Synthesis, spectral characterization and biological evaluation of phosphorylated derivatives of galanthamine

Article abstract of DOI:10.1016/j.ejmech.2009.09.045

A series of novel phosphorylated derivatives of galanthamine 6-11 and 12-17 were synthesized in two step process with high yields. In the first step galanthamine 1 was reacted with bis (2-chloroethyl) phosphoramidic dichloride 2/4-nitrophenyl phosphorodichloridate 3 in presence of triethylamine (TEA) in dry tetrahydrofuran (THF) yielded the intermediates 4/5. They were further reacted with various compounds like 2-aminoethanol, ethyleneglycol, ethylenediamine, 2-aminoethanethiol, 2-hydroxyethanethiol, monopotassium dihydrogenphosphate to obtain the title compounds 6-11 and 12-17. The title compounds showed promising antimicrobial, antioxidant activities and was greatly influenced by the presence of different bioactive groups.

Full text of DOI:10.1016/j.ejmech.2009.09.045

European Journal of Medicinal Chemistry 45 (2010) 203–209
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, spectral characterization and biological evaluation of phosphorylated
derivatives of galanthamine
,
b
c
V. Koteswara Rao a, A. Janardhan Rao a, S. Subba Reddy a, C. Naga Raju a , P. Visweswara Rao , S.K. Ghosh
*
a Department of Chemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh 517 502, India
b Department of Biotechnology, Sri Venkateswara University, Tirupati, Andhra Pradesh 517 502, India
c Bioorganic Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra 400 085, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel phosphorylated derivatives of galanthamine 6–11 and 12–17 were synthesized in two
step process with high yields. In the first step galanthamine 1 was reacted with bis (2-chloroethyl)
phosphoramidic dichloride 2/4-nitrophenyl phosphorodichloridate 3 in presence of triethylamine (TEA)
in dry tetrahydrofuran (THF) yielded the intermediates 4/5. They were further reacted with various
compounds like 2-aminoethanol, ethyleneglycol, ethylenediamine, 2-aminoethanethiol, 2-hydroxy
ethanethiol, monopotassium dihydrogenphosphate to obtain the title compounds 6–11 and 12–17. The
title compounds showed promising antimicrobial, antioxidant activities and was greatly influenced by
the presence of different bioactive groups.
Received 31 July 2009
Received in revised form
23 September 2009
Accepted 28 September 2009
Available online 6 October 2009
Keywords:
Gram-positive bacteria
Gram-negative bacteria
Antimicrobial activity
Antioxidant activity
Ó 2009 Elsevier Masson SAS. All rights reserved.
Phosphorylated derivatives of galanthamine
1. Introduction
these observations and applications of galanthamine, we have
focused on the synthesis of a series of novel phosphorylated
Galanthamine commercially available ReminylÒ [1] is an
approved AChE inhibitor in USA by the FDA, and in Europe by the
European registration bureau for the symptomatic treatment of
Alzheimer’s disease (AD) [2]. Galanthamine, the naturally occurring
alkaloid isolated from the bulbs of different species of the Amar-
yllidaceae family [3] and the Caucasian snow-drop (Galanthus
woronowii). Its structure was determined on the basis of spectral
properties. Galanthamine has been found to possess potent, wide
spread spectrum of activities like reversible competitive anti-
cholinesterase inhibitor [4], anti-anesthetic [5], analeptic [6],
analgesic [7] and antioxidant property [8]. Alzheimer’s disease [9–
11] is a progressive neurodegenerative disorder and one of the most
common causes of mental deterioration in the elderly people. Other
major and the most developed class of drugs approved for AD
therapy, such as tacrine (CognexÒ) [12], donepezil (AriceptÒ)
[13,14], rivastigmine (ExelonÒ) [15].
derivatives of galanthamine by incorporating the bioactive groups
attached to phosphorus atom, which fortunately resulted with
noticeable antioxidant and antimicrobial activities. The oxidative
stress of galanthamine derivatives was assessed by estimating the
activities of lipid peroxidation (LPO), reduced glutathione content
(GSH), superoxide dismutase (SOD) and catalase (CAT). The
obtained results were compared with the natural antioxidants like
Vitamin E and Vitamin C. Antimicrobial activity of phosphorylated
derivatives of galanthamine have been investigated against Gram-
positive bacteria: Bacillus subtilis, Staphylococcus aureus, and Gram-
negative bacteria: Escherichia coli and Klebsiella pneumoniae. The
structures of all the newly synthesized compounds have been
established by elemental analyses and spectral data (IR, 1H, 13C, 31
P
NMR and mass). All the compounds have been screened for their
antioxidant and antimicrobial activities.
These potent biological activities [16] has stimulated great
interest in the synthesis of such compounds for extensive studies
related to their biological activities. Previously galanthamine sulfur
analogs are devoid of noticeable AChE inhibitory activity. In view of
2. Chemistry
The synthesis of phosphorylated derivatives of galanthamine
was carried out by the following process. The hydrobromide salt of
galanthamine (Nivalin) was neutralized with ammonium
hydroxide to produce pure galanthamine 1 in 89% yield. Galanth-
amine 1 was reacted with bis (2-chloroethyl) phosphoramidic
* Corresponding author. Tel.: þ91877 2249666x479; fax: þ91877 2225211.
E-mail address: naga_raju04@yahoo.co.in (C. Naga Raju).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.09.045

204
V. Koteswara Rao et al. / European Journal of Medicinal Chemistry 45 (2010) 203–209
dichloride 2 in the presence of triethylamine (TEA) in dry tetrahy-
drofuran (THF) at ꢀ10 ꢁC for 2 h. The reaction was monitored by
thin layer chromatography (TLC). Formation of an intermediate 4
was ascertained after 2 h. Further, the intermediate 4 was reacted
with various compounds namely 2-aminoethanol, ethyleneglycol,
ethylenediamine, 2-aminoethanethiol, 2-hydroxyethanethiol,
monopotassium dihydrogenphosphate to obtain 6–11 in high yields
(68–82%). The same protocol was employed for the synthesis of
12–17 by reacting galanthamine 1 with 4-nitrophenyl phosphor-
odichloridate 3 under same reaction conditions to obtain the
intermediate 5. Further, the intermediate 5 was reacted with the
same compounds as shown in Scheme 1 to obtain 12–17 in high
yields (68–80%).
shifts for compounds 6–17 were observed in their expected regions.
31P NMR signals were observed in the region
[19,21].
d
ꢀ7.11 to ꢀ19.39 ppm
As shown in Table 4 most of the compounds showed high
activities for CAT, SOD, LPO and GSH inhibition compared to gal-
anthamine. The potency of these enzymes inhibition was mainly
influenced by the fragments attached to the phosphorylated gal-
anthamine. The fragments bis (2-chloroethyl) amine and 2-
hydroxyethanethiol linked to phosphorus led to a great change in
CAT, SOD, LPO and GSH inhibitory potency (i.e-9). Bis (2-chloro
ethyl) amine and 2-hydroxyethanethiol moieties were found to be
the most potent fragments for the enzymes inhibition. N and S
chains of phosphorus atom in compound 9 showed high inhibitory
potency when compared with other moieties.
