Bioactivity of novel transition metal complexes of N′-[(4-methoxy)thiobenzoyl]benzoic acid hydrazide

Article abstract of DOI:10.1016/j.ejmech.2007.04.017

Cu(II), Fe(III), and Mn(II) complexes of a novel ligand N′-[(4-methoxy)thiobenzoyl]benzoic acid hydrazide (H₂mtbh) have been synthesized and characterized by elemental analyses, IR, UV-vis, NMR, mass, EPR and Moessbauer spectroscopy. The results suggest a square planar structure for [Cu(Hmtbh)Cl] and [Cu(mtbh)] whereas an octahedral structure for [Mn(Hmtbh)₂] and [Fe(Hmtbh)(mtbh)]. Mn(II) and Fe(III) complexes were found to inhibit proliferation of HT29 cells. [Mn(Hmtbh)₂] and [Fe(Hmtbh)(mtbh)] inhibited proliferation of HT29 cells with half maximal inhibition (IC₅₀) of 8.15 ± 0.87 and 68.1 ± 4.8 μM, respectively, whereas H₂mtbh showed growth inhibition with IC₅₀ of 90.9 ± 7.8 μM and were able to inhibit NMT activity in vitro. Mn(II) and Fe(III) complexes inhibited NMT activity in a dose dependent manner with IC₅₀ values of 20 ± 2.2 and 60 ± 7.2 μM, respectively, whereas ligand (H₂mtbh) displayed IC₅₀ of 3.2 ± 0.5 mM.

Full text of DOI:10.1016/j.ejmech.2007.04.017

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 577e583
http://www.elsevier.com/locate/ejmech
Original article
Bioactivity of novel transition metal complexes of
N0-[(4-methoxy)thiobenzoyl]benzoic acid hydrazide
Anuraag Shrivastav a,b, Pratibha Tripathi c, Ajay K. Srivastava c,
a,b,
c,**
*
Nand K. Singh
, Rajendra K. Sharma
a Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan,
20 Campus Drive, Saskatoon, SK, Canada S7N 4H4
b Saskatchewan Cancer Agency, Saskatoon, SK, Canada S7N 4H4
c Department of Chemistry, Banaras Hindu University, Varanasi 221005, India
Received 10 October 2006; received in revised form 22 March 2007; accepted 19 April 2007
Available online 21 May 2007
Abstract
Cu(II), Fe(III), and Mn(II) complexes of a novel ligand N0-[(4-methoxy)thiobenzoyl]benzoic acid hydrazide (H2mtbh) have been synthesized
and characterized by elemental analyses, IR, UVevis, NMR, mass, EPR and Mossbauer spectroscopy. The results suggest a square planar struc-
¨
ture for [Cu(Hmtbh)Cl] and [Cu(mtbh)] whereas an octahedral structure for [Mn(Hmtbh)2] and [Fe(Hmtbh)(mtbh)]. Mn(II) and Fe(III) com-
plexes were found to inhibit proliferation of HT29 cells. [Mn(Hmtbh)2] and [Fe(Hmtbh)(mtbh)] inhibited proliferation of HT29 cells with
half maximal inhibition (IC50) of 8.15 ꢀ 0.87 and 68.1 ꢀ 4.8 mM, respectively, whereas H2mtbh showed growth inhibition with IC50 of
90.9 ꢀ 7.8 mM and were able to inhibit NMT activity in vitro. Mn(II) and Fe(III) complexes inhibited NMT activity in a dose dependent manner
with IC50 values of 20 ꢀ 2.2 and 60 ꢀ 7.2 mM, respectively, whereas ligand (H2mtbh) displayed IC50 of 3.2 ꢀ 0.5 mM.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Cytotoxic activity; Transition metal complexes; N-Myristoyltransferase
1. Introduction
structural and kinetic properties involved in biological activity
[5,6]. However, significant problems are still extant including
side effects, toxicity, cancer specificity and specially acquired
resistance. The toxicity associated with platinum drugs limits
its extensive use in the treatment of cancer. These facts pro-
voke the search for a new drug with a different mode of action
[7,8]. Earlier transition metals are less toxic and have shown
promising antitumor activity [9e13]. These challenges are
exemplified by the problems encountered in the use of general
anticancer drugs. Recently, N-myristoyltransferase has emerged
as a novel therapeutic target for cancer. N-myristoylation is re-
ferred to the addition of myristic acid to the N-terminal gly-
cine residue of the protein [14,15]. Many proteins involved in
a wide variety of a signal cascade, cellular transformations
and oncogenesis are myristoylated [14e16]. Examples in-
clude the catalytic subunit of cAMP-dependent protein
kinase [17], the b-subunit of calcineurin [18], the a-subunit
of several G-proteins [19], the cellular and transforming
The study of biologically important compounds has led to
the development of several chemotherapeutic drugs in past
few decades, the most notable example of which is cisplatin.
