A new antimetabolite, 5-morpholinomethyl-2-thiouracil - Spectral properties, thermal profiles, antibacterial, antifungal and antitumour studies of some of its metal chelates

Article abstract of DOI:10.1039/b202127g

A new antimetabolite of thymine, 5-morpholinomethyl-2-thiouracil (MMTU) has been synthesized via the Mannich reaction of 2-thiouracil, formaldehyde and morpholine. The product was chemically and structurally characterized using various techniques ranging

Full text of DOI:10.1039/b202127g

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A new antimetabolite, 5-morpholinomethyl-2-thiouracil—spectral
properties, thermal profiles, antibacterial, antifungal and
antitumour studies of some of its metal chelates†
a
a
b
P. Kamalakannan, D. Venkappayya* and T. Balasubramanian
a
Department of Chemistry, Regional Engineering College, Tiruchirappalli-620015, India.
E-mail: dvenka@rect.ernet.in
Department of Physics, Regional Engineering College, Tiruchirappalli-620015, India
b
Received 28th February 2002, Accepted 18th June 2002
First published as an Advance Article on the web 26th July 2002
A new antimetabolite of thymine, 5-morpholinomethyl-2-thiouracil (MMTU) has been synthesized via the Mannich
reaction of 2-thiouracil, formaldehyde and morpholine. The product was chemically and structurally characterized
using various techniques ranging from microelemental analysis to XRD studies. About thirteen metal ion complexes
of MMTU have been isolated and were found to exhibit five different types of bonding pattern such as unidentate
dimeric, bidentate, bidentate binuclear, tridentate binuclear and tetradentate binuclear. To gain theoretical support
for the experimental findings regarding the donor atoms proposed based on spectral studies of the complexes semi-
empirical quantum mechanical calculations were carried out using MOPAC. The anomalous behaviour exhibited by
a few complexes was explained based on steric constraint and stabilization energy gain upon six membered chelate
formation. Thermodynamic and kinetic parameters were evaluated. Antimicrobial, antifungal and antitumour
studies have also been carried out. Structure, thermal stability and biological activity of the complexes have been
correlated.
Introduction
the study of the interaction of metal ions with the above
derivative would be useful in understanding the effect of free
heteroatoms on biological activity.
The present objective is to synthesize a more potent anti-
metabolite and to explore preliminarily the possibility of its use
in chemotherapy. This work is in continuation of our systematic
study of the synthesis and characterization of new antimetabol-
Results and discussion
1
–6
ites derived from pyrimidine and their metal ion complexes.
MMTU crystallised from methanol registers a single R value
f
The substituted (especially at the 5 position) uracils and thio-
uracils are known to play a vital role in many metabolic
on an HPLC run confirming its purity. The compound melts at
Ϫ1
2
08.3 ЊC with a heat of absorption of 103.3 J g . The C, H, N
7–20
21–23
processes
most of which are also mediated by metal ions.
and S analytical results were found to be 47.47, 5.71, 18.50,
4.23 and 14.09% respectively, which are in close agreement
with the calculated values of 47.58, 5.73, 18.50, 14.10 and
4.10%. Elemental analysis indicates the molecular formula as
C H N O S.
Many of the drugs have been known to exhibit increased anti-
cancer activity when administered as metal complexes.
Considering the structural requirements for significant cyto-
1
2
4–27
1
2
8–33
toxic activity, carrier moieties such as heterocyclic amines
9
13
3
2
have been introduced so as to readily react with plasma pro-
teins. In particular, morpholine substituted anthracyclines and
adriamycin have been found to show greater antitumour activ-
Ϫ3
The UV-Vis spectrum of a 10 M MMTU in DMF
registered bands at 260 and 290 nm which are assignable to the
n
π* and π
π* transitions, respectively of the thiocarbonyl
34,35
ity than the parent compound.
In view of the above it was
group. The above two bands occur at slightly longer wave-
considered appropriate to introduce morpholine through a
methylene bridge at the 5 position of 2-thiouracil via the
Mannich route and isolate the new product, MMTU. The
thiouracils have the potential to bind to the metal ion through
40
lengths compared with those for 2-thiouracil which indicates
the derivatisation of the compound.
The IR spectrum of MMTU registers absorption bands at
Ϫ1
3
200 and 3140 cm which are the split bands of a symmetric
36–39
sulfur, nitrogen, oxygen or a combination of these.
Such
Ϫ1
stretch of NH and the other bands at 3040 and 2960 cm arise
bindings are likely to leave some potential donor atoms free and
these free donor atoms enhance the biological activity. Apart
from the thiouracil donor atoms, morpholine nitrogen and
oxygen atoms can also act as binding sites. It was thus felt that
due to νCH of ‘CH’ and ‘CH ’ groups, respectively. The sharp
2
Ϫ1
absorption bands at 1690, 1571 and 1473 cm are due to νC(4)O
,
δN(1)H and δN(3)H, respectively. Compared with the IR spectrum
Ϫ1
of 2-thiouracil, a new sharp and intense band at 1102 cm is
seen which can be assigned to νC–N–C of the morpholine moiety.
This band confirms the insertion of the amino moiety into
†
Electronic supplementary information (ESI) available: thermograms,
Ϫ1
infrared, NMR and mass spectra of MMTU; table comparing bond
lengths in MMTU with other thiouracil derivatives; tables of data for
MMTU including: least-squares plane calculations, hydrogen bond dis-
tances, results of the PM3 calculation, eigen values for the M.O. levels,
thiouracil. The absorption band at 1225 cm is assigned to νC ᎐᎐ S.
1
The H NMR spectrum of MMTU (Fig. S1, ESI) in
DMSO-d contained four signals, two singlets and two doub-
6
41
lets. Comparing this NMR spectrum with that for thiouracil,
S
A.O. electron populations using the PM3 Hamiltonian, the bonding
it is observed that the resonance signal due to C(5)H is absent
and three new signals are observed at highfield. The chemical
shift values of the signals are δ 7.32 (s), 5.84 (s), 3.56 (t), 3.16 (s)
contribution of each M.O., dipole, point charge and hybrid values and
net atomic charges and dipole contributions. See http://www.rsc.org/
suppdata/dt/b2/b202127g/
DOI: 10.1039/b202127g
J. Chem. Soc., Dalton Trans., 2002, 3381–3391
This journal is © The Royal Society of Chemistry 2002
3381

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and 2.38 (t) which can be assigned to C(6)H, C(2)SH, –(CH ) –
2
2
O, –CH – and –N–(CH ) –, respectively. These signals evidence
2
2 2
the incorporation of an aminomethyl moiety at the 5 position
of 2-thiouracil. Offset scanning beyond δ 10 to capture the
N(1)H and N(3)H resonances was unsuccessful due to the
exceedingly fast exchange rates and therefore this proton could
not be identified using the CW-60 MHz NMR spectrometer.
