Synthesis and antimicrobial activity of 5,5′-dimethyl-2-oxido-[1,2,3] -dioxaphosphorinane-2-yl-amino carboxylates

Article abstract of DOI:10.1002/hc.20426

Synthesis of several 5,5-dimethyl-2-oxido-[1, 3,2]-dioxaphosphorinane-2-yl- amino car-boxylates (4a-j) was accomplished through a two-step process. This involves prior preparation of the intermediate monochloride (2), 2-chloro-5,5-dimethyl [1,3,2]dioxaphosphorinane-2-oxide and its subsequent reaction with various amino acid esters (3a-j) in dry tetrahydrofuran in the presence of triethyl amine at room temperature. They were characterized by elemental analysis, IR, ¹H, ¹³C, ³¹P NMR, and mass spectral data. Their antifungal and antibacterial activity is also evaluated. Majority of these compounds exhibited moderate antimicrobial activity in the assay.

Full text of DOI:10.1002/hc.20426

Heteroatom Chemistry
Volume 19, Number 3, 2008
S
ynthesis and Antimicrobial Activity of
ꢀ
5
,5 -Dimethyl-2-Oxido-[1,3,2]-Dioxaphos-
phorinane-2-yl-Amino Carboxylates
B. Hari Babu, G. Syam Prasad, C. Suresh Reddy, and C. Naga Raju
Department of Chemistry, Sri Venkateswara University, Tirupati 517 502, India
Received 25 August 2006; revised 16 June 2007
moieties exhibited enhanced anti-HIV activity [6].
ABSTRACT: Synthesis of several 5,5-dimethyl-
-oxido-[1,3,2]-dioxaphosphorinane-2-yl-amino car-
ꢀ
5
-Phosphorylated AZT with tryptophan ethyl ester
2
attached at phosphorus exhibited 8-fold increase in
anti-HIV activity compared to free AZT without any
symptoms of toxicity [7]. Furthermore, hydrolysis
of these novel heterocycles may release products of
limited toxicity in the host system [8]. In view of
this background, the title compounds were designed
incorporating active pharmacophoric structural fea-
tures, synthesized, characterized, and screened for
their antimicrobial activity.
boxylates (4a–j) was accomplished through a two-
step process. This involves prior preparation of the
intermediate monochloride (2), 2-chloro-5,5-dimethyl
[1,3,2]dioxaphosphorinane-2-oxide and its subse-
quent reaction with various amino acid esters (3a–j)
in dry tetrahydrofuran in the presence of triethyl amine
at room temperature. They were characterized by el-
1
13
31
emental analysis, IR, H, C, P NMR, and mass
spectral data. Their antifungal and antibacterial ac-
tivity is also evaluated. Majority of these compounds
exhibited moderate antimicrobial activity in the as-
say. ꢁC 2008 Wiley Periodicals, Inc. Heteroatom Chem
RESULTS AND DISCUSSION
1
9:256–260, 2008; Published online in Wiley InterScience
(
www.interscience.wiley.com). DOI 10.1002/hc.20426
The synthetic route (Scheme 1) involves the cycliza-
tion of 2,2-dimethyl-1,3-propanediol (1) with phos-
phorusoxychloride in dry toluene in the presence of
triethylamine (TEA), and we obtained the interme-
diate monochloride (2). It was reacted with amino
acid methyl/ethyl ester hydrochlorides (3a–j) in dry
tetrahydrofuran (THF) in the presence of TEA to get
title compounds 4a–j in good yields.
INTRODUCTION
Phosphoramides substituted with an amino acid es-
ter are an important class of rationally designed ther-
apeutics especially with antineoplastic [1–5]. The
attachment of an amino acid group to the phosphate
moiety is expected to increase their cellular uptake
and thus enhance their chemotherapeutic prop-
erties [3,4]. Recently, McGuigan and Narasimhan
reported that phosphorus triester derivatives of
The second step of the reaction was completed
at room temperature with stirring for 5–6 h to get
4a–j. Progress of the reaction was monitored by
thin layer chromatography (TLC) analysis of the
reaction mixture. The title compounds 4a–j were pu-
rified by flash chromatographic method using ethy-
lacetate:methanol (7:3) step gradient mixtures as an
eluent. Their structures were established by elemen-
3
-azido-3-deoxythymidine (AZT) bearing amino acid
Correspondence to: C. Naga Raju; e-mail: naga raju04@yahoo.
1
13
31
tal analysis, IR, H, C, P NMR, and mass spectral
data (Tables 1–4).
co.in.
ꢀ
c 2008 Wiley Periodicals, Inc.