Since cisplatin emerged as the most important anticancer
drug [1,2] number of coordination compounds of other transi-
tion metal ions have been synthesized and characterized [3,4]
to study the effect of metal, ligand and other substituents on
* Corresponding author. Department of Pathology and Laboratory Medicine,
College of Medicine, University of Saskatchewan, 20 Campus Drive, Saska-
toon, SK, Canada S7N 4H4. Tel.: þ1 306 966 7733; fax: þ1 306 655 2635.
** Corresponding author. Department of Chemistry, Faculty of Science,
Banaras Hindu University, Varanasi 221 005, UP, India. Tel.:þ91 542
2307321; fax: þ91 542 2368174.
E-mail addresses: nksingh@bhu.ac.in (N.K. Singh), rsharma@scf.sk.ca
(R.K. Sharma).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.04.017

578
A. Shrivastav et al. / European Journal of Medicinal Chemistry 43 (2008) 577e583
forms of pp60src [20], several tyrosine kinases and proteins im-
portant for the assembly, maturation and infectivity of mature
virus particles, such as the murine leukemia virus Pr65gag
precursor [21] and poliovirus VPO polypeptide precursor [22].
Myristoylation of proteins is catalyzed by the eukaryotic
enzyme NMT. Earlier we reported for the first time in a rat
model that NMT is more active in colonic epithelial neoplasms
than in the corresponding normal appearing colonic tissue, and
that an increase in NMT activity appears at an early stage in
colonic carcinogenesis [23]. Increased NMT activity was
also observed in human colonic tumors and was predomi-
nantly cytosolic [24]. Furthermore, colorectal tumors showed
increased immunohistochemical staining for NMT compared
to normal mucosa [24]. In addition, gallbladder carcinoma
displayed strong cytoplasmic positivity for NMT with an in-
creased intensity in the invasive component. Normal gallblad-
der mucosa showed weak to negative cytoplasmic staining
[25].
A large number of diverse structures and proteins are capable
of inhibiting NMT [26e31]. Earlier, we have demonstrated that
early transition metal complexes of sulfur and nitrogen donor
ligands have dual mechanism of action in tumor regression
[12]. These metal complexes have protective effect on normal
cells and selectively induced apoptosisin tumor cells[12]. There-
fore, we synthesized novel sulfur and nitrogen donor ligand and
its metal complexes. Various ligands with a eC(S)NHNH(O)Ce
skeleton have been prepared in order to understand the phar-
macophore responsible for anti-proliferative and anti-NMT
activity. Present paper aims to study the anti-proliferative and
anti-NMT activity of Cu(II), Fe(III) and Mn(II) complexes of
a novel nitrogen and sulfur donor ligand N0-[(4-methoxy)-
thiobenzoyl]benzoic acid hydrazide (H2mtbh) (Fig. 1).
Fig. 1. Tentative structure of the ligand N0-[(4-methoxy)thiobenzoyl]benzoic
acid hydrazide (H2mtbh).
The magnetic moment of 2.1 and 1.89 BM for
[Cu(Hmtbh)Cl] and [Cu(mtbh)], respectively, are normal as ex-
pected for the presence of one unpaired electron. Both the com-
plexes display a broad UVevis band at 16 130 and 17 200 cmꢂ1
assigned to the envelope of 2B1g / 2A1g, 2B2g and 2Eg transi-
tions and suggest a square planar geometry for Cu(II) [12].