However, in the FT-400 MHz NMR spectrometer the N(3)H
13
signal appeared as a broad band at δ 12.10. The C NMR
signals at δ 176.111, 161.479, 140.167, 113.361, 105.362, 66.261
and 52.949 (Fig. S2, ESI) were assigned to C(2)SH, C(4)O,
C(6)H, C(5)H, N–(CH ) , (CH ) –O and –CH – respectively.
2
2
2
2
2
The observed carbon chemical shift values are in close agree-
ment with the structure proposed on the basis of H NMR
1
Fig. 1 ORTEP structure of MMTUؒHCl.
data. The above observation is further substantiated by the fact
that 2-thiouracil exists as the thiol rather than as the thione
isomer.
The solution state high resolution NMR spectrum exhibits a
signal corresponding to the presence of N(3)H and the absence
of N(1)H, whereas the solid state IR study indicates the pres-
differences in molecular dimensions because of several factors:
42
the conjugation of the lone electron pair on N(3) with C(4)᎐O,
᎐
or on S(7) with C(5)᎐C(6), electron delocalisation of different
᎐
sorts and hydrogen bonding. The remarkable shortening
47–50
reported for C(4)᎐O(8) in saturated pyrimidines
was also
᎐
observed in the present dimeric structure. The C(2)᎐S(7) dis-
᎐
ence of C(2)᎐S and N(1)H. It is therefore inferred that in the
᎐
tance observed is shorter than for similar bonds in the thione
solid state MMTU exists in the thione form (I). In DMSO solu-
tion, it displays thione–thiol equilibrium, the latter being the
predominant isomer.
43,45,47
form of thiopyrimidine.
The valence angles at N1 and N3
are larger than 120Њ and these N atoms have extra-annular H
51
atoms, in accordance with observations cited elsewhere. The
least-squares plane calculation for the morpholine ring on
MMTU (Table S2, ESI) shows that N10 and O13 are displaced
on opposite sides, i.e. O13 and N10 are in endocyclic and exo-
cyclic positions respectively in molecule A, whereas in molecule
B O13 and N10 are in exocyclic and endocyclic positions,
respectively. In both molecules S7, O8 and C9 of the thiouracil
moiety significantly deviate from the mean plane of N1–C2–
N3–C4–C5–C6. The thiouracil and morpholine rings are not in
the same plane. The total puckering amplitude for thiouracil
and morpholine was found to be 0.0375 and 0.5849 Å for
molecule A and 0.0420 and 0.5818 Å for molecule B. In this
structure we observe a different type of hydrogen bonding
The mass spectrum of MMTU (Fig. S3, ESI) exhibits a
molecular ion peak at m/z 227 which corresponds to the
proposed molecular mass of the compound. The likely frag-
mentation pattern of MMTU is depicted in the following
scheme.
Molecular topology of MMTU
The crystal data and structure refinement parameters for
MMTU are given in Table 9. An ORTEP view of MMTU with
the atom numbering scheme is shown in Fig. 1. There are two
pseudo-symmetric molecules with two water oxygens and two
chlorides in the asymmetric unit. Table S1 (ESI) compares the
bond lengths of MMTU with those of 5,6-dihydro-2-
pattern (Fig. 2, Table S3, ESI) which differs from the character-
4
3
44
45
46
52,53
thiouracil, 5,6-dihydrouracil, 2,4-dithiouracil, uracil and
6
istic pattern exhibited by pyrimidine analogs.
The molecules
47
-amino-2-thiouracil monohydrate. The C(5)–C(6) distances
A and B are connected by hydrogen bonds C11–H11Ј ؒ ؒ ؒ O8Ј
and C15Ј–H15 ؒ ؒ ؒ O8 of 2.527 and 2.531 Å, respectively form-
ing random HB arrays. Two inter-molecular contacts
H6 ؒ ؒ ؒ S7 and H9 ؒ ؒ ؒ S7 of 2.744 and 2.964 Å are seen which
of 1.339(8) Å for molecule A and 1.359(8) Å for molecule B are
3
48
significantly shorter than the sp single bond value of 1.533 Å.
It is difficult to derive any general conclusion about the
3
382
J. Chem. Soc., Dalton Trans., 2002, 3381–3391

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Table 1 Elemental analyses and molar conductance data for cobalt(), nickel() and zinc() complexes of MMTU
Elemental analyses (%)
a
Ϫ1
Yield Carbon
(%)
Hydrogen
Nitrogen
Sulfur
Metal
Anion
ΛM /Ω
2
Ϫ1
Compound
ؒ ؒ [N SO C H CoCl C H N SO ] ؒ ؒ ؒ
Obs. (calc.) Obs. (calc.) Obs. (calc.) Obs. (calc.) Obs. (calc.) Obs. (calc.) cm mol
ؒ
80
78
71
82
80
75
67
74
77
80
78
75
73
30.25
30.27)
24.32
24.23)
18.79
18.76)
26.35
26.35)
25.85
25.84)
27.50
27.51)
18.79
18.77)
24.24
24.23)
25.92
25.86)
28.37
28.33)
23.90
23.89)
25.95
25.94)
27.80
27.81)
3.70
(3.67)
2.98
(2.94)
2.95
(2.97)
3.18
(3.19)
4.07
(4.10)
4.35
(4.36)
2.95
(2.95)
3.82
(3.84)
4.10
(4.10)
3.98
(3.96)
2.91
(2.90)
3.15
(3.14)
3.35
11.78
(11.77)
9.40
(9.42)
7.28
9.00
(8.98)
7.20
(7.19)
5.56
(5.57)
7.81
(7.82)
15.31
(15.33)
8.15
(8.16)
5.58
(5.56)
7.18
(7.18)
15.35
(15.34)
8.42
(8.40)
7.10
16.51
(16.50)
13.23
(13.21)
10.25
(10.23)
14.40
(14.37)
14.10
(14.09)
14.95
(14.94)
10.21
(10.20)
13.15
(13.14)
14.05
(14.04)
17.15
(17.14)
14.45
(14.45)
15.71
(15.69)
16.80
19.87
(19.85)
35.86
(35.83)
44.00
(44.06)
—
(30.25)
22.95
(22.96)
18.06
(18.05)
44.12
(44.10)
—
(30.27)
22.98
(22.98)
18.56
(18.58)
35.33
(35.32)
—
45
67
3
2
9
13
2
9
13
3
2
(
(
(
(
(
(
(
(
(
(
(
(
(
[
[
[
[
[
[
[
[
[
[
[
[
C H N SO CoBr ]
9 13 3 2 2
C H N SO CoI (H O) ]
67
9
13
3
2
2
2
2
(7.29)
17.08
(17.07)
10.06
(10.04)
10.69
(10.69)
7.29
(7.30)
15.70
(15.70)
10.00
(10.05)
11.00
(11.01)
9.31
C H N SO Co(NO ) ]
83
9
13
3
2
3 2
C H N SO ؒCoSO (H O) ]
39
9
13
3
2
4
2
2
C H N SO ؒNiCl (H O) ]
30
9
13
3
2
2
2
2
C H N SO ؒNi(H O) ]
155
81
9
13
3
2
2
2
C H N SO ؒNi(NO ) (H O) ]
9
13
3
2
3
2
2
2
C H N SO ؒNiSO ؒ(H O) ]
64
9
13
3
2
4
2
2
(C H N SO ) ZnCl ؒH O]
26
9
13
3
2
2
2
2
(C H N SO ) ؒZn ]Br
4
162
30
9
13
3
2
2
2
(9.29)
16.83
(16.81)
10.80
(10.81)
(7.09)
7.70
(7.70)
16.50
(16.50)
(C H N SO ) ؒZn (NO ) ]
9
13
3
2
2
2
3 4
(29.78)
24.73
(24.71)
C H N SO ؒZn (SO ) ]
25
9
13
3
2
2
4 2
(3.37)
(16.82)
a
In DMF solution.
