,2]oxaza phosphol-2-yl)guanidine (5a): This
was reacted with thiophosphoryl chloride (2) in the presence compound was prepared in 80% yield by the protocol de-
of Et3N in THF to get a monochloride, 2-choloro-3H-[1,3,2]- scribed in the general procedure. mp 165–167°C. IR (KBr)
oxazaphospholo[4,5-b] p y r i d i n e - 2 - s u l fi d e 3 after stirring at cm−1: 3380 (N–H), 3342 (C=NH), 1624 (C=N), 756 (P=
1
room temperature for 2h. Formation of monochloride 3 was S), 914 (P-N). H-NMR (DMSO-d6) δ: 8.31 (1H, s, C=NH),
ascertained by TLC analysis run in 3:2 mixture of ethyl ace- 7.83–6.97 (7H, m, Ar-H), 5.41 (1H, s, Ar′-NH), 3.93 (1H, s
tate and hexane. TEA hydrochloride was removed from the re- Ar-NH) 3.49 (1H, s, P-NH). 13C-NMR (DMSO-d6) δ: 138.2
action mixture by filtration. The compound was characterized (C-2), 117.2 (C-3), 124.8 (C-4), 139.4 (C-5), 148.2 (C-9), 162.7
by spectral analysis. Yield: 88%; mp 118–120°C. IR (KBr) (C-11), 172.2 (C-13), 151.6 (C-15), 116.4 (C-16), 126.0 (C-17),
1
cm−1: 789 (P=S), (P-N). H-NMR (DMSO-d6) δ: 7.83–6.97 124.6 (C-18), 120.2 (C-19), 128.4 (C-20). 31P-NMR (DMSO-d6)
+
(3H, m, Ar-H), 3.49 (1H, s, P-NH). 13C-NMR (DMSO-d6) δ: δ: 11.1. m/z (%): 362 (100) [M ], 361 (52), 260 (41), 152 (35),
·

January 2013
31
89 (21). Anal. Calcd for C13H11N6OS2P: C, 43.09; H, 3.06; N,
23.19. Found: C, 43.07, H, 3.04; N, 23.17.
N-(2,4-Difluorophenyl)-N′-(2-thioxo-2,3-dihydro-2λ5-
pyrido[2,3-d][1,3,2]oxazaphosphol-2-yl)guanidine (5f): This
N-(4-Chloro-1,3-benzothiazol-2-yl)-N′-(2-thioxo-2,3-di- compound was prepared in 79% yield by the protocol de-
hydro-2λ5-pyrido[2,3-d][1,3,2]oxazaphosphol-2-yl)guanidine scribed in the general procedure. mp 182–184°C. IR (KBr)
(5b): This compound was prepared in 78% yield by the pro- cm−1: 3452 (N–H), 3352 (C=NH), 1638 (C=N), 747 (P=S),
1
tocol described in the general procedure. mp 174–176°C. IR 960 (P-N): H-NMR (DMSO-d6) δ: 10.12 (1H, s, C=NH),
(KBr) cm−1: 3412 (N–H), 3348 (C=NH), 1632 (C=N), 724 7.84–6.58 (6H, m, Ar-H), 5.3 (1H, s, Ar′-NH), 4.8 (1H, s,
1
(P=S), 921 (P-N). H-NMR (DMSO-d6) δ: 8.43 (1H, s, C= Ar-NH), 3.34 (1H, s, P-NH). 13C-NMR (DMSO-d6) δ: 139.4
NH), 7.83–7.06 (6H, m, Ar-H), 5.2 (1H, s, Ar′-NH), 4.5 (1H, (C-2), 116.4 (C-3), 125.6 (C-4), 141.5 (C-5), 150.6 (C-9), 165.6
s, Ar-NH), 3.12 (1H, s, P-NH) . 13C-NMR (DMSO-d6) δ: 137.4 (C-11), 126.4 (C-13), 158.6 (C-14), 106.4 (C-15), 152.6 (C-16),
(C-2), 118.4 (C-3), 125.2 (C-4), 138.6 (C-5), 149.4 (C-9), 164.2 118.5 (C-17), 124.2 (C-18). 31P-NMR (DMSO-d6) δ: 14.2. m/z
+
·
(C-11), 173.4 (C-13), 152.4 (C-15), 120.4 (C-16), 127.2 (C-17), (%): 341 (100) [M ]. Anal. Calcd for C12H10F2N5OSP: C,
126.2 (C-18), 121.6 (C-19), 129.7 (C-20). 31P-NMR (DMSO- 42.23; H, 2.95; N, 20.52. Found: C, 42.20, H, 2.94; N, 20.48.