The magnetic moment of 6.0 and 6.01 BM for [Mn(Hmtbh)2]
and [Fe(Hmtbh)(mtbh)], respectively, suggest their high spin
nature. [Mn(Hmtbh)2] shows two UVevis bands at 18 020 and
24 690 cmꢂ1 assigned to the 6A1g / 4T1g, 4T2g transitions, re-
spectively, in an octahedral geometry around high spin Mn(II).
A magnetic moment of 6.01 BM and the presence of an UVe
vis band at 19 800 cmꢂ1 attributed to the 6A1g / 4T2g transition
is in accord with the octahedral geometry of [Fe(Hmtbh)(mtbh)]
[32].
1
The H NMR spectrum of H2mtbh exhibits two signals at
d 11.9 and 11.1 ppm for the amide and thioamide protons,
which disappear on D2O exchange. The signal at d 3.9 ppm
is attributed to eOCH3 protons of 4-methoxythiobenzoyl
part. The protons due to the two aromatic rings appear as mul-
tiplets between d 8.01 and 7.0 ppm. The 13C NMR spectrum of
H2mtbh shows 11 signals due to 15 carbon atoms, out of which
signals at d 195.43 and 164.81 ppm are due to pC]S and
pC]O carbons, respectively. The assignment of the ring car-
bons for 4-methoxythiobenzoyl moiety has been made by
comparing the spectrum of the H2mtbh with benzoic acid hy-
drazide [33].
2. Results and discussion
2.1. Chemistry
Elemental analyses reveal that H2mtbh and its complexes
are of good quality. From the analytical data it is clear that
H2mtbh reacts with metal salts either in 1:1 or 2:1 molar pro-
portion by losing either one or both the hydrazinic protons
from each ligand. The complexes are soluble in polar coordi-
nating solvents such as DMSO and are colored. The formation
of the complexes is given below:
Important IR spectral data of the ligands and complexes are
summarized in Table 1. The IR spectrum of H2mtbh shows
bands at 3164 and 3105 cmꢂ1 for the two eNH groups present
in the ligand. The bands occurring at 1657, 1433, 1343, 902
and 833 cmꢂ1 are assigned to n(C]O), thioamide
I
[b(NH) þ n(CN)], thioamide II [n(CN) þ b(NH)], n(NeN)
and n(C]S), respectively [34]. An exhaustive comparison of
the IR spectra of the ligand and complexes gave information
about the mode of bonding of the ligand in metal complexes.
The IR spectrum of [Cu(mtbh)] when compared with H2mtbh,
indicates that bands due to n(NH), n(C]O) and n(C]S) are
absent, but new bands appear at ca. 1602 and 763 cmꢂ1 due
to n(N]C) of NCO and n(CeS), respectively, suggesting
removal of both the hydrazinic protons via enolisation and thi-
oenolisation and bonding of the resulting enolic oxygen and
thiolato sulfur takes place with Cu(II). Furthermore, the ligand
bands due to thioamide I, thioamide II and n(NeN) undergo
a positive shift of 46, 57 and 60 cmꢂ1, respectively. The mag-
nitude of the positive shift supports that enolic oxygen,
CuCl2$2H2O þ H2mtbh / ½CuðHmtbhÞClꢁ þ HCl þ 2H2O
CuðOAcÞ2$H2O þ H2mtbh / ½CuðmtbhÞꢁ þ 2AcOH
þ H2O
MnðOAcÞ2$4H2O þ 2H2mtbh / ½MnðHmtbhÞ2ꢁ þ 2AcOH
þ 4H2O
½FeðacacÞ3ꢁ þ 2H2mtbh / ½FeðHmtbhÞðmtbhÞꢁ þ 3Hacac

A. Shrivastav et al. / European Journal of Medicinal Chemistry 43 (2008) 577e583
579
Table 1
Important IR spectral bands (cmꢂ1) and their assignments
Assignments
[H2mtbh]
[Cu(mtbh)]
[Cu(Hmtbh)Cl]
[Mn(Hmtbh)2]
[Fe(Hmtbh)(mtbh)]
n(NH)
3164, 3105
1657
1433
e
3106
1624 (ꢂ33)
1460 (þ27)
1394 (þ51)
952 (þ50)
789
3128
1620 (ꢂ37)
1470 (þ37)
1400 (þ57)
952 (þ50)
792
3123
1622, 1600
1473 (þ40)
1403 (þ40)
952 (þ50)
780
n(C]O)/n(NCO)
Thioamide(I), n[bNH þ n(CN)]
Thioamide(II), n[(CN) þ b(NH)
n(NeN)
1602
1479 (þ46)
1400
1343
902
962 (þ60)
763
n(C]S)/(CeS)
833
Figures in parentheses indicate shifts in band position.