iodide and zinc() bromide complexes negative shifts in respect
of νC(2)S, δN(3)H, νC(4)O and νC–N–C by about 104–108, 17–88,
Ϫ1
3
0–100 and 27–91 cm respectively, are observed. The stretch-
ing frequencies of the thiocarbonyl and carbonyl group exhibit
Ϫ1
negative shifts by about 85–107 and 40–80 cm respectively, in
the copper() sulfato and zinc() nitrato and sulfato complexes.
In the IR spectrum of the zinc() chloro complex νC(2)S
,
Ϫ1
νC(4)O and νC–N–C bands are lowered by 104, 29 and 26 cm ,
respectively. The cobalt() chloro complex exhibits negative
shifts only in respect of C(4)O and N(1)H stretching and bend-
Ϫ1
ing vibrations by about 85 and 62 cm , respectively.
The diagnostic bands of the nitrato group in the case of
cobalt() and zinc() complexes show a separation of (ν Ϫ ν )
5
1
Ϫ1
=
100 cm which indicates the monodentate mode of coordin-
ation. The IR spectrum of the nickel() nitrato complex
Ϫ1
exhibits bands at ∼1500, ∼1300 and ∼1000 cm which do not
have any corresponding bands in the free ligand. These are
Fig. 2 Hydrogen bonding and stacking interactions of MMTUؒHCl.
assigned to ν , ν and ν modes of the coordinated nitrato group.
1
5
2
54
are lower than the H ؒ ؒ ؒ S van der Waals distance of 3.05 Å.
The other two contacts of S7 with H11 and H14 are 3.102 and
.005 Å, respectively. Pyrimidine oxygen atom O8 is only
The magnitude of ν Ϫ ν clearly indicates that the nitrato group
5 1
55
is coordinated bidentately in these complexes. All the sulfato
complexes register new strong bands in the range of 1200–1290,
3
Ϫ1
involved in the C–H ؒ ؒ ؒ O type interaction.
1125–1170 (ν ); 860–980 (ν ); 600–650 (ν ) and 540–580 cm
3
1
4
(
ν ) which point to a bidentate mode of coordination for
2
55
Microelemental analyses and conductance measurements
the sulfato group. The cobalt() iodo and sulfato; nickel()
chloro, sulfato and iodide and zinc() chloro complexes exhibit
new non-ligand bands at 3400, 1620, 860, 820 and 760 cm in
Ϫ3
The ΛM values for 10 M DMF solutions of the nickel()
Ϫ1
iodide and zinc() bromide complexes suggest them to be 1 : 2
electrolytes and the remaining complexes are non-electrolytes.
The analytical data for C, H, N, S, metal and anion content in
the complexes are given in Table 1.
55
the complexes indicating the presence of coordinated water.
In a few cases the νOH band has been masked by other broad
bands in that region. However, the wagging and rocking modes
Ϫ1
of water occurring at 605 and 400 cm respectively show the
presence of coordinated water.
Infrared spectra
The above IR data indicate that MMTU exhibits six types of
coordination pattern viz., bidentate through C(4)O and C–N–
C; bidentate binuclear through C(2)S and C–N–C and C(2)S
and C(4)O; tridentate binuclear through C(2)S, C(4)O and
C–N–C; polymer of unidentate dimeric arrays involving N(1)H
and C(4)O atoms; and tetradentate binuclear through C(2)S,
N(3)H, C(4)O and C–N–C. Far IR band assignments further
Comparison of the IR spectrum of MMTU with those of its
complexes (Table 2, also see Figs. S4–S6, ESI) suggests the fol-
lowing. Perceptible shifts by about 50 to 100 and 52 to 90 cm
are observed in the νC(4)O and νC–N–C respectively in the case of
cobalt() bromo, iodo, nitrato and sulfato and nickel() chloro,
nitrato and sulfato complexes. In the IR spectra of the nickel()
Ϫ1
J. Chem. Soc., Dalton Trans., 2002, 3381–3391
3383

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Ϫ1
Table 2 Important IR absorption bands (cm ) of cobalt(), nickel() and zinc() complexes of MMTU
Compound
C H N SO
2
νNH
νCH
νC᎐O
δN(1)H
δN(3)H
νC᎐S
νC–N–C
3280m
3458m
3396b
3100m
3090m
1690s
1605s
1594s
1591s
1595s
1595s
1620s
1645s
1640s
1590s
1661s
1660s
1610s
1615s
1571m
1473m
1469m
1476m
1468m
1465m
1475m
1470m
1456m
1470m
1473m
1470m
1430m
1470m
1475m
1225s
1228s
1229s
1194s
1220s
1129s
1220s
1117s
1120s
1125s
1121s
1120s
1140s
1130s
1102s
1101s
9
13
3
ؒ
ؒ ؒ [N SO C H CoCl C H N SO ] ؒ ؒ ؒ
1509m
1547m
1542m
1545m
1570m
1575m
1586m
1575m
1575m
1560m
1562m
1565m
1562m
3
2
9
13
2
9
13
3
2
[
[
[
[
[
[
[
[
[
[
[
[
C H N SO CoBr ]
1013m
1012m
1015m
1020m
1025m
1053m
1050m
1040m
1076m
1075m
1100m
1100m
9
13
3
2
2
C H N SO CoI (H O) ]
3420b
3423b
3420b
9
13
3
2
2
2
2
C H N SO Co(NO ) ]
9
13
3
2
3 2
C H N SO ؒCoSO (H O) ]
9
13
3
2
4
2
2
C H N SO ؒNiCl (H O) ]
3460m
3422m
3420b
3050m
3222m
9
13
3
2
2
2
2
C H N SO ؒNi(H O) ]I
9
13
3
2
2
2
2
C H N SO ؒNi(NO ) (H O) ]
9
13
3
2
3
2
2
2
C H N SO ؒNiSO ؒ(H O) ]
3485b
9
13
3
2
4
2
2
(C H N SO ) ZnCl ؒH O]
3800m
3400m
3400m
3400m
3029m
3058m
3060m
3050m
9
13
3
2
2
2
2
(C H N SO ) ؒZn ]Br
9
13
3
2
2
2
4
(C H N SO ) ؒZn (NO ) ]
9
13
3
2
2
2
3 4
C H N SO ؒZn (SO ) ]
9
13
3
2
2
4 2
s = Sharp, m = medium b = broad.