3. Pharmacology
Attachment of different moieties to phosphorus atom could
affect the ability of the molecule to interact with the peripheral and
active cites of the enzymes simultaneously and there by influence
the CAT, SOD, LPO and GSH inhibitory potency. The bis (2-chloro
ethyl) amino fragment series (i.e 6–11) compounds 6, 8 and 9
showed high inhibitory potency for CAT, SOD, LPO and GSH when
compared to 7 and 10. However, the same trend was not observed
in the 4-nitrophenoxy series (i.e 12–17) compounds 14 and 15
showed high inhibitory potency of GSH and SOD respectively when
compared to compounds 12, 13, 16 and 17.
The significant loss in potency was observed when 4-nitro-
phenoxy moiety as R fragment in series attached to phosphorus
atom with different R1 moieties when compared to bis (2-chloro
ethyl) amino fragment as R series with different R1 moieties, sug-
gested that the two free chlorine atoms of aliphatic chain are highly
reactive when compared with the nitro group attached to an
aromatic ring, which led to a slight drop in enzyme inhibitory
potency of derivatives containing 4-nitrophenoxy moiety.
Replacement of the terminal R1 fragments in the phosphory-
lated derivative series 6–11 and 12–17 shows significant inhibitory
potency when phosphorus atom directly attached with N and S
aliphatic chains with terminal O–H group when compared with O,
S, N directly attached to phosphorus atom without having terminal
O–H group.
The antimicrobial activity of the phosphorylated derivatives of
galanthamine has been investigated and their results are presented
in Table 1. Antimicrobial activity has been evaluated against Gram-
positive bacteria: B. subtilis, S. aureus, and Gram-negative bacteria:
E. coli, K. pneumoniae. The antioxidant activity was evaluated by
Lipid Peroxidation, Reduced Glutathione Content, Superoxide Dis-
mutase and Catalase and their results are presented in Table 2.
Chloramphenicol was measured as a standard for antimicrobial
activity and Vitamine E and Vitamine C were measured as stan-
dards for antioxidant activity respectively.
4. Results and discussion
The chemical structures of all the title compounds 6 –17 were
characterized by IR, 1H, 13C, 31P NMR and APCI-MS studies and their
data are presented in the experimental section. Characteristic IR
stretching absorptions were observed in the regions
1212–1231 cmꢀ1, 3375–3385 cmꢀ1 and 3412–3416 cmꢀ1 for P]O
[17], N–H [18], O–H [19] respectively. In the 1H NMR spectra of
compounds 6–17, the chemical shifts of aromatic hydrogens of the
phenyl ring appeared as doublets in the region
N–H hydrogen resonated as a broad singlet in the region
4.86 and P–O–H protons were resonated as singlets at 7.27–7.35.
The allylic proton H6 was observed as a broad triplet because H7
and H5 protons are in the same chemical environment with
d
6.62–6.70 [20]. The
d
4.72–
The phosphorylated derivative 9 showed significant MIC against
all represented micro organisms similarly derivative 8 exhibited
significant MIC against BS, SA, KP micro organisms. Minimum
Inhibitory Concentration (MIC) in microbiology is the lowest
concentration of an antimicrobial that will inhibit the visible
growth of microorganism after over night incubation. These results
d
b
respect to H6 [2]. The H12 protons were observed as doublets in the
region 3.19–3.33 and 3.67–3.75 respectively. The H9 proton was
observed as a multplet in the region 1.50–1.78. In 13C NMR chemical
CH3
CH3
N
CH3
N
N
10
11
12
12a
9
1
8
O
P
O
P
7
R
12b
R
8a
O
P
THF, TEA 2
R1-H
6
R1
THF, TEA
4a
O
O
+
Cl
R
-10 ºC- reflux
3a
Cl
5
3
OH
O
O
4
Cl
2, 3
O
1
O
O
H3C
H
H3C
H
O
4, 5
6-11, 12-17
H
CH3
13
14
Cl
R=
N
Compound
R1
Compound
R1
15
16
O
Cl
6-11
OH
6, 12
O
P
OH
9, 15
S
S
15
OH
14
13
1
16
1
1
R=
O
NO2
OH
2
NH2
NH2
7, 13
8, 14
O
10, 16
11, 17
18
17
12-17
OH
N
H
N
H
Scheme 1. Synthesis of title compounds 6–17.

V. Koteswara Rao et al. / European Journal of Medicinal Chemistry 45 (2010) 203–209
205
Table 1
Table 2
Minimum inhibitory concentrations (MIC in
m
g/ml) of the compounds and
EC50 values for galanthamine and its derivatives along with standard antioxidants.
galanthamine against the respective microorganisms.
S.No
Compound
CAT
g/
mL)
5.06 4.93 3.84 4.21
LOP
g/
mL)
SOD GSH
g/ g/
mL) mL)
Gal Chlo 6 10 11 12 13 14 15 16 17
7
8
9
(
m
(
m
(
m
(m
BS 0.18 0.15 0.16 0.28 0.11 0.13 0.17 0.12 0.19 0.25 0.21 0.10 0.09 0.15
SA 1.0 0.20 0.38 0.42 0.29 0.21 0.55 0.49 0.76 1.09 0.81 0.29 0.22 0.75
KP 0.62 0.13 0.48 0.57 0.15 0.59 0.51 0.33 0.28 0.39 0.42 0.36 0.14 0.38
EC 0.7 0.25 0.42 0.50 0.35 0.26 0.28 0.55 0.46 0.67 0.44 0.34 0.23 0.62
Galanthamine
–
Cl
Cl
O
N
P
Abbreviations: Gal – galanthamine Bs – B. subtilis; Sa – Staphylococcus aureus; Kp –
Klebsiella pneumoniae; Ec – E. coli. Chlo – chloramphenicol.
O
P
O
OH
6
2.18 1.98 1.26 2.68
OH
clearly indicate that phosphorus atom directly attached to sulfur
and nitrogen with their aliphatic chains ending with OH and Cl
were found to possess promising MIC when compared with other
moieties at their ends. The derivatives 6, 15 and 16 also showed
high MIC values for BS and SA respectively, which indicates that
their activity was enhanced by the presence of terminal –OH group.
Cl
Cl
O
P
N
O
7
2.21 2.87 1.98 2.02
1.52 1.42 1.28 1.03
1.67 1.33 1.62 1.11
2.66 2.91 2.31 2.66
OH
5. Conclusion
Cl
Cl
O
N
P
In summary, a series of novel phosphorylated galanthamine
derivatives were synthesized and evaluated their antimicrobial and
antioxidant properties. Structure activity studies showed that the
antioxidant and antimicrobial potency was mainly influenced by
the functional groups at the end of the aliphatic chain, as well as the
nature of atom attached directly to phosphorus atom and the two
different types of R fragments attached to phosphorus atom, which
provided additional sites of interactions between the inhibitor and
the enzyme, which constitutes a key element for enhanced affinity.