thiolato sulfur and both hydrazinic nitrogens are involved in
coordination and H2mtbh behaves as binegatively charged
quadridentate species in [Cu(mtbh)] [35]. The IR spectra of
[Cu(Hmtbh)Cl] and [Mn(Hmtbh)2] show a band at 3106e
3128 cmꢂ1 due to n(NH), suggesting loss of either of the
two hydrazinic protons upon complexation. A negative shift
of 33e37 cmꢂ1 in n(C]O), the disappearance of n(C]S)
and appearance of a new band at 789e92 cmꢂ1 due to n(CeS),
indicate bonding through carbonyl oxygen and thiolato sul-
fur. Bands due to thioamide I, thioamide II and n(NeN) un-
dergo positive shift of 27e37, 51e57 and 50 cmꢂ1
suggesting the participation of above described sites in bond-
ing and also either of the two hydrazinic nitrogens. The diva-
lent metal ions Mn(II) and Cu(II) involve mononegative
tridentate ligand bonding through carbonyl oxygen, thiolato
sulfur and one of the two hydrazinic nitrogens. The spectrum
of [Fe(Hmtbh)(mtbh)] shows a band at 3123 cmꢂ1 due to
n(NH). Presence of two peaks at 1622 and 1600 cmꢂ1 for
n(C]O) and n(C]N) of NCO, respectively, indicates that
one of the two ligands is bonding in the keto form while the
other in enolic form. The disappearance of n(C]S) and ap-
pearance of a new band at 780 cmꢂ1 due n(CeS) indicates
bonding through thiolato sulfur. Further, a positive shift of
50 cmꢂ1 in n(NeN) suggests bonding through hydrazine nitro-
gen. Thus, out of the two ligands, one acts as a uninegative
tridentate, bonding through thiolato sulfur, hydrazine nitrogen
and carbonyl oxygen, while other acts as dinegetive tridentate,
the bonding sites being thiolato sulfur, hydrazine nitrogen and
enolic oxygen [12,33].
The Mossbauer spectrum of [Fe(Hmtbh)(mtbh)] has been
¨
recorded at liquid nitrogen temperature. The isomer shift,
d ¼ 0.047 mm/s and quadrupole splitting, DEq ¼ 0.374 mm/s
supports an octahedral geometry around high spin Fe(III)
and is undoubtely a single species formed by one dinegative
and an uninegative ligand [36]. On the basis of above physio-
chemical studies the tentative structures of the complexes are
given in Fig. 2.
2.2. Bio-evaluation of novel metal complexes
To investigate the antitumor activity of H2mtbh and its metal
complexes, their effect on the growth of HT29 cells in vitro was
measured by MTT assay. It was observed that treatment of
tumor cells with [Mn(Hmtbh)2] caused maximum growth
inhibition followed by [Fe(Hmtbh)(mtbh)] and the ligand.
[Mn(Hmtbh)2] and [Fe(Hmtbh)(mtbh)] showed anti-
proliferative effect with half maximal inhibition (IC50) of
8.15 ꢀ 0.87 and 68.1 ꢀ 4.8 mM, respectively, whereas H2mtbh
showed growth inhibition with IC50 of 90.9 ꢀ 7.8 mM. Cu(II)
complexes precipitated out while giving treatments hence its
bioactivity could not be determined. The reason for the ob-
served inhibition of tumor cell growth by the metal complexes
is unclear, however, several possibilities could be considered.
The cytostatic activity of the metal complexes could be a direct
result of the interaction of the metal complexes with DNA, thus
interfering with the process of DNA replication. Indeed, DNA
binding of metal complexes has been documented [37].
Furthermore, inhibition/activation of various enzymes di-
rectly/indirectly involved in DNA replication is not ruled out.
These results may also indicate a possible decline of the overall
metabolic activity of the tumor cells with a concomitant inhibi-
tion of the activity of various enzymes involved in respiration.