256

ꢀ
Synthesis and Antimicrobial Activity of 5,5 -Dimethyl-2-Oxido-[1,3,2]-Dioxaphosphorinane-2-yl-Amino Carboxylates 257
SCHEME 1
1
In the H NMR spectra (Table 2), aromatic pro-
tons resonated at δ 6.91–7.90. The singlet at δ 10.20–
1
ANTIMICROBIAL ACTIVITY
The compounds were diluted in dimethylformamide
13
1.34 is assigned to P-NH. C NMR chemical shifts
Table 3) were assigned based on the reported lit-
erature data [9]. P NMR spectra of 4a–j showed
only one phosphorus resonance signal in the range
of –5.89 to 4.19 ppm [10].
(DMF) for bioassay. Solvent control was included al-
(
though no antimicrobial activity has been noted in
the solvent employed. Ciprofloxacin controls were
included to compare with compounds 4a–j. All
31
TABLE 1 Physical, IR and 31P NMR Spectral Data of 4a–j
Elemental Analysis
Found (Calcd) (%)
a
−1
)
IR (cm
P O C O NH 31P NMRb,c
Melting
Yield
Molecular
Formula
◦
Compound Point ( C) (%)
C
H
N
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
104–105
111–112
118–119
123–124
119–120
114–115
127–128
117–118
116–117
134–135
68
59
61
57
62
72
64
68
71
68
C H16NO5P 40.34 (40.49) 6.68 (6.74) 5.79 (5.90) 1213 1644 3410
2.36
2.69
2.91
4.19
4.24
2.23
2.03
–4.83
–4.62
–5.89
8
C H18NO5P 42.85 (43.01) 7.21 (7.16) 5.43 (5.57) 1213 1635 3407
9
C12H24NO5P 49.14 (48.96) 8.19 (8.14) 4.77 (4.71) 1216 1648 3418
C11H22NO5P 47.42 (47.28) 7.76 (7.88) 5.13 (5.01) 1210 1644 3418
C12H24NO5P 49.21 (49.14) 8.26 (8.19) 4.62 (4.77) 1211 1659 3413
C11H22NO5P 47.18 (47.31) 7.80 (7.88) 4.91 (5.01) 1217 1649 3415
g
h
i
C H18NO5P 43.14 (43.01) 7.09 (7.16) 5.43 (5.57) 1214 1626 3431
9
C15H22NO5P 55.09 (55.01) 6.58 (6.72) 4.20 (4.27) 1215 1624 3410
C14H20NO5P 53.69 (53.64) 6.31 (6.38) 4.38 (4.47) 1212 1648 3436
j
C17H24NO5P 57.69 (57.76) 6.70 (6.79) 3.86 (3.96) 1210 1658
–
a
Recorded as KBr pellets.
b
c
Recorded in CDCl
3
.
Chemical shifts are in ppm from 85% phosphoric acid.
Heteroatom Chemistry DOI 10.1002/hc

258 Hari Babu et al.
TABLE 2 1H NMR Spectral Dataa,b of 4a–j
Compound CH (4 and 6) 5,5-(CH
2
3
)
2
N-H
Ar-H
Amino Acid Ester-H
3.15 (s, 3H, OCH ), 4.03 (s, 2H, CH )
4a
b
3.95–4.03 (m, 4H) 1.02, 0.90 (2s, 6H) 11.25 (s, 1H)
3.80–4.03 (m, 4H) 1.02, 0.90 (2s, 6H) 11.16 (s, 1H)
–
–
3
2
4
3.95 (m, 2H, OCH ), 3.48 (s, 2H,
2
CH ), 3.14 (s, 3H, CH )
2
3
4
4
4
c
d
e
3.81–4.16 (m, 4H) 1.06, 0.80 (2s, 6H) 11.24 (s, 1H)