The results suggest that the compounds 6, 8, 9, 14, 15 showed
promising antioxidant activities with different R1 fragments con-
taining terminal –OH groups and also with different R fragments
(i.e.) bis (2-chloroethyl) amino and 4-nitrophenoxy moieties.
Majority of the derivatives (6–17) exhibited higher antimicrobial
activity and lower MIC values when compared to galanthamine and
chloramphenicol. The compounds 6, 8, 9, 15, 16 exhibited higher
antimicrobial activities when compared with other derivatives. The
results provided a foundation for future design and development of
antioxidant, antimicrobial compounds and also more potent drugs
for AD.
HN
8
OH
Cl
O
P
N
Cl
S
9
OH
Cl
Cl
O
N
P
S
10
NH2
Cl
O
P
N
Cl
HN
11
12
2.73 2.88 2.77 2.43
NH
2
6. Experimental protocols
O
P
6.1. Chemistry
NO
2
O
O
O
OH
Chemicals were procured from Sigma–Aldrich, Merck and Lan-
caster, and were used as such without further purification. All
solvents used for spectroscopic and other physical studies were
reagent grade and were further purified by literature methods [22].
Melting points (m.p.) were determined using a calibrated ther-
mometer by Guna Digital Melting Point apparatus. They expressed
in degrees centigrade (ꢁC) and are uncorrected. Infrared Spectra (IR)
were obtained on a Perkin-Elmer Model 281-B spectrophotometer.
Samples were analyzed as potassium bromide (KBr) disks.
Absorptions were reported in wave numbers (cmꢀ1). 1H and 13C
NMR spectra were recorded as solutions in DMSO-d6 on a Bruker
AMX 400 MHz spectrometer operating at 400 MHz for 1H, 100 MHz
for 13C and 161.9 MHz for 31P NMR. The 1H and 13C chemical shifts
were expressed in parts per million (ppm) with reference to tet-
ramethylsilane (TMS) and 31P chemical shifts to 85% H3PO4. Optical
rotations (in degrees, ꢁ) were recorded in methanol on a Perkin-
Elmer Model 241 polarimeter at the sodium D line. APCI mass
spectra were recorded on a Jeol SX 102 DA/600 Mass spectrometer.
2.92 3.33 2.69 2.15
P
OH
O
NO2
3.28 3.17 3.28 2.82
P
O
O
13
14
OH
O
NO2
P
HN
O
4.94 2.81 2.57 1.08
OH
(continued on next page)

206
V. Koteswara Rao et al. / European Journal of Medicinal Chemistry 45 (2010) 203–209
Table 2 (continued)
Table 4
Antioxidants in brain homogenates of young rats using galanthamine derivatives
6–17.
S.No
Compound
CAT
g/
mL)
LOP
g/
mL)
SOD GSH
g/ g/
mL) mL)
(
m
(
m
(
m
(m
Parameters
LPO(m
g/mL)
GSH(
mg/mL)
SOD(
m
g/mL)
CAT(mg/mL)
Galanthamine
6
7
8
9
10
11
12
13
14
15
16
70.68 ꢄ 1.46
74.86 ꢄ 2.32
68.24 ꢄ 3.69
72.32 ꢄ 1.76
71.23 ꢄ 2.38
72.39 ꢄ 2.09
72.46 ꢄ 1.23
72.17 ꢄ 1.94
72.11 ꢄ 1.79
72.48 ꢄ 1.68
72.77 ꢄ 1.47
75.49 ꢄ 1.36
75.92 ꢄ 1.28
4.69 ꢄ 0.25 29.37 ꢄ 0.62 283.44 ꢄ 6.32
4.72 ꢄ 0.74 28.14 ꢄ 0.35 278.33 ꢄ 5.65
4.67 ꢄ 0.92 29.02 ꢄ 0.65 291.27 ꢄ 10.52
4.73 ꢄ 0.87 29.86 ꢄ 0.97 287.59 ꢄ 7.67
4.92 ꢄ 1.73 29.55 ꢄ 1.16 285.32 ꢄ 4.76
5.02 ꢄ 1.44 30.55 ꢄ 1.43 285.43 ꢄ 5.27
4.84 ꢄ 1.91 30.29 ꢄ 1.76 285.48 ꢄ 4.39
4.22 ꢄ 1.83 29.55 ꢄ 1.42 287.38 ꢄ 4.88
5.21 ꢄ 1.62 29.41 ꢄ 1.39 286.43 ꢄ 4.62
4.72 ꢄ 1.90 29.37 ꢄ 1.47 284.32 ꢄ 4.21
O
NO2
P
O
S
15
16
17
4.25 2.64 1.55 1.49
2.03 1.86 1.74 1.56
1.81 1.99 1.91 1.89
OH
O
P
NO2
O
S
4.77 ꢄ 1.64
29.69 ꢄ 1.86 288.58 ꢄ 4.47
5.08 ꢄ 1.72 29.85 ꢄ 1.71 292.83 ꢄ 4.89
5.06 ꢄ 1.88 29.73 ꢄ 1.66 295.92 ꢄ 4.75
NH2
17
Ascorbic acid
Vitamin E
88.06 ꢄ 1.28 31.34 ꢄ 0.74 Unaffected
385.46 ꢄ 5.25
82.78 ꢄ 1.43 43.62 ꢄ 1.54 32.30 ꢄ 1.04 403.27 ꢄ 4.76
O
Abbreviations: LPO – lipid peroxidation; GSH – reduced glutathione content; SOD –
superoxide dismutase; CAT – catalase.
NO2
P
HN
O
NH2
stirred for 30 min at 0 ꢁC, and then for 2 h at ambient temperature.
Ether (200 mL) was added, followed by addition of solid NaCl
results in formation of two layers. The aqueous layer was extracted
with ether (3 ꢂ 200 mL). The extract was washed with brine solu-
tion, dried over anhydrous Na2SO4. The solvent was removed under
reduced pressure to get crude galanthamine free base and then
purified by silica gel column chromatography eluting with acetone:
methanol (95:5) to afford pure galanthamine 1 as white crystals
Vitamin E
Vitamin C
–
1.48 1.22 1.03 0.99
1.37 1.02 0.99 0.98
Ascorbic acid
Abbreviations: LPO – lipid peroxidation; GSH – reduced glutathione content; SOD –
superoxide dismutase; CAT – catalase.
Elemental analyses were performed by Central Drug Research
Institute, Lucknow, INDIA.
(4.52 g, 85% yield), m.p. ½a D25
ꢃ
f114.4ꢁ, IR (KBr); (O–H) 3400 cmꢀ1
.
6.1.1. Procedure for the preparation of galanthamine 1
6.1.2. Procedure for the preparation of compound 6
Galanthamine hydrobromide (6.80 g, 18.5 mmol) was dissolved
in water (400 mL), the solution was cooled to 0 ꢁC and ammonium
hydroxide (70 mL) was slowly added. The reaction mixture was
To a stirred solution of galanthamine (1, 0.200 g, 0.876 mmol) in
dry tetrahydrofuran (THF) (17 mL) was added bis (2-chloroethyl)
phosphoramidic dichloride (2, 0.2269 g, 0.876 mmol) at ꢀ10 ꢁC in
the presence of triethylamine (0.0886 g, 0.876 mmol) (TEA). After
completion of the addition, the reaction mixture was stirred for 2 h
at 0 ꢁC. The reaction progress was monitored by thin layer chro-
matography (TLC) acetone: methanol (95: 5). After completion of
the reaction, it was filtered to remove triethylamine hydrochloride.