Interaction of metal complexes with protein components of
viable cells has been reported [38]. Binding of metal complexes
with protein may cause alterations in the structural and func-
tional organization of proteins. Studies from various laborato-
ries including ours have established N-myristoyltransferase
(NMT) as a putative therapeutic target for cancer. We reported
earlier increased activity and expression of NMT during the
early stages of colon cancer. Very recently we had demon-
strated that Cu(II) and Mn(III) complexes of N0-[(2-hydro-
xyphenyl)carbonothioyl]pyridine-2-carbohydrazide inhibited
NMT activity in vitro and specifically reduced the levels of
NMT2 [39]. It has been demonstrated that NMT plays a crucial
role in the apoptosis [40,41]. Furthermore it has been
The powder EPR spectrum of [Cu(Hmtbh)Cl] obtained in the
X-band region displays two peaks with g ¼ 2.175 and
k
gt ¼ 2.053, while that of [Cu(mtbh)] shows an axial spectrum
withg ¼ 2.152andgt ¼ 2.02. Theobtainedspectralparameters
k
are in agreement with square planar geometry around Cu(II).
[Fe(Hmtbh)(mtbh)] shows two peaks with g1 ¼ 2.076 and
g2 ¼ 2.04 as observed for a distorted octahedral Fe(III) complex.
The ligand H2mtbh is identified by FAB mass spectrum,
which shows many peaks due to various fragments. The mo-
lecular ion peak [MH]þ is observed at m/z ¼ 287. Other im-
portant peaks are those at m/z ¼ 253 (65% intensity) and
151 (90% intensity) corresponding to fragments formed
from the MHþ after the release of H2S and PhCONHN, re-
spectively. FAB mass spectral analyses were carried out for
the complexes [Mn(Hmtbh)2] and [Fe(Hmtbh)(mtbh)] which
show the presence of the corresponding molecular ion peak
and confirms the composition of the complexes.

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A. Shrivastav et al. / European Journal of Medicinal Chemistry 43 (2008) 577e583
Fig. 2. Tentative structures of the Cu(II) and Fe(III) complexes.
demonstrated that ablation of NMT1 and NMT2 results in the
apoptotic cell death [42]. Therefore, we also investigated the ef-
fect of these metal complexes on NMT activity. The anti-NMT
activity of [Cu(mtbh)] could not be evaluated due to its precip-
itation in the assay reaction mixture. However, Mn(II) and
Fe(III) complexes displayed anti-NMT activity in vitro.
Mn(II) and Fe(III) inhibited NMT activity in a dose dependent
manner with IC50 values of 20 ꢀ 2.2 and 60 ꢀ 7.2 mM, respec-
tively (Fig. 3a and b), whereas, ligand (H2mtbh) displayed IC50
of 3.2 ꢀ 0.5 mM. These results suggest that complexation with
metals increased the anti-proliferative and anti-NMTactivity of
the newly synthesized ligand. Increased anti-proliferative activ-
ity upon complex formation of the nitrogen and sulfur donor li-
gands with transition metal complexes have been reported
earlier [12,13]. Transition metal complexes of nitrogen and sul-
fur donor ligands are emerging as a new class of NMT inhibi-
tors. Further work is in progress to understand the interaction of
metal complexes with NMT and its mode of action.
benzaldehyde (24 ml) in EtOH (40 ml) at 65 ꢃC with freshly
prepared ammonium polysulfide solution (135 ml). The reac-
tion mixture was refluxed for 30 min and immediately cooled
in ice. A red solution was separated from the red resinous mass
and acidified with conc. HCl in the presence of Et2O under
ice-cooled condition. 4-Methoxydithio benzoic acid separated
as red oil in the Et2O layer. The remaining resinous mass was
refluxed for 8 h in EtOH (40 ml) and KOH (5 g), and then
cooled in ice, acidified with conc. HCl in the presence of
Et2O. Both the ethereal extracts were washed with water
(3 ꢄ 40 ml) and then shaken with 1 N NaOH (3 ꢄ 50 ml) in or-
der to extract the sodium salt of 4-methoxydithiobenzoate. To
this sodium salt was added a solution of ClCH2COOH (10 g)
already neutralized with sodium carbonate and maintained the
pH of the solution at 7.0. After standing the mixture overnight
at room temperature, it was acidified with conc. HCl to precip-
itate the ester, (4-methoxyphenyl)carbonothioylthioacetic
acid. The orange yellow colored ester was recrystallised
from MeOH in the presence of charcoal. Yield, 9 g (21%);
m.p. 127 ꢃC (lit. 122e25 ꢃC).