3.82–4.27 (m, 4H) 1.20, 0.90 (2s, 6H) 11.34 (s, 1H)
3.87–4.24 (m, 4H) 1.18, 0.89 (2s, 6H) 11.10 (s, 1H)
–
–
–
3.92 (m, 2H, OCH ), 4.51 (m, 1H,
NH-CH-), 2.26 (m, 2H,
2
CH CH CH ), 2.16 (m, 2H,
2
2
3
CH CH CH ), 1.41 (t, CH CH CH )
2
2
3
2
2
3
3.72 (s, 3H, OCH ), 3.67–3.71 (m, 1H,
3
CH-(CH ) ), 0.98 (d, 6H, J = 12.7
3
2
Hz, CH(CH ) , 1.20–1.31 (m, 1H,
3
2
CH-CH(CH ) )
3
2
3.60 (s, 3H, OCH ), 4.07–4.11 (m, 1H,
3
NH-CH), 3.07–3.19 (m,
1
H-CH-CH(CH )-CH CH ), 1.17 (d,
3 2 3
J = 12.1 Hz, 3H,
CH-CH(CH )-CH -CH ), 2.17–2.35
3
2
3
(
0
m, 2H, CH-CH(CH )-CH -CH ),
3 2 3
.97 (t, 3H, CH-CH(CH )-CH -CH )
3 2 3
4
f
3.81–4.20 (m, 4H) 1.17, 0.91 (2s, 6H) 10.20 (s, 1H)
3.61–3.95 (m, 4H) 1.24, 0.90 (2s, 6H) 11.24 (s, 1H)
–
–
3.26 (s, 3H, OCH ), 4.46–4.52 (m, 1H,
NH-CH), 1.42–1.55 (m,
3
2
H-CH-CH -CH(CH ) , 1.22–1.31
2 3 2
(m, 1H, CH-CH -CH(CH ) ), 1.10
2
3 2
(
d, 6H, J = 12.7 Hz,
CH-CH -CH(CH )
2
3 2
4
4
4
4
g
h
i
3.17 (s, 3H, OCH ), 3.72 (m, 1H, CH),
3
0.83 (d, 3H, CH3)
3.69–4.14 (m, 4H) 1.25, 0.90 (2s, 6H) 11.04 (s, 1H) 7.07–7.20
m, 5H)
3.93–4.44 (m, 4H) 1.30, 0.88 (2s, 6H) 11.28 (s, 1H) 7.21–7.36
m, 5H)
7.27–7.36
m, 5H)
3.69 (s, 3H, OCH ), 3.95 (d, 2H, CH ),
3
2
(
4.14–4.41 (m, 1H, CH-CH2)
3.72 (s, 3H, OCH ), 5.15 (s, 1H,
3
(
CHCOOCH3)
j
3.78–4.25 (m, 4H) 1.17, 0.86 (2s, 6H)
–
5.12–5.20 (s, 2H, OCH ), 4.07 (m, 1H,
2
ꢀ
(
CH (C-2 ), 1.94–2.08 (m, 4H,
CH CH (C-3 , C-4 ), 3.42 (m, 2H,
ꢀ
ꢀ
2
2
ꢀ
CH (C-5 )
2
a
Recorded in CDCl
Chemical shifts are in ppm.
3
b
TABLE 3 13C NMR Chemical Shifts of Some Members of 4a,b
Compound C-5 C-4 and C-6 5,5-(CH
3
)
2
Chemical Shifts (Amino Acid Ester)
173.1 (COOCH ), 47.2 (CH COOCH ), 57.1 (OCH )
4
a
d
31.6
31.6
78.4
78.9
21.8
21.8
19.3
19.7
3
2
3
3
4
171.6 (COOCH ), 45.9 (CHCOOCH ), 57.4 (OCH ), 29.6 (CH(CH ) ,
3
3
3
3 2
2
1.6, 21.1 (CH(CH ) )
3 2
1
1
4
4
4
h
31.8
32.2
31.8
79.0
77.8
79.1
21.8
21.8
21.8
19.8
21.3
21.2
172.3 (COOCH ), 54.5 (OCH ), 135.0 (s, 2C, C and C ), 127.1
3 3
5
3
1
1
1
(
s, 2C, C and C ), 129.8 (s, 1C, C ), 55.3 (CH-CH -C H5)
2 6
2 6 4
1
3
i
172.5 (COOCH ), 52.7 (OCH ), 55.3 (CHCOOCH ), 129.8 (s, 2C, C
3
3
3
1
1
1
1
and C ), 12.3 (s, 2C,C and C ), 135.0 (s, 1C, C
4
5
2
6
j
172.0 (COOCH C H5), 66.8 (COOCH C H5) 58.3 (S, 1C, C ), 31.8
2 6 2 6 2
1
(
1
S, 1C, C ), 24.3 (S, 1C, C ) 46.0 (d, 1C, C5), 135.4 (S, 1C, C ,
3 4
1
1
1
1
1
28.7 (S, 2C, C ) 127.4 (S, 2C, C and C ), 128.5 (S, 1C, C )
6 3 4
5
a
3
Recorded in CDCl .