support the metal–heteroatom and metal–anion bonding
suggested by the IR data.
spectra of zinc() nitrato and sulfato complexes suggest
coordination through the C(4)O and C(2)S and accordingly no
major shifts in the positions of NMR signals are observed in
these cases. The zinc() chloro complex registers an additional
signal at δ 4.08–4.18 confirming the presence of coordinated
water in this complex.
Electronic spectra
Cobalt(II) complexes. The UV-Vis spectra of cobalt()
chloro, bromo and nitrato complexes exhibit absorption bands
Based on microelemental analyses, conductance measure-
(
Table 3) in the visible and near IR region due to the transition
1
ments, IR, UV-Vis, magnetic moment measurements and H
4 4 4
from A to T (P) and T (F), respectively. These transitions are
characteristic of cobalt() in a tetrahedral environment. The
2
1
1
NMR the following are the tentative structures proposed for
the complexes:
56
magnetic moments of the complexes lie in the range of 4.41–
4
.73 µB which again supports the tetrahedral environment
57
Semi-empirical quantum mechanical calculations
around cobalt(). The percentage covalency for the cobalt()
chloro, bromo and nitrato complexes are 35, 41 and 40, respect-
ively. This observation can be explained on the basis of the
polarisabilities of the anion. The LFSE (Table 3) for the com-
plexes were found to be in the order: nitrato > chloro > bromo
complexes. Electronic spectra of the cobalt() iodo and sulfato
complexes exhibit bands corresponding to the transitions from
The optimized geometry of MMTU is shown in Fig. 3. The
4
4
4
4
T (F) to T , A and T (P). The µ values fall in the range
1g
2g
2g
1g
eff
5
.30–5.34 µ and the electronic spectral band positions indicate
B
an octahedral coordination geometry for these complexes. The
various ligand field parameters for the complexes are listed in
Table 3. Among the halide complexes the ligand field splitting
Ϫ
Ϫ
Ϫ
trend observed is I > Cl > Br . Among tetrahedral complexes
Ϫ
Ϫ
the order of crystal field splitting is found to be Br < Cl
<
Ϫ
NO3 which is in accordance with their positions in the
spectrochemical series.
Fig. 3 Optimized geometry of MMTU for M.O. calculations.
results of the calculations (Table S4, ESI) show that there are
Nickel(II) complexes. The chloro, iodide, sulfato and nitrato
4
1 filled levels, the HOMO has an energy of 9.20756 eV. The
complexes have a magnetic moment of 3.34 to 3.65 µ suggest-
B
negative value of this energy level is the ionization potential, in
accordance with Koopman’s theorem. Analysis of the corre-
sponding eigenvector shows that it is mainly due to the lone
pair on oxygen. The eigenvector forms an orthonormal set. The
presence of a z-dipole indicates that the molecule is not flat or
planar. The barriers of rotation were calculated by twisting the
molecule about the bond or bonds in question and at each
point, the geometry with respect to all other geometric variables
was optimized. From the magnitude of energy involved in
rotation, it was possible for us to correlate with the thermal
decomposition patterns. The bonds, which require less energy,
are cleaved first and vice versa. The gradient normalization
energy for various bonds, bond angles and dihedral angles also
correlate well with the thermal decomposition pattern (Fig. S8,
ESI). Comparison of the magnitude of energy involved for a
variation in bond length and angles gives an indication that the
decomposition has to originate from the carbon atom of the
amino moiety which has a weak bond. An analogous trend was
also observed in the mass spectral fragmentation pattern.
The electron density order for the ligand was found to be
C(4)O > C(2)S > C–O–C > N(3)H > N(1)H > C–N–C. Based
ing an octahedral environment. Three electronic absorption
bands are seen (Table 3) in all the nickel() complexes. Their
3
energy positions characterise the ground state as A2 thereby
g
supporting the distorted octahedral geometry which is also
reinforced by their ν /ν values. The coordination environment
2
1
of the iodide and chloro complexes are 2N, 3O and 1S and 1N,
O and 2Cl, respectively. Apart from O and N, the other donor
3
atoms are S and Cl. The position of S is lower compared to Cl
in the spectrochemical series and hence the Dq value is lower
for the iodide complex. The ν /ν ratios for the various nickel()
2
1
complexes are in very close agreement with the values expected
for nickel() in an octahedral environment.
1
H NMR spectra
1
In the H NMR of zinc() chloro (Fig. S7, ESI) and bromide
complexes, the –N–(CH ) signal experiences a downfield shift
2
2
due to the deshielding effect and a consequent electron drift
from the nitrogen to the metal centre upon coordination,
thereby supporting the coordination of the ligand through
C–N–C of the amino moiety as suggested from IR data. IR
3
384
J. Chem. Soc., Dalton Trans., 2002, 3381–3391

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on the above order for a bidentate mode of chelation, the donor
atoms are C(4)O and C(2)S. For a tridentate mode of coordin-
ation one of the options for the donor atom is morpholine
oxygen and the involvement of this atom in complex formation
may not be feasible because the oxygen atom is exocyclic with
respect to the thiouracil plane and a conformational change
has to occur if oxygen has to be involved in coordination. The
next obvious choice is N(3)H and for other higher denticities
preferences will be N(1)H, C–N–C in that order. The above
prediction regarding the donor atoms holds good for the com-
10
plexes having a d configuration. The discrepancies in the pre-
7
8
dictions occur for d and d metal ions, they follow a random
order. This behaviour might be due to inter-electron repulsion,
however, the involvement of the pair C(4)O and C–N–C
(
morpholine nitrogen) is due to the additional stability achieved
by the formation of a six membered chelate even though C–N–
C is a poor electron donor among the predicted donor
atoms.
Calculations of thermodynamic quantities using determined
vibrational frequencies indicates some deviations when com-
pared with the experimentally observed vibrational frequencies.