The filtrate was reacted with mono potassium dihydrogen phos-
phate (0.1191 g, 0.876 mmol) in THF at reflux temperature for 12 h.
The progress of the reaction was monitored by TLC acetone:
methanol (95: 5). After completion of the reaction, solvent was
removed in a rota-evaporator to obtain crude product. It was
purified by silica gel column chromatography eluting with acetone:
methanol (95:5) mixture to afford the title compound, 2-dihy-
drogenphosphato (3-methoxy-11-methyl-5, 6, 9, 10, 11, 12-hex-
Table 3
Antimicrobial activity of the phosphorylated derivatives of galanthamine 6–17.
Samples
Concentration (mg/mL)
Inhibition zone (in mm)
BS
SA
KP
EC
Galanthamine
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
2.0
1.0
2.0
1.65
2.25
2.89
4.49
2.36
4.89
1.75
3.46
1.53
3.01
1.90
4.82
1.84
3.90
1.63
3.78
1.57
3.71
1.81
4.16
1.62
3.06
1.14
3.03
1.53
4.91
1.02
2.99
1.39
2.66
1.75
4.65
2.43
5.02
1.69
4.78
1.47
3.09
2.38
4.92
1.67
3.51
2.09
4.56
1.42
4.27
1.53
3.94
1.82
4.91
1.32
4.47
2.42
5.09
2.67
3.08
1.83
2.21
2.08
3.68
2.69
4.76
2.78
4.45
3.03
6.46
2.35
4.65
2.50
4.80
2.73
5.45
1.72
4.96
2.61
5.44
1.93
5.01
1.57
6.33
2.33
6.11
3.62
4.02
1.47
2.02
1.88
4.08
1.65
3.18
1.72
3.86
1.55
2.21
1.59
2.43
2.12
4.90
2.22
4.97
2.05
5.01
1.70
4.44
1.26
4.38
1.76
4.84
1.62
3.73
2.02
3.27
6
7
8
9
ahydro-4aH-4-oxa-11-azacyclohepta
[de]
fluoren-6-yl)
bis
10
(2-chloroethyl) amino phosphate 6. The same experimental
procedure was adopted for the preparation of the remaining title
compounds 7–17.
11
12
6.1.2.1. Synthesis of 2-dihydrogenphosphato (3-methoxy-11-methyl-
5, 6, 9, 10, 11, 12-hexahydro-4aH-4-oxa-11-azacyclohepta [de] fluo-
ren-6-yl) bis (2-chloroethyl) amino phosphate 6. Yield: 78%;
13
14
m.p.151–153 ꢁC; ½a D25
ꢃ
ꢀ117.2ꢁ; IR (KBr) 3415 (O–H), 1228 (P]O),
cmꢀ1 1H NMR (DMSO-d6)
; d 1.50 –1.59 (m, 2H, H9), 2.39 (s, 3H,
15
NMe), 2.51–2.60 (m, 1H, H5
2H, H10), 3.23(d, 1H, J ¼ 13.6 Hz, H12
H16), 3.62 (t, 4H, J ¼ 13.6 Hz, H13, H15), 3.67(d, 1H, J ¼ 14.9 Hz,
H12 ), 3.82 (s, 3H, OMe), 4.07–4.11 (m, 1H, H4a), 4.62 (br t, 1H,
a
), 2.62–2.78(m, 1H, H5b), 2.99–3.10 (m,
16
a), 3.26 (t, 4H, J ¼ 13.6 Hz, H14,
17
b
J ¼ 6.0 Hz, H6), 6.00 (d, 1H, J ¼ 10.4 Hz, H7), 6.06 (d, 1H, J ¼ 10.4 Hz,
Chloramphenicol
H8), 6.62 (d, 1H, J ¼ 8.4 Hz, H1), 6.66 (d, 1H, J ¼ 8.0 Hz, H2), 7.27 (s,
2H, P–OH) ppm; 13C NMR (DMSO-d6)
d 30.1(C-9), 33.9(C-5), 41.8(C-
Abbreviations: Bs – B. subtilis; Sa – Staphylococcus aureus; Kp – Klebsiella pneumo-
niae; Ec – E. coli.
14), 42.2(NMe), 48.3(C-13), 55.9(C-8a), 56.1(OMe), 56.0(C-10),

V. Koteswara Rao et al. / European Journal of Medicinal Chemistry 45 (2010) 203–209
207
60.7(C-12), 62.2(C-6), 88.8(C-4a), 111.4(C-2), 122.2(C-1), 127.8(C-
150 ꢁC; ½a 2D5
ꢃ
ꢀ125.0ꢁ; IR (KBr) 3385 (N–H), 1231 (P]O), cmꢀ1
;
1H
12a), 127.6 (C-7), 129.4(C-8), 133.2(C-12b), 144.2(C-3), 145.9(C-3a);
NMR (DMSO-d6) d 1.59–1.65 (m, 2H, H9), 2.39 (s, 3H, NMe), 2.69–
ꢅ
31P (DMSO-d6)
d
ꢀ7.11, ꢀ19.39; APCI-MS m/z (%) 571[MHþ ]; Anal.
2.75 (m, 1H, H5
a
), 2.73 (t, 2H, J ¼ 15.6 Hz, H10), 2.98–3.04 (m, 2H,
Calcd. C21H30Cl2N2O8P2: C 44.15, H 5.29, N 4.90; Found: C 44.11, H
5.27, N 4.89.
