Synthesis and cytotoxic evaluation of uracil C-Mannich bases

Article abstract of DOI:10.3906/kim-1201-6

This study covers the design, synthesis, characterization, and cytotoxic activity of a series of uracil C-Mannich bases. Among them, one hybrid compound (1), a molecular combination of the potential antimetabolite substituted uracil and nitrogen mustard, having potential alkylating capability, was prepared as a Mannich base. The other compound was synthesized with the replacement of chlorines in the ethyl chains with hydroxyl groups for testing for anticancer activity. Some of Mannich bases having several amino groups with different pKa values were also synthesized and investigated in terms of cytotoxic activity. Their chemical structures were confirmed by means of their UV, IR, ¹H-NMR, ¹³C-NMR, and MS data. Compounds 6 with diethylamine and 8 with piperazine are reported for the first time in the literature and compounds 1, 4, and 5 containing nitrogen mustard, pyrolidine, and diethanolamine, respectively, as amine function are reported for first time with detailed spectral data herein. Morpholine, piperidine, and dimethylamine were used in Mannich reactions for the synthesis of compounds 2, 3 and 7. We assessed their biological activities using MTT assays on 3 human cell lines: HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma), and A431 (skin epidermoid carcinoma). While compounds 2-8 have the potential to deaminate, forming ortho-quinone methides, which would be capable of alkylating cellular thiols, compound 1 has the potential to give aziridinium ion for nucleophilic alkylation. Our results are discussed in terms of the significance of these compounds in pharmaceutical use. TUeBITAK.