Chemical shifts are in ppm.
b
Heteroatom Chemistry DOI 10.1002/hc

ꢀ
Synthesis and Antimicrobial Activity of 5,5 -Dimethyl-2-Oxido-[1,3,2]-Dioxaphosphorinane-2-yl-Amino Carboxylates 259
TABLE 4 Mass Spectral Data of Important Ions of 4i and 4j
Compound m/z (Relative Abundance)
+
+
4
4
i
j
314 (M + 1), 313 (M , 26) (12), 268 (18), 247 (14), 228 (8), 179 (18), 167 (30), 152 (18), 136 (16), 118 (6),
1
02 (79), 99 (16), 91 (12)
+
+
354 (M + 1, 100), 353 (M , 11), 352 (21), 315 (12), 306 (6), 273 (4), 262 (12), 234 (32), 218 (96), 216 (9),
1
50 (42), 136 (16), 102 (28)
samples were tested in triplicate, and average results
were recorded.
Compounds 4a–j were screened for their an-
tibacterial activity against E. coli and S. aureus (10
6
The compounds were assayed for antibacte-
rial activity against six registered bacterial iso-
lates, which were obtained from the National
Collection of Industrial Microorganisms, National
Chemical Laboratories, Pune, India. The antibac-
terial activity of all the title compounds 4a–j
was evaluated [11,12] against the growth of
Staphylococcus aureus (Gram-positive bacteria) and
Escherichia coli (Gram-negative bacteria) at concen-
trations (100, 50, 25 ppm). The bacteria were rejuve-
nated on Hi-media nutrient agar and subcultured as
needed.
The compounds 4a–j (Table 5) were screened
for their antifungal activity against Aspergillus
niger and Helminthosporium oryzae along with the
standard fungicide bavistin. The disk diffusion
method [13] was followed for screening of the
compounds at three different concentrations (25,
cell/mL) by the disk diffusion method in nutrient
agar medium at various concentrations (25, 50, 100
µg/disk) in dimethylformamide (DMF). The solu-
tions were added to each filter disk, and DMF was
used as the control. The plates were incubated at
◦
35 C and examined for the zone of inhibition around
each disk after 24 h. The results were compared with
the activity of the standard antibiotic penicillin (25,
50, 100 µg/disk). Their antifungal activity was evalu-
ated against A. niger and H. oryzae at concentrations
of 25, 50, 100 µg/disk. Bavistin was used as a ref-
erence compound. Fungal cultures were grown on
◦
potato dextrose broth at 25 C, and finally spore sus-
5
pension was adjusted to 10 spores/mL.
It is interesting to observe that all compounds
4a–j exhibited more antifungal and antibacterial ac-
tivity when compared with that of standard. The
highlight is that all the compounds exhibited very
high activity against fungi, and some compounds
were twice more effective than the standard bavistin.
Similarly, it is gratifying to observe that these com-
pounds are extremely more effective against bacteria
S. aureus.
50, 100 ppm). Their antibacterial activity was also
evaluated according to the disk diffusion method at
three different concentrations against E. coli and S.
aureus by comparing with standards streptomycin
and penicillin.
TABLE 5 Antifungal and Antibacterial Activities of Compounds 4 a–j in Terms of Zone of Inhibition (mm)
Fungi Bacteria
Heliminthosporium oryzae Staphylococcus aureus
Aspergillus niger
Escherichia coli
1
(
00
µg/
Compound disk)
50
(µg/
disk)
25
(µg/
disk)
100
(µg/
disk)
50
(µg/
disk)
25
(µg/
disk)
100
(µg/
disk)
50
(µg/
disk)
25
(µg/
disk)
100
(µg/
disk)
50
(µg/
disk)
25
(µg/
disk)
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
12
10
10
12
9
9
8
8
9
5
5
9
7
9
8
5
–
–
4
5
8
3
–
6
4
6
6
2
–
12
13
12
13
11
10
12
11
11
13
12
–
6
10
11
10
9
–
5
7
5
6
–
8
5
6
4
3
–
13
12
11
13
8
10
12
8
8
8
9
9
3
5
9
5
4
8
–
6
5
3
5
3
–
–
3
–
–
2
–
3
11
11
12
14
10
9
11
–
12
12
–
8
8
9
11
7
3
8
3
10
9
5
6
5
4
–
–
5
–
–
3
–
2
9
4
g
h
i
14
13
13
10
8
10
10
8
10
9
9
12
–
j
Bavistin
–
5
Penicillin
–
–
10
9
All concentrations are expressed in ppm.
indicates no activity.