H20), 2.91–3.02(m, 1H, H5
b), 3.10–3.14 (m, 2H, H10), 3.16 (d, 1H,
J ¼ 13.2 Hz, H12
H16), 3.64 (t, 4H, J ¼ 13.6 Hz, H13, H15), 3.71 (d, 1H, J ¼ 15.2 Hz,
H12 ), 3.84 (s, 3H, OMe), 4.07–4.11 (m, 1H, H4a), 4.61 (br t, 1H,
a
), 3.19 (s, 2H, NH2), 3.28 (t, 4H, J ¼ 13.6 Hz, H14,
6.1.2.2. Synthesis of 2-hydroxyethoxy (3-methoxy-11-methyl-5, 6, 9,
10, 11, 12-hexahydro-4aH-4-oxa-11-azacyclohepta [de] fluoren-6-yl)
bis (2-chloroethyl) amino phosphate 7. Yield: 82%; m.p.172–174 ꢁC;
b
J ¼ 6.2 Hz, H6), 6.01 (dd, 1H, J ¼ 10.4 Hz, H7), 6.07 (d, 1H, J ¼ 10.4 Hz,
H8), 6.63(d, 1H, J ¼ 8.4 Hz, H1), 6.67 (d, 1H, J ¼ 8.0 Hz, H2), ppm; 13C
½
a 2D5
ꢃ
ꢀ117.8ꢁ; IR (KBr) 3416 (O–H), 1231 (P]O), cmꢀ1
;
1H NMR
NMR (DMSO-d6) d
28.3(C-10), 31.2(C-9), 34.7(C-5), 37.7(C-20),
(DMSO-d6)
IH, H5
J ¼ 13.9 Hz, H12
d
1.60 –1.66(m, 2H, H9), 2.40 (s, 3H, NMe), 2.70–2.79 (m,
41.1(C-14), 43.1(NMe), 47.1(C-13), 54.9(C-8a), 56.2(OMe), 56.7(C-
10), 61.3(C-12), 62.5(C-6), 86.9(C-4a), 114.8(C-2), 119.3(C-1),
a
), 2.80–2.86 (m,1H, H5
), 3.32 (t, 4H, J ¼ 13.6 Hz, H14, H16), 3.61 (t, 4H,
), 3.80 (s, 3H,
b), 3.14–3.20 (m, 2H, H10), 3.26(d,1H,
a
126.8(C-7), 127.4(C-12a), 131.3(C-8), 134.2(C-12b), 144.4(C-3),
ꢅ
J ¼ 13.6 Hz, H13, H15), 3.70(d, 1H, J ¼ 15.2 Hz, H12
b
146.5(C-3a); 31P (DMSO-d6)
d
ꢀ12.46; APCI-MS m/z (%) 550[MHþ ].
OMe), 3.85–3.88 (m, 2H, H20), 3.99 (t, 2H, J ¼ 15.2 Hz, H10), 4.09–
4.14 (m, 1H, H4a), 4.62 (br t, 1H, J ¼ 5.5 Hz, H6), 5.02 (s, 1H, OH)6.03
(d, 1H, J ¼ 10.4 Hz, H7), 6.05 (d, 1H, J ¼ 10.4 Hz, H8), 6.64 (d, 1H,
J ¼ 8.4 Hz, H1), 6.67 (d, 1H, J ¼ 8.0 Hz, H2) ppm; 13C NMR (DMSO-
6.1.2.6. Synthesis of 2-aminoethylamino (3-methoxy-11-methyl-5, 6,
9, 10, 11, 12-hexahydro-4aH-4-oxa-11-azacyclohepta [de] fluoren-6-
yl) bis (2-chloroethyl) amino phosphate 11. Yield: 78%; m.p.152–
d6)
d
28.4(C-9), 34.7(C-5), 41.5(NMe), 43.5(C-14), 46.9(C-13),
154 ꢁC; ½a 2D5
ꢃ
ꢀ119.2ꢁ; IR (KBr) 3375 (N–H), 1228 (P]O), cmꢀ1
;
1H
54.1(OMe), 55.4(C-8a), 56.7(C-10), 61.3(C-20), 63.2(C-12), 67.3(C-10),
74.2(C-6), 86.1(C-4a), 114.8(C-2), 119.4(C-1), 126.4(C-12a), 127.3(C-
NMR (DMSO-d6)
d
1.56–1.65 (m, 2H, H9), 2.40 (s, 3H, NMe), 2.69–
2.80 (m, 1H, H5
a
), 3.02–3.10 (m, 1H, H5
b
), 3.12–3.17 (m, 2H, H10),
7), 131.4(C-8), 134.9(C-12b), 144.8(C-3), 146.5(C-3a); 31P (DMSO-d6)
3.19 (s, 3H, NH), 3.20–3.23 (m, 2H, H20), 3.25–3.29 (m, 2H, H10),
ꢅ
d
ꢀ14.59; APCI-MS m/z (%) 535[MHþ ] C23H33Cl2N2O6P: C 51.60, H
3.28 (t, 4H, J ¼ 13.6 Hz, H14, H16), 3.32(d, 1H, J ¼ 13.7 Hz, H12
a
b
),
),
6.21, N 5.23; Found: C 51.55, H 6.18, N 5.22.
3.60 (t, 4H, J ¼ 13.6 Hz, H13, H15), 3.71(d, 1H, J ¼ 15.2 Hz, H12
3.84 (s, 3H, OMe), 4.04–4.08 (m, 1H, H4a), 4.63(br t, 1H, J ¼ 5.7 Hz,
H6), 5.05 (s, 1H, OH), 6.05 (dd, 1H, J ¼ 10.4 Hz, H7), 6.08 (d, 1H,
J ¼ 10.4 Hz, H8), 6.60 (d, 1H, J ¼ 8.4 Hz, H1), 6.64 (d, 1H, J ¼ 8.0 Hz,
6.1.2.3. Synthesis of 2-hydroxy ethylamino (3-methoxy-11-methyl-5,
6, 9, 10, 11, 12-hexahydro-4aH-4-oxa-11-azacyclohepta [de] fluoren-
6-yl) bis (2-chloroethyl) amino phosphate 8. Yield: 76%; m.p.180–
H2) ppm; 13C NMR (DMSO-d6)
d
28.2(C-10), 32.1(C-9), 33.2(C-5),
182 ꢁC; ½a 2D5
ꢃ
ꢀ125.1ꢁ; IR (KBr) 3412 (O–H), 3385 (N–H), 1228 (P]O),
41.3(C-14), 42.9(C-20), 44.3(NMe), 46.9(C-13), 55.2(C-8a), 56.2(C-
10), 56.7(OMe), 61.2(C-12), 64.2(C-6), 86.7(C-4a), 114.7(C-2),
119.5(C-1), 126.6(C-7) 127.2(C-12a) 131.4(C-8), 134.9(C-12b),
cmꢀ1; 1H NMR (DMSO-d6)
d 1.52–1.57 (m, 2H, H9), 2.38 (s, 3H, NMe),
2.70–2.81 (m, 1H, H5a), 2.84–2.89 (m, 1H, H5b), 2.96–3.08 (m, 2H,
H10), 3.19 (s, 1H, NH), 3.24 (d, 1H, J ¼ 13.5 Hz, H12
a
), 3.28 (t, 4H,
144.3(C-3), 145.1(C-3a); 31P (DMSO-d6)
d
ꢀ14.89; APCI-MS m/z (%)
ꢅ
J ¼ 13.6 Hz, H14, H16), 3.49 (q, 2H, H10), 3.48–3.54 (m, 2H, H20), 3.60
533[MHþ ].