Full text of DOI:10.3906/kim-1201-6

Turk J Chem
36 (2012) , 583 – 592.
¨
˙
c
ꢀ TUBITAK
doi:10.3906/kim-1201-6
Synthesis and cytotoxic evaluation of uracil C-Mannich
bases
1
2
1
1,∗
˙
´
¨
¨
˙
Hu¨seyin ISTANBULLU , Istva´n ZUPKO , Vildan ALPTUZUN , Erc¸in ERCIYAS
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ege University,
˙
Izmir, 3510, TURKEY
e-mail: ercin.erciyas@ege.edu.tr
2Department of Pharmacodynamics and Biopharmacy, University of Szeged,
Szeged, H-6270, HUNGARY
Received: 03.01.2011
This study covers the design, synthesis, characterization, and cytotoxic activity of a series of uracil
C-Mannich bases. Among them, one hybrid compound (1), a molecular combination of the potential
antimetabolite substituted uracil and nitrogen mustard, having potential alkylating capability, was prepared
as a Mannich base. The other compound was synthesized with the replacement of chlorines in the ethyl chains
with hydroxyl groups for testing for anticancer activity. Some of Mannich bases having several amino groups
with different pKa values were also synthesized and investigated in terms of cytotoxic activity. Their chemical
structures were confirmed by means of their UV, IR, 1 H-NMR, 13 C-NMR, and MS data. Compounds 6
with diethylamine and 8 with piperazine are reported for the first time in the literature and compounds 1,
4, and 5 containing nitrogen mustard, pyrolidine, and diethanolamine, respectively, as amine function are
reported for first time with detailed spectral data herein. Morpholine, piperidine, and dimethylamine were
used in Mannich reactions for the synthesis of compounds 2, 3 and 7. We assessed their biological activities
using MTT assays on 3 human cell lines: HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma),
and A431 (skin epidermoid carcinoma). While compounds 2-8 have the potential to deaminate, forming
ortho-quinone methides, which would be capable of alkylating cellular thiols, compound 1 has the potential
to give aziridinium ion for nucleophilic alkylation. Our results are discussed in terms of the significance of
these compounds in pharmaceutical use.
Key Words: Uracil, Mannich base, antiproliferative effect, anti-cancer compounds
∗Corresponding author
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Synthesis and cytotoxic evaluation of uracil C-Mannich bases, H. ISTANBULLU, et al.
Introduction
Mannich bases are pharmacologically important molecules as they have been reported to show a wide range of
bioactivities such as antineoplastic,1−5 diuretic,6 antipsychotic,7 anticonvulsant,8−10 central acting muscle
relaxant,11 antibacterial,12−14 antimalarial,15,16 and antiviral17 activities. A number of modifications of
Mannich bases gave rise to improved aqueous solubility and hydrolytic stability.18 Among the modifications,
alkylation is of special importance as it plays an important role in the biogenesis of nucleic acids. Therefore,
incorporation of alkyl groups received increasing attention for chemotherapeutic control of neoplastic diseases.
In addition, uracil molecule occupies an important position inasmuch as it is a primary component of the nucleic
acid moiety. Hence to combine uracil and alkylating compounds in a unique molecule seemed to us a logical
approach for designing some new cytotoxic compounds. It has been reported that nitrogen mustard, known as an
alkylating agent, varies in its alkylating properties depending on whether it is directly attached to an aromatic or
pseudoaromatic ring system or to an aliphatic system.19,20 The results concerning the comparative alkylating
activities of the studied Mannich bases clearly indicated that all the nitrogen mustards reacted similarly to
mechlorethamine but differently from uracil mustard showing enamine conjugation.21 In the case of the uracil
mustard, the reactivity of the alkylating functional group was similar to the findings reported by Bardos and
co-workers for aromatic nitrogen mustards.22 They proposed that in the aromatic nitrogen mustard series the
alkylating activity is strikingly dependent on the basicity of the nitrogen atom. It seems to us that the methylene
bridge between uracil and mustard moieties will contribute to aziridinium ion formation. It has been reported
that uracil-5-mustard having methylene bridge (C-Mannich base of uracil) between the alkylating nitrogen
mustard group and uracil moiety (1) inhibits the mitotic division in HeLa cells by at least 2 mechanisms: (i) its
incorporation into DNA, and (ii) its capacity to injure mitotic processes.23,24 As a tymine derivative 5-((bis(2-
chloroethyl)amino)methyl)uracil hydrochloride exhibited a marked cytostatic activity.25 These data prompted
researchers to compare the alkylating activity of compounds with or without methylene bridge between the
alkylating functional group and uracil moiety.
Over the years there has been continuing interest in analogs of thymine that might have cytotoxic
activity. Thus, compounds such as 5-fluorouracil,26 5-trifluoromethyluracil,27 and 5-mercaptomethyluracil28
are effective as inhibitors of cell growth. In view of the biological significance of these 5-substituted uracils we
became interested in extending the derivatization at the 5 position of uracil by the use of the Mannich reaction.
There exists a close parallel between in vivo thymidine 5-phosphate synthesis and the Mannich reaction. The
Mannich reaction has been applied to 6-methyluracil29 in addition to uracil.30,31 Accordingly, we synthesized a
series of 5-substituted aminomethyluracil derivatives (Scheme 1) in order to ascertain the scope of the reaction
as well as any differences the side chain might have on biological activity.
The aim of this study was to synthesize and biologically evaluate a number of Mannich bases derived
from uracil ring (Scheme 1). Firstly, bis(2-chloroethyl)amine was chosen as an alkylating agent (1) since
nitrogen mustard derivatives like mechloretamine are very well known compounds having alkylating potency
upon aziridinium ion.32 To preserve the aliphatic character of the nitrogen mustard group, we did not intend to
attach the mustard nitrogen directly to the uracil ring. Several aminomethylation positions of the pyrimidine
ring can be expected, since there are several possible alkylation sites that exhibit quite different acidities and
basicities. C-aminomethylation is favored by acidic conditions, whereas N-Mannich bases are produced when
free amine and formaldehyde in anhydrous solvents are employed as aminomethylating agents.31 Thus the
584