Many of the errors in calculating vibrational frequencies were
systematic, similar deviations occur for a given type of
vibration in the molecule. However, for a few vibrations the
deviations may be both because of their large number and the
complexities which arise from the numerous combination and
overtone bands possible in the system. Since a systematic vari-
ation exists between the calculated and observed vibrational
frequencies, a rigorous quantum mechanical scaling method
needs to be applied for correcting the vibrational frequencies
and to calculate the various thermodynamic parameters.
Thermal decomposition and kinetic studies
The important features of the TG/DTA curves and the various
stages of decomposition are given in Table 4. The examination
of thermograms indicates a multistage decomposition involving
loss of coordinated water molecules, oxidation of the organic
moiety and formation of metal oxides. The derivative thermo-
grams of all the complexes are characterized by an intense
sharp peak for the last stage decomposition (see Fig. S9, ESI,
for a representative example). The thermal parameters for the
various decomposition stages are given in Table 5. In the major-
ity of the metal complexes, the last stage decomposition corre-
sponds to the formation of metal oxide. The activation energies
for the last stage of decomposition in the complexes are higher
than for all the other earlier stages of thermal decomposition.
The difference in the activation energy values for the different
stages may be due to the different types of decomposition
mechanism operating in different temperature zones. The mag-
nitude of the activation energy is primarily influenced by the
mechanistic pathway of the decomposition. In general, a chem-
ically controlled reaction mechanism is known to exhibit a
higher activation energy and a pore diffusion controlled reac-
tion mechanism, a lower value. Therefore, in the present study
the initial stages of the thermal decomposition of the com-
pounds may correspond to a mechanistic pathway involving the
diffusion-controlled mechanism thereby resulting in a lower
58
activation energy value for the decay. Once the water/organic
moiety is eliminated from the complex, many pores may be
formed over the surface and at higher temperature, the reaction
mechanism is chemically controlled resulting in higher acti-
vation energies for the formation of metal oxides. The negative
#
values of ∆S show that the complexes are more ordered in the
activated state than the reactants and the reactions are slower as
indicated by the TG curve at the corresponding temperature.
#
The positive ∆S of the complexes indicates that the activated
state is less ordered in structure compared with the reactants
and the reactions may be fast as indicated by rapid mass loss in
the thermograms.
J. Chem. Soc., Dalton Trans., 2002, 3381–3391
3385

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Biological studies
be more active. The steric constraints are less for
a
tetrahedral than for an octahedral complex and we observed
that the tetrahedral complexes are more active than the
octahedral ones.
Antibacterial activity. Among the compounds tested, the
cobalt() bromo complex was found to be the most active
against S. aureus and E. coli (Table 6). The activity of the
cobalt() complex may be due to its interaction with RNA.
Also cobalt() is an essential micronutrient during transcrip-
tion and transformation. The order of activity was found to be
cobalt() bromo complex > cobalt() chloro complex > zinc()
sulfato complex > zinc() chloro complex > nickel() iodide
complex > standard > MMTU. It was found that the metal ion
complexes were more biologically active than MMTU in all
cases. It was also observed that there existed some relationship
between the lability (as discerned from thermal studies) of
M–O, M–N, M–S and M–X (X = anionic counter part of the
metal ion) and the biological activities; viz. the activity
increased with increasing lability of the metal complexes.
Among the complexes the thermally labile ones were found to
Antifungal studies. MMTU was found to be less active com-
pared to its metal ion complexes with Co(NO ) ؒMMTU being
3
2
found to be the most active. The zones of inhibition exhibited
by these complexes are presented in Table 7. A possible mechan-
ism of antifungal activity may be speculated in the light of
59
chelation theory. Chelation considerably reduces the polarity
of the metal ion mainly because of partial sharing of its posi-
tive charge with donor groups and possible π-delocalisation
over the chelate ring. This increases the lipophilic character of
the neutral chelate which favours its permeation through the
lipid layers of fungus membranes. Furthermore, the mechanism
of action of the compounds may involve the formation of
3
386
J. Chem. Soc., Dalton Trans., 2002, 3381–3391

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Table 4 TG/DTG/DSC data, activation energy, entropy for cobalt(), nickel() and zinc() complexes of MMTU
Decomposition
temperature range/
K (ЊC)
Nature of the
DSC peak
DTG
peak/K
a
Compound
C H N SO
2
Stages
Initial
Final
Probable intermediate (calc. %) obs. %
Phase transformation
Stable up to
Stage I
Stage II
Stage III
Stable up to
Stage I
Stage II
Stable up to
Stage I
473 (200)
Endothermic
9
13
3
473 (200) 523 (250) Four membered cyclic compound (R)
673 (400) 713 (440) Oxidative degradation (G)
713 (440) 853 (580) Volatilisation (R)
508
688
788
Exothermic
Exothermic
Endothermic
[
C H N SO CoBr ]
353 (80)
353 (80)
723 (500) 848 (575) CoO (16.81) 17.00 (R)
353 (80) NiI ؒMMTU; (93.33) 93.00; (G)
Phase transformation
9
13
3
2
2
393 (120) Reductive elimination of Br; (82.07) 82.00; (S) Exothermic
533
763
Exothermic
Endothermic
Exothermic
Exothermic
Exothermic
Endothermic
Exothermic
Exothermic
[
C H N SO ؒNi(H O) ]I
9
13
3
2
2
2
2
2
468 (198) 573 (300) Oxidative degradation (G)
583 (310) 693 (420) (G)
723 (450) 773 (500) NiO (13.84) 14.00 (G)
493
628
753
Stage II
Stage III
Stable up to
Stage I
[
(C H N SO ) ZnCl ؒH O]
413 (140)
ZnCl ؒMMTU; (95.28) 95.00
9
13
3
2
2
2
2
2
498 (225) 773 (500) Oxidative degradation (S)
813 (540) 873 (600) ZnO (21.33) 21.00 (R)
588
843
Stage II
a
R = Rapid mass loss, G = gradual mass loss, S = slow mass loss.