+
·
+
·
d6) δ: 12.1. m/z (%): 397 (100) [M +1], 399 (33) [M +2],
246 (35), 172 (19), 105 (14). Anal. Calcd for C13H10N6OS2P: C, pyrido[2,3-d][1,3,2]oxazaphosphol-2-yl)guanidine (5g): This
39.35; H, 2.54; N, 21.18. Found: C, 39.32, H, 2.50; N, 21.14. compound was prepared in 75% yield by the protocol de-
N-(2-Chloro-4-fluorophenyl)-N′-(2-thioxo-2,3-dihydro-2λ5-
N-(6-Methoxy-1,3-benzothiazol-2-yl)-N′-(2-thioxo-2,3-di- scribed in the general procedure. mp 168–170°C. IR (KBr)
hydro-2λ5-pyrido[2,3-d][1,3,2]oxazaphosphol-2-yl)guanidine cm−1; 3452 (N–H), 3352 (C=NH), 1638 (C=N), 734 (P=
1
(5c): This compound was prepared in 82% yield by the pro- S), 960 (P-N). H-NMR (DMSO-d6) δ: 8.62 (1H, s, C=NH),
tocol described in the general procedure. mp 181–183°C. IR 7.84–6.58 (6H, m, Ar-H), 5.44 (1H, s, Ar′-NH), 4.8 (1H, s,
(KBr) cm−1: 3418 (N–H), 3342 (C=NH), 1624 (C=N), 710 Ar-NH), 3.34 (1H, s, P-NH). 13C-NMR (DMSO-d6) δ: 138.6
1
(P=S), 934 (P-N). H-NMR (DMSO-d6) δ: 8.66 (1H, s, C= (C-2), 118.2 (C-3), 124.8 (C-4), 140.8 (C-5), 151.6 (C-9), 166.2
NH), 8.24–7.08 (7H, m, Ar-H), 5.45 (1H, s, Ar′-NH), 3.94 (C-11), 127.4 (C-13), 138.2 (C-14), 116.2 (C-15), 132.6 (C-16),
(1H, s, Ar-NH), 3.06 (1H, s, P-NH), 2.83 (3H, s, O–CH3). 119.2 (C-17), 120.6 (C-18). 31P-NMR (DMSO-d6) δ: 13.8.
+
13C-NMR (DMSO-d6) δ: 135.2 (C-2), 116.5 (C-3), 125.2 (C-4), m/z (%): 357 (100) [M ], 236 (54), 195 (21). Anal. Calcd
·
138.6 (C-5), 149.4 (C-9), 164.2 (C-11), 173.4 (C-13), 152.4 for C12H10ClFN5OSP: C, 40.29; H, 2.82; N, 19.58. Found: C,
(C-15), 120.4 (C-16), 127.2 (C-17), 126.2 (C-18), 121.6 (C-19), 40.25, H, 2.79; N, 19.52.