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Synthesis and cytotoxic evaluation of uracil C-Mannich bases, H. ISTANBULLU, et al.
O
O
H
R2
R1
R2
O
HN
HN
N
HN
R1
H
H
O
N
H
O
N
H
Compound
R Groups
Compound
R Groups
I
R1=R2= -CH2CH2Cl
V
R1=R2= -CH2CH2OH
R1=R2= -CH2CH3
R1=R2= -CH3
II
R1=R2= -C2H4OC2H4 -
VI
III
IV
R1=R2= -C5H10
R1=R2= -C4H8 -
-
VII
VIII
R1=R2= -C2H4NHC2H4 -
Scheme 1. The synthesis pathway of the compounds by Mannich reaction.
corresponding heterocyclic C-Mannich base can be obtained by arranging reaction time, reaction solvent, and
temperature. Our investigations were not extended to N-1, N-3 substituted Mannich base considering these
derivatives cannot form hydrogen bonds with the uracil-specific carriers or enzymes. Secondly, we prepared
different Mannich bases not only including mustard function giving aziridinium ion as an alkylating agent but
also using some other secondary amines that were not able to give aziridinium ions. In this case the action
mechanism of the compounds should be different from that of nitrogen mustard derivatives. The deamination
reaction might be observed in the case of practically all C-Mannich bases; it possesses both theoretical and
practical interest. Usually Mannich base derived aliphatic or aryl aliphatic ketones give rise to activated α,β-
unsaturated ketones by deamination in the first step, which is very sensitive for Michael addition to alkylate
certain cellular constituents like thiol function.33 In addition, deamination of Mannich bases on the phenyl ring
to the corresponding ortho-quinone methides is conceivable.1 These enones and ortho-quinone methides have
high affinities for thiols, which are absent in nucleic acids. Hence in contrast to currently available alkylating
agents used in cancer chemotherapy, interactions with nucleic acids may be absent and side effects such as
mutagenicity and carcinogenicity may be prevented. Another reason for undertaking this study was selective
toxicity, because certain cancer cells have a lower pH than the corresponding normal cells and hence molecules
that form alkylating species under acidic conditions may have selective toxicity towards neoplastic tissues.34
The compounds described in this report were designed as prodrugs of alkylators except compounds 1 and 2.
Our proposed alkylation mechanism of the compounds is illustrated in Scheme 2. According to the mechanism,
compounds might generate carbocation and quinone methide intermediates having alkylator properties. It can
be expected that pKa values of amine functions might have an effect on the deamination process of Mannich
bases. Finally, another objective of the present investigation was to evaluate their cytotoxic efficacy.
585

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Synthesis and cytotoxic evaluation of uracil C-Mannich bases, H. ISTANBULLU, et al.
OH
CH2Nu
N
H
HO
N
O
NuH
O
O
R1
R1
CH2
N
CH2
N
H
CH2
R1
R2
HN
HN
HN
R2
R2
HN
H
+
O
O
N
H
N
H
O
N
H
OH
OH
OH
N
CH2
CH2
CH2
H
N
N
N
HO
O
N
N
HO
NuH
SHCH 2CH2OH
OH
OH
H2
CH2Nu
1,6-Additio n
SCH2CH2OH
C
N
N
HO
N
O
N
Scheme 2. The putative pathways for the formation of alkylating species carbocation and quinone-methide of uracil-
Mannich bases in acidic medium.
Experimental
Chemistry
Melting points were determined with a capillary melting point apparatus (Buchi 510, BUCHI, Switzerland) and
are uncorrected. UV spectra were recorded on a spectrophotometer in methanol solution (UV-160, Shimadzu,
Japan). The IR spectra were obtained using a Jasco FT/IR-430 spectrophotometer (JASCO, Japan) as
potassium bromide pellets. The NMR spectra (400 MHz for 1 H and 100 MHz for 13 C) were recorded in
appropriate solvents without TMS (AS 400 Mercury Plus NMR Varian, USA). Chemical shifts were measured
as parts per million (δ) and the J values given in Hz. A Memmert water bath with shaker attachment
586