(t, 4H, J ¼ 13.6 Hz, H13, H15), 3.75(br d,1H, J ¼ 15.4 Hz, H12
b), 3.84 (s,
3H, OMe), 4.07–4.12 (m, 1H, H4a), 4.65 (br t, 1H, J ¼ 6.6 Hz, H6), 6.01
(dd, 1H, J ¼ 10.4 Hz, H7), 6.07 (d, 1H, J ¼ 10.4 Hz, H8), 6.62 (d, 1H,
J ¼ 8.4 Hz, H1), 6.64 (d, 1H, J ¼ 8.0 Hz, H2), ppm; 13C NMR (DMSO-d6)
6.1.2.7. Synthesis of 2-dihydrogenphosphato (3-methoxy-11-methyl-
5, 6, 9, 10, 11, 12-hexahydro-4aH-4-oxa-11-azacyclohepta [de] fluo-
ren-6-yl) (4-nitrophenyl) phosphate 12. Yield: 75%; m.pꢀ.1161–163 ꢁC;
;
1H NMR
d
28.4(C-9), 32.3(C-10), 34.7(C-5), 42.9(C-14), 43.5(NMe), 47.2(C-13),
½
a 2D5
ꢃ
ꢀ116.9ꢁ; IR (KBr) 3424 (O–H), 1230 (P]O), cm
56.2(OMe), 55.4(C-8a), 56.7(C-10), 61.3(C-20), 65.1(C-12), 74.5(C-6),
86.5(C-4a), 114.3(C-2), 119.2(C-1), 126.3(C-12a), 127.1(C-7), 131.5(C-
(DMSO-d6)
1H, H5
1H, J ¼ 13.4 Hz, H12
d 1.58–1.69 (m, 2H, H9), 2.39 (s, 3H, NMe), 2.66–2.80 (m,
a
), 2.89–2.94 (m, 1H, H5
b
), 3.22–3.29 (m, 2H, H10), 3.34(d,
), 3.80 (s, 3H,
8), 134.8(C-12b), 144.1(C-3), 146.5(C-3a); 31P (DMSO-d6)
d
ꢀ14.56;
a
), 3.71 (d, 1H, J ¼ 15.2 Hz, H12
b
ꢅ
APCI-MS m/z (%) 534[MHþ ]; Anal. Calcd. C23H34Cl2N3O5P: C 51.69, H
OMe), 4.09–4.13 (m, 1H, H4a),4.52 (br t, 1H, J ¼ 6.5 Hz, H6), 6.04 (dd,
1H, J ¼ 10.4 Hz, H7), 6.06 (d, 1H, J ¼ 10.4 Hz, H8), 6.64 (d, 1H,
J ¼ 8.4 Hz, H1), 6.67 (d, 1H, J ¼ 8.0 Hz, H2), 7.35 (s, 2H, POH) 7.38 (d,
2H, J ¼ 8.0 Hz, H14, H18), 8.10 (d, 2H, J ¼ 7.9 Hz, H15, H17), ppm; 13C
6.41, N 7.86; Found: C 51.65, H 6.39, N 7.83.
6.1.2.4. Synthesis of 2-hydroxy ethylmercapto (3-methoxy-11-
methyl-5, 6, 9, 10, 11, 12-hexahydro-4aH-4-oxa-11-azacyclohepta [de]
fluoren-6-yl) bis (2-chloroethyl) amino phosphate 9. Yield: 72%;
NMR (DMSO-d6)
d 31.3(C-9), 34.7(C-5), 42.9(NMe), 55.4(C-8a),
56.4(C-10), 56.7(OMe),61.3(C-12), 69.8(C-6), 86.1(C-4a), 114.8(C-2),
119.4(C-14 & C-18), 122.7 (C-1), 125.1 (C-15 & C-17),127.4 (C-12a),
131.1 (C-7),134.2 (C-8),135.5 (C-12b),141.2 (C-16),144.1 (C-3),146.2
m.p.147–149 ꢁC; ½a D25
ꢃ
ꢀ127.6ꢁ; IR (KBr) 3411 (O–H), 1230 (P]O),
cmꢀ1; 1H NMR (DMSO-d6)
d 1.56–1.62 (m, 2H, H9), 2.38 (s, 3H, NMe),
2.72–2.81 (m, 1H, H5
a
), 2.71 (t, 2H, J ¼ 15.6 Hz, H10), 3.02–3.09 (m,
(C-3a), 154.2 (C-13); 31P (DMSO-d6)
d
ꢀ7.83, ꢀ19.37; APCI-MS m/z
1H, H5
b
), 3.12–3.17 (m, 2H, H10), 3.19 (d, 1H, J ¼ 13.6 Hz, H12
a), 3.28
(%) 568 [MHþ]; Anal. Calcd. C23H26N2O11P2: C 48.60, H 4.61, N 4.93;
Found: C 48.55, H 4.58, N 4.91.
(t, 4H, J ¼ 13.6 Hz, H14, H16), 3.60 (t, 4H, J ¼ 13.6 Hz, H13, H15),
), 3.76 (s, 3H, OMe), 3.81–3.87 (m, 2H,
3.71(d, 1H, J ¼ 15.2 Hz, H12
b
H20), 4.09–4.15 (m,1H, H4a), 4.70 (br t,1H, J ¼ 5.9 Hz, H6), 5.04 (s,1H,
OH), 6.02(dd, 1H, J ¼ 10.4 Hz, H7), 6.07 (d, 1H, J ¼ 10.4 Hz, H8), 6.60
(d, 1H, J ¼ 8.4 Hz, H1), 6.63(d, 1H, J ¼ 8.0 Hz, H2), ppm; 13C NMR
6.1.2.8. Synthesis of 2-hydroxyethoxy (3-methoxy-11-methyl-5, 6, 9,
10, 11, 12-hexahydro-4aH-4-oxa-11-azacyclohepta [de] fluoren-6-yl)
(4-nitrophenyl) phosphate 13. Yield: 71%; m.p.158–160 ꢁC; ½a D25
ꢃ
(DMSO-d6)
d
28.4(C-10), 32.7(C-9), 35.9(C-5), 41.5(C-14), 42.9(NMe),
ꢀ112.4ꢁ; IR (KBr) 3416 (O–H), 1231 (P]O), cmꢀ1; 1H NMR (DMSO-
46.3(C-13), 54.3(C-8a), 56.5(C-10), 60.5(OMe), 61.3(C-20), 66.2(C-12),
74.6(C-6), 86.3(C-4a), 113.4(C-2), 119.3(C-1), 126.5(C-12a), 127.4(C-7),
d6)
H5
J ¼ 13.6 Hz, H12
d 1.55–1.62 (m, 2H, H9), 2.39 (s, 3H, NMe), 2.70–2.89 (m, 1H,
a
), 2.95–3.08(m, 1H, H5
b), 3.15–3.20 (m, 2H, H10), 3.23 (d, 1H,
131.4(C-8), 134.8(C-12b), 144.2(C-3), 145.3(C-3a); 31P (DMSO-d6)
a
), 3.70(d, 1H, J ¼ 15.2 Hz, H12
b), 3.72–3.79 (m, 2H,
ꢅ
d
ꢀ7.82; APCI-MS m/z (%) 551[MHþ ].