–
Heteroatom Chemistry DOI 10.1002/hc

260 Hari Babu et al.
EXPERIMENTAL
the TLC analysis. Triethylamine hydrochloride was
separated by filtration, and the solvent was evapo-
rated under reduced pressure. The residue was pu-
rified by flash chromatography on silica gel, using
All melting points were determined on a Mel-Temp
apparatus and were uncorrected. Elemental analyses
were performed by the Central Drug Research Insti-
tute, Lucknow, India. The IR spectra were recorded
ethyl acetate: methanol (7:3) as an eluent, to yield
◦
0
.48 g (68%) of 4j, mp 134–135 C. All the other com-
1
as KBr pellets on a Perkin-Elmer 1000 unit. The H,
pounds (4a–j) were prepared by adopting the above
procedure.
13
31
C, and P spectra were taken on AMX 400 MHz
1
NMR spectrometer operating at 400 MHz for H,
13
31
1
00 MHz for C, and 161.9 MHz for P NMR. Com-
pounds were dissolved in CDCl , and chemical shifts
3
ACKNOWLEDGMENTS
1
13
were referenced to TMS ( H and C NMR) and 85%
The authors thank Prof. C. Devendranath Reddy for
helpful discussion and to the Director of SIF, IISC,
Bangalore, India, for the spectral data.
31
H
3
PO ( P NMR).
4
Synthesis of 2-Chloro-5,5-dimethyl[1,3,
]dioxaphosphorinane 2-oxide (2)
2
REFERENCES
A solution of phosphorus oxychloride (0.76 g,
mmol) in 25 mL of dry toluene was added dropwise
over a period of 20 min to a stirred solution (0.52 g,
mmol) of 2,2-dimethyl-1,3-propanediol (1) and tri-
[
1] Fest, C.; Schmidt, K. J. The Chemistry of
Organophosphorus Pesticides; Springer-Verlag:
Berlin, 1982.
5
5
[2] Szekerke, M. Cancer Treat Rep 1976, 60, 347.
3] Cox, P. J. Biochem Pharmacol 1979, 28, 2045.
[
ethylamine (1.01 g, 10 mmol) in 50 mL of toluene at
◦
[4] Dewine, K. G.; McGuigan, C.; O’Cunnor, T. J.;
Nicholis, S. R.; Kinchington, D. AIDS 1990, 4,
5
–10 C. After the addition, the reaction mixture was
stirred at room temperature for 4 h. Completion of
the reaction was monitored by the TLC analysis. The
reaction mixture was filtered, and the solvent from
the filtrate was evaporated under reduced pressure.
3
71.
[
5] McGuigan, C.; Narasimhan, P. Synthesis 1993, 311.
[6] Siddiqui, A. Q.; Ballatore, C.; McGuigan, C.; Clercq,
E. D.; Balzarini, J. J Med Chem 1999, 42, 393.
[
[
[
7] Wagner, C. R.; McIntee, E. J.; Schinazi, R. F.;
Abraham, T. W. Bioorg Med Chem Lett 1995, 5,
The residue was washed with petroleum ether (60–
◦
8
0 C) and used for the second step reactions without
1
819.
further purification.
8] Derine, K. G.; McGuigan, C.; O’Connor, T. V.;
Nicholis, S. R.; Kinchington, D. AIDS 1990, 4,
3
71.
Synthesis of (5,5-Dimethyl-2-oxido-[1,3,
9] Chakrvarthy, M.; Srinivas, B.; Muthiah, C.; Kumara
Swamy, K. C. Synthesis 2003, 15, 2368.
2
]dioxaphosphorinane-2-yl)pyrrolidine-2(5)-
[
10] Raghu, K. V.; Devendranath Reddy, C.; Naga Raju, C.
benzyl carboxylate (4j)
Asian J Chem 1996, 8(1), 45.
A mixture of proline benzyl ester hydrochloride
[11] Cruickshan, K. R. Medical Microbiology, A Guide to
Diagnosis and Control of Infection, 2nd ed.; E & G
Livingston: Edinburgh, UK, 1968; p. 888.
(
[
3j; 0.483 g, 2 mmol) and 2-chloro-5,5-dimethyl
1,3,2]dioxaphophorinane 2-oxide (2) (0.369 g, 2
[12] Bever, A. W.; Kirby, M. M.; Sherries, J. C.; Truck, A.
Am J Clin Pathol 1969, 45, 493.
mmol) and triethylamine (0.41 g, 4 mmol) in 50 mL
of dry THF was stirred at room temperature for 6
h. The progress of the reaction was monitored by
[
13] Benson, H. J. Microbial Applications, 5th ed.; W. C.
Brown Publications, Boston, USA, 1990; p. 134.
Heteroatom Chemistry DOI 10.1002/hc