3. Experimental protocols
3.1. General materials and techniques
3.2. Preparation of the ligand
All chemicals of analytical grade were used after further
purification. Ammonium polysulfide [43] and tris(acetylaceto-
nato)iron(III) [44] were prepared by the reported procedures.
The sodium salt of (4-methoxyphenyl)carbonothioylthioacetic
acid [45] was prepared by treating a solution of 4-methoxy
N0-[(4-Methoxy)thiobenzoyl]benzoic acid hydrazide (H2mtbh)
was prepared by reacting (4-methoxyphenyl)carbonothioylthio-
acetic acid (2.42 g, 10 mmol) and benzoic acid hydrazide (BAH)
(1.38 g, 10 mmol), both were dissolved separately in aqueous
NaOH (1 N, 10 ml), mixed together and filtered. The mixture

A. Shrivastav et al. / European Journal of Medicinal Chemistry 43 (2008) 577e583
581
3.3. Preparation of complexes
A solution of H2mtbh (0.286 or 0.552 g, 1 or 2 mmol as ap-
propriate) in CHCl3 (15 cm3) was added to a solution of
CuCl2$2H2O or Cu(OAc)2$H2O or Mn(OAc)2$4H2O
(1 mmol) in 10 cm3 of MeOH. The mixture was magnetically
stirred for 2 h at room temperature. The resulting solids were
filtered off, washed thoroughly with EtOH and dried in vacuo.
Yield ranged from 45 to 65%. The complexes were character-
ized using the techniques mentioned earlier.
[Cu(Hmtbh)Cl]: dark green. Anal. Found (%): C, 47.2; H,
3.8; N, 7.8; Cl, 9.2; Cu, 16.4. Calculated (%) for C15H13ClCu-
N2O2S (384.1): C, 46.8; H, 3.4; N, 7.3; Cl, 9.2; Cu, 16.5. IR (n
cmꢂ1, KBr): 3106 m (NeH), 1624 s (C]O), 1468 s (thioa-
mide I), 1394 s (thioamide II), 952 s (NeN), 786 m (CeS).
UVevis (n cmꢂ1): 17 200, 31 250, 32 890.
[Cu(mtbh)]: black. Anal. Found (%): C, 52.0; H, 3.6; N,
8.4; Cu, 17.6. Calculated (%) for C15H12CuN2O2S (348): C,
51.8; H, 3.5; N, 8.0; Cu, 18.2. IR (n cmꢂ1, KBr): 1602 s
(NCO), 1479 s (thioamide I), 1400 s (thioamide II), 962 s
(NeN), 768 m (CeS). UVevis (n cmꢂ1): 16 130, 21 505,
26 315.
[Mn(Hmtbh)2]: yellow. Anal. Found (%): C, 57.8; H, 4.3;
N, 8.8; Mn, 9.3. Calculated (%) for C30H26MnN4O4S2
(625.4): C, 57.6; H, 4.2; N, 8.9; Mn, 8.7. IR (n cmꢂ1, KBr):
3128 m (NeH), 1620 s (C]O), 1460 s (thioamide I), 1397
s (thioamide II), 952 s (NeN), 792 m (CeS). UVevis (n
cmꢂ1): 18 020, 24 690, 33 330. MS (FAB): m/z ¼ 625 [M]þ.
[Fe(Hmtbh)(mtbh)]: orange brown. This complex was pre-
pared by refluxing a solution of H2mtbh with [Fe(acac)3] in
CHCl3 for 2 h. The resulting precipitate was filtered off,
washed successively with CHCl3 and MeOH and dried in va-
cuo. Anal. Found (%): C, 56.8; H, 4.2; N, 8.3; Fe, 8.4. Calcu-
lated (%) for C30H25FeN4O4S2 (625.2): C, 56.6; H, 3.9; N, 8.9;
Fe, 8.9. IR (n cmꢂ1, KBr): 3123 m (NeH), 1622 (C]O), 1600
s (NCO), 1462 s (thioamide I), 1393 s (thioamide II), 956 s
(NeN), 780 m (CeS). UVevis (n cmꢂ1): 19 800, 21 200,
32 400. MS (FAB): m/z ¼ 625 [M]þ.