H20), 3.82 (s, 3H, OMe), 3.95 (t, 2H,, J ¼ 15.2 Hz, H10), 4.02–4.08 (m,
1H, H4a), 4.61 (br t, 1H, J ¼ 5.4 Hz, H6), 5.06 (s,1H, OH), 6.00 (dd,1H,
J ¼ 10.4 Hz, H7), 6.06 (d, 1H, J ¼ 10.4 Hz, H8), 6.62 (d, 1H, J ¼ 8.4 Hz,
H1), 6.66 (d, 1H, J ¼ 8.0 Hz, H2), 7.40 (d, 2H, J ¼ 8.5 Hz, H14, H18),
8.11 (d, 2H, J ¼ 9.2 Hz, H15, H17), ppm; 13C NMR (DMSO-d6)
6.1.2.5. Synthesis of 2-amino ethylmercapto (3-amino-11-methyl-5,
6, 9, 10, 11, 12-hexahydro-4aH-4-oxa-11-azacyclohepta [de] fluoren-
6-yl) bis (2-chloroethyl) amino phosphate 10. Yield: 68%; m.p.148–

208
V. Koteswara Rao et al. / European Journal of Medicinal Chemistry 45 (2010) 203–209
d
28.8(C-9), 35.9(C-5), 41.1(NMe), 55.2(C-8a), 56.2(C-10),
6.1.2.12. Synthesis of 2-aminoethylamino (3-methoxy-11-methyl-5, 6,
9, 10, 11, 12-hexahydro-4aH-4-oxa-11-azacyclohepta [de]fluoren-6-
56.2(OMe), 60.1(C-20), 61.2(C-12), 67.7(C-10), 71.9(C-6), 86.0(C-4a),
114.2(C-2), 119.2(C-14 & C-18), 121.8(C-1), 125.0 (C-15 & C-17),
127.2(C-12a), 131.2(C-7), 134.9(C-8), 137.3(C-12b), 141.1(C-16),
yl) (4-nitrophenyl) phosphate 17. Yield: 80%; m.p.175–177 ꢁC; ½a D25
ꢃ
ꢀ116.9ꢁ; IR (KBr) 3382 (N–H), 1220 (P]O), cmꢀ1; 1H NMR (DMSO-
144.5(C-3),146.5(C-3a),153.6(C-13); 31P (DMSO-d6)
d
ꢀ14.35; APCI-
d6)
H5
d 1.49– 1.64 (m, 2H, H9), 2.41 (s, 3H, NMe), 2.47–2.60 (m, 1H,
ꢅ
MS m/z (%) 532 [MHþ ], Anal. Calcd. C25H29N2O9P: C 56.39, H 5.49, N
a), 2.85–3,02(m, 1H, H5b), 3.07 (s, 3H, NH), 3.11–3.19 (m, 2H,
5.26; Found: C 56.32, H 5.46, N 5.24.
H10), 3.20–3.26 (m, 2H, H10), 3.33 (d, 1H, J ¼ 13.6 Hz, H12
a), 3.35–
b), 3.84 (s, 3H, OMe),
3.39 (m, 2H, H20), 3.74 (d, 1H, J ¼ 15.2 Hz, H12
6.1.2.9. Synthesis of 2-hydroxy ethylamino (3-methoxy-11-methyl-5,
6, 9, 10, 11, 12-hexahydro-4aH-4-oxa-11-azacyclohepta [de]fluoren-6-
4.08–4.16 (m, 1H, H4a), 4.65 (br t, 1H, J ¼ 6.3 Hz, H6), 4.86 (s, 2H,
NH2), 6.08 (dd, 1H, J ¼ 10.4 Hz, H7), 6.12 (d, 1H, J ¼ 10.4 Hz, H8), 6.66
(d, 1H, J ¼ 8.4 Hz, H1), 6.69 (d, 1H, J ¼ 8.0 Hz, H2), 7.41 (d, 2H,
J ¼ 9.6 Hz, H14, H18), 8.14 (d, 2H, J ¼ 8.6 Hz, H15, H17), ppm; 13C
yl) (4-nitrophenyl) phosphate 14. Yield: 68%; m.p.154–156 ꢁC; ½a D25
ꢃ
ꢀ118.0ꢁ; IR (KBr) 3426 (O–H), 3381 (N–H), 1212 (P]O), cmꢀ1
;
1H
NMR (DMSO-d6)
d
1.51– 1.69 (m, 2H, H9), 2.39 (s, 3H, NMe), 2.60–
NMR (DMSO-d6) d
28.2(C-10), 34.3(C-9), 35.9(C-5), 41.8(NMe),
2.81 (m, 1H, H5
a), 2.94–3.01 (m, 1H, H5
b), 3.22 (d, 1H, J ¼ 13.8 Hz,
42.5(C-20), 53.2(C-8a), 54.5(C-10), 61.1(C-12), 72.2(C-6), 86.1(C-4a),
113.5(C-2), 119.4(C-14 & C-18), 121.8(C-1), 125.2(C-15 & C-17),
126.3(C-12a), 131.1(C-7), 134.4(C-8), 137.5(C-12b), 141.3(C-16),
H12a
), 3.26–3.32 (m, 2H, H10), 3.39 (q, 2H, H10), 3.58–3.66 (m, 2H,
H20), 3.71 (d, 1H, J ¼ 15.2 Hz, H12
b), 3.82(s, 3H, OMe), 4.10–4.15 (m,
1H, H4a), 4.86 (s, 1H, NH), 4.65 (br t, 1H, J ¼ 6.5 Hz, H6), 5.06 (s, 1H,
OH), 6.05 (dd, 1H, J ¼ 10.4 Hz, H7), 6.08 (d, 1H, J ¼ 10.4 Hz, H8), 6.64
(d, 1H, J ¼ 8.4 Hz, H1), 6.68 (d, 1H, J ¼ 8.0 Hz, H2), 7.38 (d, 2H,
J ¼ 9.5 Hz, H14, H18), 8.12 (d, 2H, J ¼ 8.7 Hz, H15, H17), ppm; 13C
143.6(C-3)146.5(C-3a), 153.3(C-13); 31P (DMSO-d6)
d
ꢀ14.25; APCI-
ꢅ
MS m/z (%) 403 [MHþ ].
6.2. Pharmacology
NMR (DMSO-d6)
d
30.1(C-9), 35.7(C-5), 36.8(C-10), 41.4(NMe),
54.3(C-8a), 55.7(C-10), 56.4(OMe), 62.4(C-12), 63.2(C-20), 72.1(C-6),
86.2(C-4a), 113.8(C-2), 119.8(C-14 & C-18), 121.6(C-1), 125.9(C-15 &
C-17),127.4(C-12a), 131.9(C-7), 134.5(C-8), 137.2(C-12b), 140.9(C-
6.2.1. Preparation of microbial strains and inoculums
Antimicrobial activity of phosphorylated derivatives of gal-
anthamine have been investigated against Gram-positive bacteria:
B. subtilis, S. aureus, and Gram-negative bacteria: E. coli and
K. pneumoniae. A preliminary screening for antimicrobial activity of
6–17 and galanthamine was tested against the microbial cultures.
All the bacterial species were cultured in nutrient broth before the
antimicrobial activity test performed. The inoculated flasks were
incubated at 37 ꢁC for 18 h on a rotary shaker at 150 rpm. The
extracts were tested against all the species mentioned above and
the observed results showed 90% activity against both Gram-posi-
tive and Gram-negative bacteria in the disc-diffusion assay.
16), 144.2(C-3) 146.3(C-3a), 153.2(C-13); 31P (DMSO-d6)
d
ꢀ12.38;
ꢅ
APCI-MS m/z (%) 531 [MHþ ]; Anal. Calcd. C25H30N3O8P: C 56.49, H
5.69, N 7.91; Found: C 56.44, H 5.67, N 7.88.