129.7 (C-20) 50.2 (O-Me). 31P-NMR (DMSO-d6) δ: 12.8. m/z
N-(1,3,4-Thiadiazol-2-yl)-N′-(2-thioxo-2,3-dihydro-2λ5-
(%): 393 (100) [M+1], 361 (25), 281 (19). Anal. Calcd for pyrido[2,3-d][1,3,2]oxazaphosphol-2-yl)guanidine (5h): This
C14H13N6OS2P: C, 42.85; H, 3.34; N, 21.42. Found: C, 42.75, H, compound was prepared in 72% yield by the protocol de-
3.31; N, 21.36.
scribed in the general procedure. mp 146–148°C. IR (KBr)
N-(6-Nitro-1,3-benzothiazol-2-yl)-N′-(2-thioxo-2,3-di- cm−1: 3462 (N–H), 3342 (C=NH), 1642 (C=N), 744 (P=
hydro-2λ5-pyrido[2,3-d][1,3,2]oxazaphosphol-2-yl)guanidine S), 992 (P-N). H-NMR (DMSO-d6) δ: 8.3 (1H, s, C=NH),
1
(5d): This compound was prepared in 76% yield by the pro- 8.86–7.58 (4H, m, Ar-H), 5.46 (1H, s, Ar′-NH), 4.6 (1H, s,
tocol described in the general procedure. mp 192–194°C. IR Ar-NH), 3.12 (1H, s, P-NH). 13C-NMR (DMSO-d6) δ: 137.2
(KBr) cm−1: 3445 (N–H), 3348 (C=NH), 1648 (C=N), 726 (C-2), 117.5 (C-3), 125.2 (C-4), 140.1 (C-5), 152.4 (C-9), 167.2
1
(P=S), 965 (P-N): H-NMR (DMSO-d6) δ: 10.2 (1H, s, C= (C-11), 152.2 (C-13), 148.6 (C-16). 31P-NMR (DMSO-d6) δ:
+
·
NH), 8.32–7.38 (6H, m, Ar-H), 5.62 (1H, s, Ar′-NH), 4.4 (1H, 15.2. m/z (%): 313 (100) [M ]. Anal. Calcd for C8H8N7OS2P:
s, Ar-NH), 3.24 (1H, s, P-NH). 13C-NMR (DMSO-d6) δ: 138.6 C, 30.67; H, 2.57; N, 31.29. Found: C, 30.64, H, 2.52; N, 31.26.
(C-2), 118.2 (C-3), 125.4 (C-4), 140.4 (C-5), 152.8 (C-9), 166.4
N1-(2-Thioxo-2,3-dihydro-2λ5-pyrido[2,3-d][1,3,2]oxaza-
(C-11), 175.1 (C-13), 158.2 (C-15), 120.4 (C-16), 124.6 (C-17), phosphol-2-yl)-4-(4-chlorophenyl)-1-piperazinecarboximida-
144.6 (C-18), 120.6 (C-19), 131.2 (C-20). 31P-NMR (DMSO-d6) mide (5i): This compound was prepared in 73% yield by the
+
·
δ: 9.5. m/z (%): 406 (100) [M ], 391 (45), 363 (21), 349 (18). protocol described in the general procedure. mp 122–124°C.
Anal. Calcd for C13H10N7O3S2P: C, 38.33; H, 2.47; N, 24.07. IR (KBr) cm−1: 3472 (N–H), 3348 (C=NH), 1638 (C=N), 782
1
Found: C, 38.30, H, 2.47; N, 24.04.