Table 5 Activation energy, entropy, free energy and enthalpy for the thermal decomposition data of MMTU and its complexes by the
Coats–Redfern and Madhusudanan–Krishnan–Ninan equations
CR equation
MKN equation
#
Ϫ1
#
#
#
Ϫ1
#
#
E /kJ
Stage mol
∆S /J K
∆G /kJ ∆H /kJ
E /kJ
mol
∆S /J K
∆G /kJ
∆H /kJ
a
a
Ϫ1
Ϫ1
Ϫ1
Ϫ1
Ϫ1
Ϫ1
Ϫ1
Ϫ1
Compound
C H N SO
2
mol
mol
mol
r
mol
mol
mol
r
I
259.04
159.14
144.51
303.73
11.31
Ϫ45.10
96.29
139.92
166.95
84.34
158.71
97.75
132.91
155.19
129.36
177.00
250.59
147.70
131.41
1.11
148.96
11.52
52.91
574.06
11.55
0.9992
0.9996
0.9999
0.9983
0.9989
0.9988
0.9999
0.9989
0.9998
0.9997
259.10
159.43
147.37
303.30
11.43
Ϫ44.69
96.58
140.12
169.48
85.45
162.23
99.43
135.50
158.87
131.40
181.15
250.65
147.99
134.26
1.10
148.80
11.50
52.84
573.47
11.53
0.9993
0.9996
0.9999
0.9984
0.9989
0.9988
0.9999
0.9989
0.9999
0.9998
9
13
3
II
III
I
II
I
II
III
I
[
[
C H N SO CoBr ]
7.64 Ϫ211.79
7.64 Ϫ214.63
9
13
3
2
2
161.65
Ϫ12.78
19.72 Ϫ174.90
63.35 Ϫ127.40
161.49
Ϫ17.61
19.70 Ϫ178.35
63.29 Ϫ131.62
C H N SO ؒNi(H O) ]I
2
9
13
3
2
2
2
586.58
21.33 Ϫ200.34
348.77 187.13
556.27
585.99
21.31 Ϫ203.85
348.42 181.80
550.60
[
(C H N SO ) ZnCl ؒH O]
9 13 3 2 2 2 2
II
334.75
334.40
Table 6 Antibacterial activity (% inhibition) of MMTU and its complexes against Staphylococcus aureus and Escherichia coli
S. aureus
E. coli
Concentration of compound/µg mL
Ϫ1
Ϫ1
Concentration of compound/µg mL
2
5
50
75
100
25
50
75
100
Compound
C H N SO
2
% Inhibition
% Inhibition
52
78
70
56
62
65
58
59
83
81
63
70
73
63
65
90
88
72
78
81
68
71
98
95
81
86
88
74
50
62
60
52
56
60
48
58
71
69
62
66
70
57
70
85
80
73
78
80
68
80
97
94
85
89
90
78
9
13
3
[
ؒ
C H N SO CoBr ]
ؒ ؒ [N SO C H CoCl C H N SO ] ؒ ؒ ؒ
3 2 9 13 2 9 13 3 2
9 13 3 2 2
[
[
[
C H N SO ؒNi(H O) ]I
9 13 3 2 2 2 2
(C H N SO ) ZnCl ؒH O]
9
13
3
2
2
2
2
C H N SO ؒZn (SO ) ]
9
13
3
2
2
4 2
a
Standard
a
Streptomycin for S. aureus and bezyl pencillin for E. Coli.
hydrogen bonds through the uncoordinated heteroatoms O, S
and N with the active centres of the cell constituents resulting in
interference with the normal cell processes. The presence of
cell wall preventing penetration and hence toxic reaction within
the pores. As a corollary, the complex cannot reach the desired
site of action on the cell wall to interfere with normal cell
activity.
lipophilic and polar substituents such as C᎐O, C᎐S, and NH
᎐
᎐
are expected to enhance the fungal toxicity and therefore
these compounds have a greater chance of interaction with the
nucleotide bases, biologically essential metal ions present in
the biosystem, coordinatively unsaturated metals present in the
metal ion complexes or with some of the enzymatic functional
groups. The low activity of some of the complexes may be due
to a mismatching of the geometry and charge distribution
around the molecule with that around the pores of the fungal
Antitumour screening. The results obtained for MMTU and
its complexes of cobalt(), nickel() and zinc() on the growth
of P-338 leukemic and Dalton’s lymphoma cells are given in
Table 8. In general, an increase in growth inhibition was
observed as the concentration of test compound increased.
Here also metal ion complexes show more activity than the
ligand. The increase of activity on complexation is considerable
J. Chem. Soc., Dalton Trans., 2002, 3381–3391
3387

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Table 7 Antifungal activity of MMTU and its complexes
Zone of
inhibition/mm
Zone of
inhibition/mm
Ϫ1
Ϫ1
Compound
C H N SO
Conc./µg mL
0.2
Compound
Conc./µg mL
12
16
24
12
22
28
12
22
26
17
29
34
[C H N SO ؒNi(H O) ]I
2
0.2
0.4
0.6
0.2
0.4
0.6
0.2
0.4
0.6
0.2
0.4
0.6
14
19
26
14
18
26
13
24
30
15
27
32
9
13
3
2
9
13
3
2
2
2
0
0
.4
.6
ؒ
ؒ ؒ [N SO C H CoCl C H N SO ] ؒ ؒ ؒ
0.2
[C H N SO ؒNiCl (H O) ]
9 13 3 2 2 2 2
3
2
9
13
2
9
13
3
2
0
0
.4
.6
[
[
C H N SO CoI (H O) ]
0.2
[(C H N SO ) ZnCl ؒH O]
9 13 3 2 2 2 2
9
13
3
2
2
2
2
0
0
.4
.6
C H N SO Co(NO ) ]
0.2
[C H N SO ؒZn (SO ) ]
9 13 3 2 2 4 2
9
13
3
2
3
2
0
0
.4
.6
in the case of the zinc() complex whereas the other complexes
show moderate activity but higher than for free MMTU.
The percentage increase of life span (%ILS) of mice bearing
leukemic and lymphoma cells due to MMTU and its cobalt(),
nickel() and zinc() complexes is given in Table 8. The zinc()
sulfato complex was found to be most active among the com-
plexes. Hydrogen bonding may also be an important factor in
the antitumour mechanism. Adenine, guanine and cytosine
have amine groups adjacent to an unprotonated ring nitrogen
atom and are well positioned to form hydrogen bonds with
these compounds (drugs) and thereby inhibit normal cell
metabolism. Moreover, the presence of an endogenous recep-
tor site, viz. oxygen, is free in the terminal position of the
compound and hence the activity is increased.
All the complexes were isolated from non-aqueous media.
The cobalt(), and nickel() bromo and nitrato; cobalt() iodo
and nickel() iodide complexes were synthesized by mixing a
2-propanolic solution of the ligand with an ethanolic solution
of the metal salt in a 2 : 1 molar ratio at 70 ЊC. The complexes
precipitated in the form of insoluble products on cooling.
The precipitated complexes were washed first with hot (70 ЊC)
2-propanol and then ethanol and dried in a vacuum oven.
7
8
Ethanol was used as the medium for synthesizing the d and d
metal sulfato and chloro complexes. Ethanol and methanol
10
were used as the solvents for the ligand and the d metal salts,
respectively.