6.1.2.10. Synthesis of 2-hydroxy ethylmercapto (3-methoxy-11-
methyl-5, 6, 9, 10, 11, 12-hexahydro-4aH-4-oxa-11-azacyclohepta
[de]fluoren-6-yl) (4-nitrophenyl) phosphate 15. Yield: Yield: 78%;
m.p.168–170 ꢁC; ½a D25
ꢃ
ꢀ119.0ꢁ; IR (KBr) 3432 (O–H), 1220 (P]O),
cmꢀ1 1H NMR (DMSO-d6)
; d 1.49– 1.57 (m, 2H, H9), 2.39 (s, 3H,
NMe), 2.66 (t, 2H, J ¼ 15.4 Hz, H10), 2.54–2.72 (m, 1H, H5
a), 2.85–
2.99 (m, 1H, H5
b
), 3.18–3.22 (m, 2H, H10), 3.25 (d, 1H, J ¼ 13.6 Hz,
6.2.2. Antimicrobial activity screening
H12
a
), 3.70 (d, 1H, J ¼ 15.2 Hz, H12
b
), 3.82 (s, 3H, OMe), 4.01–
Antimicrobial studies were performed according to agar disc-
diffusion method [23,24]. To obtain more significant information of
antimicrobial potency of galanthamine derivatives 6–17 against S.
aureus, B. subtilis, K. pneumoniae and E. coli, the following test
conditions were applied; all the compounds were dissolved in
dimethylformamide (DMF, Merck). Nutrient agar (Oxoid) plates
were prepared and dried at 35–36 ꢁC for about 30 min in an incu-
bator. Test strains were spread on solid nutrient agar surface by
using sterile spreader. At the same time, absorbent paper discs
were placed on agar surface (5 mm for compounds and 6 mm for
antibiotic) and impregnated with known concentrations which
4.09(m, 2H, H20), 4.10–4.13 (m, 1H, H4a), 4.64 (br t, 1H, J ¼ 6.3 Hz,
H6), 5.06 (s, 1H, OH), 6.02 (dd, 1H, J ¼ 10.4 Hz, H7), 6.08 (d, 1H,
J ¼ 10.4 Hz, H8), 6.61 (d, 1H, J ¼ 8.4 Hz, H1), 6.62 (d, 1H, J ¼ 8.0 Hz,
H2), 7.39 (d, 2H, J ¼ 8.7 Hz, H14, H18), 8.12 (d, 2H, J ¼ 8.2 Hz, H15,
H17), ppm; 13C NMR (DMSO-d6)
d
28.1(C-10), 34.1(C-9), 35.1(C-5),
42.9(NMe), 55.2(C-8a), 55.7(C-10), 56.7(OMe), 61.3(C-12), 71.9(C-
6), 86.1(C-4a), 112.3(C-2),119.4(C-14 & C-18), 121.3(C-1), 126.1(C-15
& C-17), 127.5(C-12a), 130.6(C-7), 134.8(C-8), 137.1(C-12b), 141.9(C-
16), 143.8(C-3), 146.45(C-3a), 154.1(C-13); 31P (DMSO-d6)
d 7.82;
ꢅ
LCMS m/z (%) 548 [MHþ ].
determined previously by MIC tests (500 mg for each disc). Blank
6.1.2.11. Synthesis of 2-amino ethylmercapto (3-amino-11-methyl-5,
6, 9, 10, 11, 12-hexahydro-4aH-4-oxa-11-azacyclohepta [de]fluoren-6-
test showed that, DMF used in the preparations of the test solutions
does not affect the test organisms, they were inverted and allowed
to incubate at 37 ꢁC. The inhibition zone around the disc was
calculated edge to edge zone of confluent growth which is usually
corresponds to the sharpest edge of the zone and was measured in
millimeters. All tests were repeated three times and average data
has taken as final result (Table 3).
yl) (4-nitrophenyl) phosphate 16. Yield: 68%; m.p.168–170 ꢁC; ½a D25
ꢃ
ꢀ117.7ꢁ; IR (KBr) 3376 (N–H), 1222 (P]O), cmꢀ1; 1H NMR (DMSO-
d6)
d 1.54– 1.78 (m, 2H, H9), 2.40 (s, 3H, NMe), 2.68 (t, 2H,
J ¼ 15.5 Hz, H10), 2.62–2.83 (m, 1H, H5
a
), 2.94–3.09(m, 1H, H5
b
),
3.07–3.15 (m, 2H,H20), 3.23 (d, 1H, J ¼ 13.6 Hz, H12
a), 3.27–3.39 (m,
b), 3.80 (s, 3H, OMe), 4.05–4.11
2H, H10), 3.72 (d,1H, J ¼ 15.2 Hz, H12
(m, 1H, H4a), 4.86 (s, 2H, NH2), 4.62 (br t, 1H, J ¼ 6.0 Hz, H6), 6.11
(dd, 1H, J ¼ 10.4 Hz, H7), 6.14 (d, 1H, J ¼ 10.4 Hz, H8), 6.68 (d, 1H,
J ¼ 8.4 Hz, H1), 6.70 (d, 1H, J ¼ 8.0 Hz, H2), 7.40 (d, 2H, J ¼ 9.4 Hz,
H14, H18), 8.15 (d, 2H, J ¼ 8.6 Hz, H15, H17), ppm; 13C NMR (DMSO-
6.2.3. Evaluation of antioxidant potential
Adult Wistar albino rats (90 days) were used in the study. The
rats were fed a standard pellet diet (Hindustan Lever Ltd., India)
and were given water ad libitum. The animals were maintained
under proper temperature (25–30 ꢁC), ventilation and hygienic
conditions. They were exposed to 12 h each of light and dark. The
phosphorylated derivatives of galanthamine were used to estimate
the antioxidant activity in vitro. The oxidative stress was assessed
by estimating lipid peroxidation (LPO), reduced glutathione
content (GSH) and the activities of superoxide dismutase (SOD)
d6)
d
28.8(C-10), 35.2(C-9), 36.1(C-5), 41.1(C-2), 42.4(NMe), 53.6(C-
8a), 54.5(C-10) 56.2(OMe), 62.6(C-12), 71.4(C-6), 85.4(C-4a),
114.1(C-2), 118.6(C-14 & C-18), 121.7(C-1), 124.8(C-15 & C-17),
126.5(C-12a), 130.1(C-7), 133.7(C-8), 136.8(C-12b), 140.5(C-16),
143.7(C-3), 146.3(C-3a), 155.0 (C-13), 31P (DMSO-d6)
d
ꢀ14.12; APCI-
ꢅ
MS m/z (%) 547 [MHþ ].

V. Koteswara Rao et al. / European Journal of Medicinal Chemistry 45 (2010) 203–209
209
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Net Rate ꢀ Intercept
GSH ¼
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This study is supported from the BRNS (DAE) research project
(2007/37/46/BRNS/2916), Mumbai, INDIA.
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