(P=S), 952 (P-N). H-NMR (DMSO-d6) δ: 8.7 (1H, s, C=
N-[2-(1H-3-Indolyl)ethyl]-N′-(2-thioxo-2,3-dihydro-2λ5- NH), 8.02–7.68 (7H, m, Ar-H), 4.92 (1H, s, Ar′-NH), 3.42
pyrido[2,3-d][1,3,2]oxazaphosphol-2-yl)guanidine (5e): This (1H, s, P-NH), 3.26 (4H, t, J=5.8Hz, CH2–NAr), 2.98 (4H, t,
compound was prepared in 74% yield by the protocol de- J=4.2Hz, CH2–N aliphatic). 13C-NMR (DMSO-d6) δ: 139.5
scribed in the general procedure. mp 156–158°C. IR (KBr) (C-2), 116.4 (C-3), 125.6 (C-4), 140.4 (C-5), 151.7 (C-9), 163.5
cm−1: 3432 (N–H), 3342 (C=NH), 1636 (C=N), 713 (P= (C-11), 45.2 (C-13), 44.8 (C-13′), 52.2 (C-14), 52.6 (C-14′),
1
S), 945 (P-N). H-NMR (DMSO-d6) δ: 8.4 (1H, s, C=NH), 147.5 (C-16), 116.4 (C-17), 128.6 (C-18), 127.2 (C-19), 128.6
7.28–6.79 (8H, m, Ar-H), 5.2 (1H, s, Ar′-NH), 4.6 (1H, s, (C-20), 116.2 (C-21). 31P-NMR (DMSO-d6) δ: 12.4; m/z (%):
+
·
Ar-NH), 3.73 (1H, s, P-NH), 1.17 (2H, t, –CH2), 3.05–2.54 408 (100) [M ], 378 (62), 340 (54), 287 (36), 181 (19). Anal.
(2H, m, –CH2–NH). 13C-NMR (DMSO-d6) δ: 137.2 (C-2), Calcd for C16H18ClN6OSP: C, 47.00; H, 4.44; N, 20.56. Found:
114.5 (C-3), 125.2 (C-4), 140.7 (C-5), 150.2 (C-9), 166.6 (C-11), C, 46.98, H, 4.42; N, 20.51.
48.2 (C-13), 22.6 (C-14) 116.2 (C-15), 128.4 (C-16), 118.2
N1-(2-Thioxo-2,3-dihydro-2λ5-pyrido[2,3-d][1,3,2]oxazaph
(C-17), 132.2 (C-18), 114.2 (C-19), 121.4 (C-20), 120.5 (C-21), osphol-2-yl)-4-(4-fluorophenyl)-1-piperazinecarboximidamide
118.4 (C-22) 127.6 (C-23). 31P-NMR (DMSO-d6) δ: 6.9. m/z (5j): This compound was prepared in 71% yield by the pro-
+
·
(%): 372 (100) [M ], 354 (46), 176 (21). Anal. Calcd for tocol described in the general procedure. mp 132–134°C. IR
C16H17N6OSP: C, 51.61; H, 4.60; N, 22.57. Found: C, 51.58, H, (KBr) cm−1: 3458 (N–H), 3338 (C=NH), 1642 (C=N), 762
1
4.56; N, 22.54.
(P=S), 962 (P-N). H-NMR (DMSO-d6) δ: 8.5 (1H, s, C=

32
Vol. 61, No. 1
NH), 7.98–6.86 (7H, m, Ar-H), 4.72 (1H, s, Ar′-NH), 3.46
(1H, s, P-NH), 3.16 (4H, t, J=6.4Hz, CH2–NAr), 2.82 (4H, t,
J=4.8Hz, CH2–N aliphatic). 13C-NMR (DMSO-d6) δ: 139.2
(C-2), 115.4 (C-3), 126.2 (C-4), 141.5 (C-5), 150.4 (C-9), 164.6
(C-11), 44.8 (C-13), 43.6 (C-13'), 51.4 (C-14), 51.5 (C-14′), 146.2
(C-16), 115.7 (C-17), 118.8 (C-18), 157.4 (C-19), 118.2 (C-20),
References
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(2000).
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M. J., Shankley N. P., Watt G. F., Wright P. T., J. Med. Chem., 43,
2362–2370 (2000).
115.6 (C-21). 31P-NMR (DMSO-d6) δ: 13.4. m/z (%): 392 (100)
+
·
[M ], 272 (53), 114 (28). Anal. Calcd for C16H18FN6OSP: C,
48.97; H, 4.62; N, 21.42. Found: C, 48.92, H, 4.58; N, 21.38.
N1-(2-Thioxo-2,3-dihydro-2λ5-pyrido[2,3-d][1,3,2]oxaza-
phosphol-2-yl)-4-(2-pyrimidinyl)-1-piperazinecarboximida-
mide (5k): This compound was prepared in 77% yield by the
protocol described in the general procedure. mp 138–140°C.