Fig. 3. Dose dependent inhibition of hNMT by [Mn(Hmtbh)2] and
[Fe(Hmtbh)(mtbh)]. Purified recombinant human NMT (0.2 mg) was incubated
in the presence of various concentration of (a) [Mn(Hmtbh)2] or (b)
[Fe(Hmtbh)(mtbh)] and its activity was determined as described under Section 3.
Values are mean of three independent experiments carried out in duplica-
tes ꢀ standard deviation.
was kept at room temperature for 2 h and then acidified with
dil. AcOH (20%) whereupon a yellow precipitate formed. It
was suction filtered, washed with water, dried in vacuo and
recrystallised from MeOH. Yield, 1.5 g (52%); m. p.
138 ꢃC. Anal. Found (%): C, 62.9; H, 4.8; N, 9.3. Calculated
(%) for C15H14N2O2S (286): C, 62.9; H, 4.8; N, 9.7. IR (n
cmꢂ1, KBr): 3164 m and 3105 m (NeH), 1657 s (C]O),
1433 s (thioamide I), 1343 s (thioamide II), 902 s (NeN),
833 m (C]S). 1H NMR (DMSO-d6; d ppm): 3.9 (s, 3H,
OMe), 7.0, 7.5, 7.6, 8.0 (multiplets, 9H, phenyl), 11.1
(s, 1H, CSNeH), 11.9 (s, 1H, CONeH). 13C NMR
(DMSO-d6; d ppm): 55.53 (OMe), 132.04 (C-1), 127.57
(C-2, C-6), 128.63 (C-3, C-5), 131.09 (C-4), 132.64 (C00-1),
129.61 (C0-2, C0-6), 113.41 (C0-3, C0-5), 161.89 (C -4),
164.81 (C]O), 195.43 (C]S) (Fig. 2). UVevis (n cmꢂ1):
39 680, 35 210, 32 460. MS (FAB): m/z ¼ 287 [M þ 1]þ,
269 [M ꢂ H2O]þ, 253 [M ꢂ H2S]þ, 176 [M ꢂ (H2S þ Ph)]þ,
151 (Base Peak) [(4-CH3O)PhCS]þ, 105 [PhCO]þ. Based
on the above physico-chemical studies the tentative structure
of the ligand is depicted in Fig. 1.
3.4. Physical measurement
By following the standard procedures, the complexes were
analyzed for their metal content, after decomposition with
a mixture of conc. HNO3 and HCl, followed by conc.
H2SO4. Chloride was determined as AgCl [46]. Carbon, hy-
drogen and nitrogen were estimated on a Carlo Erba 1108
model microanalyser. Magnetic susceptibility measurements
were made at room temperature on a Cahn Faraday balance
using Hg[Co(NCS)4] as calibrant. Electronic spectra were re-
corded on a CARY 100 Varian EL01055314 UVevisible spec-
trophotometer as Nujol mulls [47]. IR spectra were recorded in
the 4000e400 cmꢂ1 region as KBr pallets on a Jasco FT/IR-
5300 spectrophotometer. 1H and 13C NMR spectra were
recorded in DMSO-d6 on a JEOL AL300 FT NMR spectrom-
eter using TMS as internal reference. ESR spectra were re-
corded on an X-band spectrometer model EPR-112 using
DPPH as a <g> marker. FAB mass spectra were obtained

582
A. Shrivastav et al. / European Journal of Medicinal Chemistry 43 (2008) 577e583
on a JEOL SX 1021 DA-6000 mass spectrometer using nitro-
benzyl alcohol (NBA) as a matrix. The Mossbauer spectrum
¨
absence of ligand or metal complexes. [Mn(Hmtbh)2] and
[Fe(Hmtbh)(mtbh)] were assayed for their inhibitory activity
against standard human NMT (hNMT). Purified recombinant
hNMT (0.2 mg/assay) was assayed [48] in the presence of
pp60src derived peptide substrate and metal complexes. A con-
trol experiment was performed in the absence of ligand or metal
complex and the hNMT activity was considered as 100%. All
other conditions were as described above.
was recorded at 77 K using a custom made cryostate (Proto-
tech Ltd.) on a Cryophysics MS-1 microprocessor-controlled
spectrometer. The source was 25 mCi, 925 MBq 57Co that
was mounted on a MVT-1000 transducer. The laser velocity
was calibrated with an MVC-450 driving angle waveform gen-
erated by an MD4-1200 driver unit and a DFG-1000 digital
function generator. Data collection utilized a PC-driven
CMCA-550 MCA/PHA card from Wissel GmbH, Germany.