Elemental analyses (C, H, N and S) were performed using a
FISONS-CHNS/O elemental analyser. The purity of the ligand
was checked using a Shimadzu LC10AT HPLC instrument fit-
ted with an ODS column in the isocratic elution mode using
1 : 1 methanol–acetonitile as eluent. The mass spectral study of
MMTU was carried out using a Finningan MAT-8230 mass
spectrometer. After decomposing the complexes with HNO3,
the metal ion contents were determined using a GBC 902 AAS
with an acetylene/air flame. The anion contents were estimated
by standard literature procedures. The infrared spectral
measurements for MMTU and its metal ion complexes were
recorded as KBr discs and on a polyethylene support for the
conventional and the far IR regions respectively using Ratio
Recording Perkin-Elmer 1430 and Brucker IFS66V FT-IR
spectrometers. The UV-Vis spectra were recorded using a
JASCO 7800 UV-Vis double beam spectrophotometer in DMF
solvent and a Hitachi U-3400 UV-Vis–NIR spectrophotometer
which also covers the near IR region. Room temperature mag-
netic measurements of the complexes were carried out using a
Gouy magnetic balance. The Gouy tube was calibrated using
mercury() tetrathiocyanatocobaltate(), as recommended by
Figgis and Nyholm. Diamagnetic corrections for various atoms
and structural units were computed from Pascal’s constants.
The effective magnetic moments were calculated from the
corrected molar magnetic susceptibilities of the complexes
The order of activity is MMTU < NiI ؒMMTU < CoBr ؒ
2
2
MMTU < ZnCl ؒMMTUؒH O < ZnSO ؒMMTU. The free
2
2
4
MMTU does not effectively reach the cancer cells and con-
sequently less active. However, among the complexes screened
the enhanced activity of the zinc() chelate can be explained as
follows. A considerable amount of zinc is needed for rapidly
dividing tumour cells, this zinc uptake tendency by cancerous
cells leads to an easy uptake of the compound by cancerous
cells. After reaching the receptor site the labile Zn–N or Zn–O
or Zn–S bonds in the complexes undergo dissociation and pro-
duce free active antimetabolite, which is active in its own right.
Comparing activities of these compounds against the two cell
systems, leukemic cells were found to be relatively more resist-
ant than lymphoma cells as the percentage of growth inhibition
is greater in the latter case. The coordinatively unsaturated
tetrahedral complex is more active than the octahedral
60,61
complex.
From the present study it is seen that there is a
relationship between the lability of M–O and M–N linkages
and biological activity. The activity is directly proportional to
the lability of the metal ion–MMTU bond.
Experimental
1
using Curie’s relation. The H NMR of MMTU and its
Materials
10
d
metal ion complexes were recorded on JEOL GSX400NB
All the reagents used for synthesizing MMTU and its com-
plexes were of A.R. grade and the solvents used were
commercial products of the highest available purity and were
further purified by distillation.
400 MHz FTNMR spectrometer employing TMS as internal
reference and DMSO-d as solvent at ambient temperature. The
6
thermal studies (TG/DSC) were carried out on a Du Pont 910
DSC system with the 1090 programmer/data analyser in an
Ϫ1
atmosphere of air at a linear heating rate of 10 ЊC min from
Synthesis of MMTU and its metal ion complexes
ambient to 1000 ЊC using alumina as the standard reference
material.
An equimolar mixture of 2-thiouracil (12.8 g), paraformalde-
hyde (3.0 g) and morpholine (8.8 g) was suspended in 400 mL
of ethanol and refluxed for about 20 h. The clear homogeneous
solution obtained was concentrated to 200 mL by distillation.
On cooling the concentrate yielded a colourless crystalline solid
XRD data collection and determination of molecular topology
MMTU was dissolved in 1 : 1 acetic acid–water and a few drops
of hydrochloric acid was added. Colourless crystalline solid
separated out after a week, which was washed first with ethanol
and then with acetone and dried under vacuum. Crystals show-
ing good extinction under a polarizing microscope were used
(
crude product 21.2 g; 93.4% yield) which was filtered, washed
first with ethanol and then with ether. The compound was
crystallised from methanol.
3
388
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Table 9 Crystal data and structure refinement for MMTUؒHCl
Identification code
Empirical formula
MMTU
C H ClN O S
9
16
3
3
Formula weight
Temperature/K
Wavelength/Å
281.80
293(2)
0.71070
Crystal system, space group
Monoclinic, P21
9.1416(14)
13.412(2)
10.6570(12)
106.647(14)
1251.9(5)
4
a/Å
b/Å
c/Å
β/Њ
3
Volume/Å
Z
Ϫ1
Absorption coefficient/mm
F(000)
0.473
591.9
Crystal size/mm
0.32 × 0.27 × 0.24
Reflections collected/unique
Refinement method
Final R indices [I > 2σ(I )]
R indices (all data)
2305/2083 [R(int) = 0.0161]
Full-matrix least-squares on F
R1 = 0.052, wR2 = 0.162
R1 = 0.057, wR2 = 0.169
2
for data collection with an ENRAF-NONIUS CAD 4 diffract-
ometer employing Mo-Kα radiation. Absorption corrections
were applied to the data using Ψ scan method. The cell param-
eters and analysis over the data set indicate that the compound
belongs to the monoclinic system and the systematic absences
of k odd reflections in the 0 k 0 series revealed a 2 screw per-
1
pendicular to the b-axis. Accordingly the space group P2 was
1
assigned to the compound. All the non-hydrogen atoms were
located from the first electron density map itself, which led to an
R value of 20.2%. After eleven cycles of full matrix least-
squares adjustment of the coordinates and isotropic temper-
ature factors the R value converged to 11.5%. The difference
map indicated the location of all the hydrogens, except water
hydrogens. Anisotropic refinement was then carried out. All
the coordinates and isotropic temperature factors were included
in the final refinement. The refinement was completed at
R = 0.0574 for all 2305 reflections with F > 4σ(F ). The weight-
o
o
ing scheme employed for the final cycle of refinement was:
2
2
2
W = 1/[σ (F ) ϩ (0.1072P) ϩ 1.52P],
o
2
2
c
where P = [max{F , 0} ϩ 2F ]/3.
o
The intensity data sets were subjected to Lorentz polarization
62
and absorption corrections using the MolEN program. The
63
structure was solved by direct methods using the SHELXS 97
64
program and refined by full-matrix least-squares methods on
2
F using the SHELXL 97 program. The geometric calculations
65,66
were performed using the program PARST.
The hydrogen
bonding and stacking interactions were calculated using
67
PLATON. The molecular structures were drawn using
68
ORTEP. All calculations were carried out using a HP-Vectra
system. Data collection and refinement details are summarised
in Table 9.
CCDC reference number 172362.
See http://www.rsc.org/suppdata/dt/b2/b202127g/ for crystal-
lographic data in CIF or other electronic format.