IR (KBr) cm−1: 3458 (N–H), 3338 (C=NH), 1642 (C=N), 732
1
(P=S), 1022 (P-N). H-NMR (DMSO-d6) δ: 8.9 (1H, s, C=
NH), 8.48–7.12 (6H, m, Ar-H), 4.92 (1H, s, Ar′-NH), 3.48
(1H, s, P-NH), 3.18 (4H, t, J=6.6Hz, CH2–NAr), 2.86 (4H, t,
J=5.2Hz, CH2–N aliphatic). 13C-NMR (DMSO-d6) δ: 138.6
(C-2), 115.7 (C-3), 125.8 (C-4), 140.6 (C-5), 151.2 (C-9), 165.2
(C-11), 45.2 (C-13), 44.8 (C-13′), 51.2 (C-14), 50.8 (C-14′),
162.5 (C-16), 156.2 (C-18), 116.4 (C-19), 157.8 (C-19). 31P-NMR
+
·
(DMSO-d6) δ: 11.7. m/z (%): 376 (100) [M ]. Anal. Calcd for
C14H17N8OSP: C, 44.68; H, 4.55; N, 29.77. Found: C, 44.64, H,
4.52; N, 29.73.
N1-(2-Thioxo-2,3-dihydro-2λ5-pyrido[2,3-d][1,3,2]oxaza-
phosphol-2-yl)-4-(4-nitrophenyl)-1-piperazinecarboximidamide
(5l): This compound was prepared in 81% yield by the
protocol described in the general procedure. mp 146–148°C.
IR (KBr) cm−1; 3462 (N–H), 3354 (C=NH), 1638 (C=N),
1
774 (P=S), 968 (P-N). H-NMR (DMSO-d6) δ: 8.58 (1H, s,
C=NH), 8.06–7.16 (7H, m, Ar-H), 4.7 (1H, s, Ar′-NH), 3.36
(1H, s, P-NH), 3.28 (4H, t, J=5.6Hz, CH2–NAr), 2.82 (4H, t,
J=4.2Hz, CH2–N aliphatic). 13C-NMR (DMSO-d6) δ: 137.8
(C-2), 116.5 (C-3), 125.2 (C-4), 140.8 (C-5), 152.4 (C-9), 166.4
(C-11), 45.6 (C-13), 45.2 (C-13′), 51.8 (C-14), 51.2 (C-14′),
156.5 (C-16), 116.4 (C-17), 126.7 (C-18), 139.2 (C-20), 125.7
13) Wagman A. S., Nuss J. M., Curr. Pharm. Des., 7, 417–450 (2001).
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supuleti V. R., Sunil K.G., Eur. J. Med. Chem., 45, 203–209 (2010).
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(1968).
17) Sadique J., Al-Rqobahs W. A., Bughaith M. F., EL Gindi A. R.,
Fitoterapia, 60, 525–532 (1989).
(C-21), 124.2 (C-21). 31P-NMR (DMSO-d6) δ: 14.7. m/z: 419
+
·
(100) [M ], 301 (67), 194 (39), 138 (25). Anal. Calcd for
C16H18N7O3SP: C, 45.82; H, 4.33; N, 23.38. Found: C, 45.76, H,
4.30; N, 23.34.
18) Winter C. A., Risly E. A., Nuss G. W., Proc. Soc. Exp. Biol. Med.,
111, 544–547 (1962).
19) Goldstein S. A., Shemano L., Demer R., Beiler J. M., Arch. Int.
Pharmacodyn. Ther., 165, 294–301 (1976).
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icals,” 4th ed., Butterworth Heinemann, Oxford, 1997.
Acknowledgment The authors express their grateful
thanks to DST-PURSE Programme, S. V. University, Tirupati,
for giving financial assistance through DST-PURSE-JRF Fel-
lowship.