The collected data were then fitted to Lorentzian line shapes
using Normos V 9.0 program.
3.8. MTT assay
The MTT assay was employed to determine the cytotoxic-
ity of the ligand and the metal complexes. The MTT assay was
performed using a standard procedure [49]. HT29 cells were
grown in a 96 well plate and treated with various concentra-
tions of H2mtbh, [Mn(Hmtbh)2] or [Fe(Hmtbh)(mtbh)]. IC50
values were calculated by plotting concentration against per-
centage cell proliferation.
3.5. Cell culture
The HT29 colon cancer cell line was obtained from ATCC.
Cells were grown in DMEM supplemented with 10% fetal calf
serum, 1% antibioticeantimycotic solution (GIBCO) contain-
ing penicillin G sodium, streptomycin sulphate and amphoter-
icin B in humidified air and 5% CO2 at 37 ꢃC. Cells were
harvested after 48 h upon treatment with various concentra-
tions of H2mtbh, [Mn(Hmtbh)2] or [Fe(Hmtbh)(mtbh)]. Cells
were lysed in RIPA buffer (50 mM TriseHCl pH 7.5,
150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate,
0.1% SDS) containing 10 mM DTT, 1 mM PMSF and 1% pro-
tease inhibitor cocktail (Sigma).
Acknowledgement
This work was supported by the Council of Scientific and
Industrial Research, India, Grant no. 01/(1835)/03/EMR-II to
N.K. Singh and Canadian Institutes of Health Research
(CIHR), Canada, Grant no. MOP-36484 to R.K. Sharma. Anu-
raag Shrivastav is a recipient of Postdoctoral Fellowship from
CIHR and Saskatchewan Health Research Foundation. Au-
thors thank the Head, RSIC, CDRI, Lucknow, India, for
CHN and FAB mass spectra; RSIC, IIT Chennai, India, for
ESR and Dr. M.J.K. Thomas, Department of Chemical and
Pharmaceutical Sciences, University of Greenwich, London,
3.6. N-Myristoyltransferase assay
NMT activity was measured as previously described [48].
Briefly, [3H]myristoyl-CoA was synthesized enzymatically ac-
cording to previously described procedure [48]. The reaction
mixture contained 40 mM TriseHCl, pH 7.4, 0.1 mM
EGTA, 10 mM MgCl2, 5 mM ATP, 1 mM LiCoA, 1 mM
[3H]myristic acid (7.5 mCi) and 0.3 unit/ml Pseudomonas
acyl-CoA synthetase in a total volume of 200 ml. The reaction
was carried out for 30 min at 30 ꢃC. The conversion to
[3H]myristoyl-CoA was generally greater than 95%. The assay
mixture contained 40 mM TriseHCl, pH 7.4, 0.5 mM EGTA,
0.45 mM 2-mercaptoethanol, 1% Triton X-100, pp60src de-
rived peptide (500 mM) and human recombinant NMT
(0.2 mg) in a total volume of 25 ml. The transferase reaction
was initiated by the addition of freshly generated [3H]myris-
toyl-CoA and was incubated at 30 ꢃC for 30 min. The reaction
was terminated by spotting 15 ml aliquots of incubation mix-
ture onto P81 phosphocellulose paper discs and drying under
stream of warm air. The P81 phosphocellulose paper discs
were washed in two changes of 40 mM TriseHCl, pH 7.3
for 60 min. The radioactivity was quantified in 7.5 ml of Beck-
man Ready Safe Liquid Scintillation mixture in a Beckman
Liquid Scintillation Counter. One unit of NMT activity was
expressed as 1 pmol of myristoyl peptide formed per minute.
for recording the Mossbauer spectrum.
¨
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