Semi-empirical quantum mechanical calculations
Calculations were performed for MMTU and its zinc() com-
plexes in order to gain theoretical support for its observed
behaviour on complex formation and to evaluate parameters
such as electronic energy, core–core repulsion, gradient norm,
dipole, number of filled levels, ionization potential, results of
SCF calculations, charge on each atom, eigen values, atomic
charges, atom electron density and atomic electron population.
The input data from the crystal structure were optimized by
minimizing their total energy with respect to the corresponding
geometric variables. The optimized geometry is in good agree-
ment with the experimental data. The error in bond lengths is
less than 0.2 Å and in bond angles less than 2Њ. Such errors are
J. Chem. Soc., Dalton Trans., 2002, 3381–3391
3389

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6
9
not of great chemical significance and attempts to reproduce
the geometry much more accurately than this are not of any
real value. Calculations were carried out using the parametric
method-3 (PM-3) Hamiltonian and the program used for
Antifungal studies
Antifungal activities of these compounds were evaluated using
the nutrient agar diffusion method and phenol as a reference
standard. Wells were made (12 mm diameter) with a sterile cork
70
performing M.O. calculation was MOPAC version 6.00.
Ϫ1
borer. To these wells 0.2, 0.4, 0.6 mL (stock 1 mg mL ) of the
The geometry was optimized using the Broyden Fletcher–
Goldfarb–Shanno (BFGS) method followed by Bartel’s/
Melver–Komornicki method and a self-consistent field is
achieved. The eigen values, the diagonal elements of the
Coulson density matrix representing the valence electron
populations from each M.O, bonding contribution of each
M.O., the charge and electron density on the atoms have also
been calculated (see Tables S5–S9, ESI).
test compound was added and the plates were allowed to cool
for an hour to facilitate the diffusion. The plates were then
incubated at 37 ± 1 ЊC for 48 h. At the end of the incubation
period, the diameter of the zone of inhibition around the wells
was measured.
Antitumour screening
Calculations of thermodynamic quantities were carried out
using vibrational frequencies determined by quantum mechan-
ical methods using the keyword FORCE. Before proceeding
to thermal calculation a check was made by calculating the
gradient norm, if significant GNORM was reduced by BFGS.
The keyword PRECISE was used to eliminate quatric
contamination. M.O. calculations were also carried out for the
zinc() complexes to explore the various bonding and physical
parameters. The optimization terminated after 36 h in a
PC with a P-IV processor. On analyzing the output geometry,
it was observed that the optimization was not complete
and required further refinement by imposing symmetry
considerations which will be looked at as part of a further
investigation.
In vitro cytotoxic studies. The in vitro cytotoxicity assay was
carried out using P-388 lymphocytic leukemic and Dalton’s
lymphoma cells and the Trypan blue dye exclusion method to
measure viability. A stock solution (1 mg mL ) of compounds
(MMTU and metal ion complexes in DMSO) was prepared.
Ϫ1
Different concentrations of complexes i.e., 25, 50, 75 and 100
Ϫ1
µg mL were added in triplicate to test tubes and diluted to 0.9
mL. The same quantity of cell suspension (0.1 mL) was added
and incubated in a CO incubator at 37 ЊC for 3 h. A series of
2
control tubes were also maintained without adding the test
compound. After incubation, 0.3 mL of 0.4% Trypan blue
solution was added to each tube and mixed gently. Since the
Trypan blue will be absorbed only by dead cells, the number
of dead cells and the percentage were estimated using a
Hemocytometer.
Thermal decomposition and kinetic studies
Growth inhibition studies. Leukemic and lymphoma cells were
used to determine the percentage growth inhibition by test
compounds. Cells were collected after 7 days of transplantation
from the tumour bearing mice and suspended in a culture
medium to a cell density of 2 × 10 cells mL . Varying concen-
trations of each complex and MMTU were added followed by
Kinetic studies of the thermal decomposition of a few selected
representative complexes have been carried out. The order
parameters are determined using the iteration method and the
kinetic parameters are evaluated using the Coats–Redfern
4
Ϫ1
71
72
(
CR)
and Madhusudanan–Krishnan–Ninan (M–K–N)
equations. The correlation coefficient ‘r’ approaches unity for
plots of the left hand side of the CR and MKN equations vs.
incubation in a CO incubator at 37 ЊC for 1 h. Cells were
2
#
#
#
counted using a Hemocytometer by the Trypan blue exclusion
method. The experiments were carried out in duplicate and the
entire operation was carried out under aseptic conditions. The
percentage growth inhibition is calculated by taking the total
number of cells in the control tube (without test compound) as
1
/T . The values of E , ∆S , ∆G and ∆H obtained by both
a
methods are in very close agreement.
Antibacterial screening
The activity of the compounds synthesized were evaluated
1
00%.
73
using a serial tube dilution technique. These tests were per-
formed in triplicate and activities of the compounds were cross-
In vivo antitumour evaluation of compounds. Antitumour
74
checked with the disc diffusion technique. The compounds
were screened for their antibacterial activity using Staphylo-
coccus aureus and Escherichia coli. The former is a gram-
positive organism, while the latter is gram-negative. The
inhibition of growth of the gram-positive organism produced
by various concentrations of test compounds was compared
under identical conditions with the inhibition of growth of the
same organism by benzyl penicillin. Similarly, the inhibition of
gram-negative organism growth produced by the test com-
pounds was compared with those for different concentrations
of streptomycin. A solution of each test compound was pre-
pared in DMF and a series of dilutions were prepared. The
evaluation experiments were performed in vivo using groups of
CDF mice and Swiss albino mice of either sex aged 6–8 weeks
and 18–22 g body mass implanted with leukemic/lymphoma
cells. A group of eight animals was treated as control and
groups containing six animals were treated with test com-
pounds. The animals were fed with normal mouse chow
(
Lipton India Ltd.) and water ad libitum and were housed in
ventilated cages with husk as bedding material in air controlled
rooms (25 ЊC). Two million cells were injected into the periton-
eal cavity of Swiss albino mice and the Ascites tumour was
allowed to grow. The test compounds were administered on the
first, third and fifth day. The results are expressed as percentage
increase in life span (%ILS) calculated as
Ϫ1
concentrations tested were 25, 50, 75 and 100 µg mL of the
compound under investigation, a standard antibiotic (strepto-
mycin/benzyl pencillin), the respective metal salt dissolved in
DMF and the blank (DMF). A control tube containing no test
compound was also included. All these operations were care-
fully carried out under aseptic conditions. The assay tubes were
incubated at 37 ± 1 ЊC. The resultant turbidities were measured
in a Nephelo-turbidity meter. The relationship used for
calculating the activity is:
[
(T Ϫ C)/C] × 100
where T = median survival time of treated animals and
C = median survival time of control animals.
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