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Synthesis and cytotoxic evaluation of uracil C-Mannich bases, H. ISTANBULLU, et al.
was used in the stability study. The MS was determined on an LC/MS Waters 2695 Alliance Micromass
ZQ. TLC was developed routinely using Merck silica-gel plates (Kieselgel 60F254) and the solvent systems of
chloroform/methanol/ammonium hydroxide (4:2:0.1) was used.
General procedure for synthesis of the compounds
Compounds were synthesized as described before21 . A suspension of uracil (0.01 mol), amine (0.02 mol), and
formalin (0.022 mol) in THF was stirred at room temperature for 24 h. The reaction mixture was heated under
reflux at 70 ◦ C for 72 h. Removal of the solvent in vacuo produced the crude residue, which was recrystallized
from methanol/ether or ethyl acetate/ether to yield the desired Mannich bases.
5-((bis(2-chloroethyl)amino)methyl)uracil hydrochloride (1)
Yield 35%; mp 222 ◦ C (lit. 224-226 ◦ C);35 UV λmax (nm) (log ε) 211 (3.921), 263 (4.066); IR νmax (cm−1
)
2978, 1714, 1675, 638, 1464, 918; 1 H-NMR (CDCl3) δ 7.79 (s, 1H, CCH NH), 4.25 (s, 2H, CCH2 N), 4.04 (t,
4H, J =5.9 Hz, CH2 CH2 Cl), 3.66 (t, 4H, J =5.9 Hz, CH2 CH2 Cl); 13 C-NMR (CDCl3) δ 166.2 (NHC OC),
152.6 (NHC ONH), 146.8 (CC HNH), 101.6 (COC CH), 55.0 (CH2C H2 Cl), 52.0 (CC H2 N), 37.7 (C H2 CH2 Cl);
ESI-MS 266.10 [M+1]+ .
5-(morpholinomethyl)uracil (2)
Yield 52%; mp 208 ◦ C (lit. 217 ◦ C); 30 UV λmax (nm) (log ε) 207 (4.295), 263 (3.972); IR νmax (cm−1) 3215,
3039, 2813, 1722, 1670, 1450, 1111, 867; 1 H-NMR (CD3 OD) δ 7.39 (s, 1H, CCH NH), 3.67 (t, 4H, J =4.8
Hz, CH2 OCH2), 3.23 (s, 2H, CCH2 N), 2.47 (t, 4H, J =4.8 Hz, CH2 NCH2); 13 C-NMR (CD3 OD) δ 165.5
(NHC OC), 152.2 (NHC ONH), 141.4 (CC HNH), 108.4 (COC CH), 66.6 (C H2 OC H2), 53.3 (CC H2 N), 53.2
(C H2 NC H2); ESI-MS 212.10 [M+1]+ .
5-(piperidinomethyl)uracil (3)
Yield 61%; mp 149 ◦ C (lit. >300 ◦ C);30 UV λmax (nm) (log ε) 208 (4.013), 263 (3.752); IR νmax (cm−1
)
3282, 2938, 1732, 1706, 1669, 1447, 1067; 1 H-NMR (DMSO-d6) δ 7.39 (s, 1H, CCH NH), 3.24 (s, 2H, CCH2 N),
2,46 (t, 4H, J =4.8 Hz, CH2 NCH2), 1.62-1.44 (m, 6H, CH2 CH2 CH2 CH2 CH2); 13 C-NMR (DMSO-d6) δ
165.7 (NHC OC), 152.5 (NHC ONH), 142.3 (CC HNH), 108.2 (COC CH), 53.8 (C H2 NC H2), 53.2 (CC H2 N),
25.6 (CH2C H2 CH2) 25.3 (C H2 CH2C H2); ESI-MS 210.00 [M+1]+ .
5-(pyrrolidinomethyl)uracil (4)
Yield 32%; mp 210 ◦ C (lit. 201 ◦ C);36 UV λmax (nm) (log ε) 215.6 (2.477), 263.8 (1.930); IR νmax (cm−1
)
3037, 2962, 1731, 1668, 1450, 1207, 856; 1 H-NMR (CD3 OD) δ 7.41 (s, 1H, CCH NH), 3.40 (s, 2H, CCH2 N),
2.61 (p, 4H, J1 =3.2, J2 =7.2 Hz, CH2 NCH2), 1.81 (p, 4H, J1 =3.2, J2 =7.2 Hz, CH2 CH2 CH2 CH2); 13 C-
NMR (CD3 OD) δ 165.6 (NHC OC), 152.8 (NHC ONH), 142.5 (CC HNH), 109.1 (COC CH), 53.4 (C H2 NC H2),
50.1 (CC H2 N), 23.0 (CH2C H2C H2 CH2); ESI-MS 196.20 [M+1]+ .
587

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Synthesis and cytotoxic evaluation of uracil C-Mannich bases, H. ISTANBULLU, et al.
5-((bis(2-hydroxyethyl)amino)methyl)uracil (5)
Yield 20%; mp >300 ◦ C (lit. >300 ◦ C);35 UV λmax (nm) (log ε) 214.8 (1.979), 266 (0.564); IR νmax (cm−1
)
3415, 2866, 1716, 1668, 1448, 1240, 1079, 1027; 1 H-NMR (CD3 OD) δ 7.48 (s, 1H, CCH NH), 3.61 (t, 4H, J =5.9
Hz, CH2 CH2 OH), 3.39 (s, 2H, CCH2 N), 2.64 (t, 4H, J =5.9 Hz, NCH2 CH2); 13 C-NMR (CD3 OD) δ 166.0
(NHC OC), 152.4 (NHC ONH), 140.9 (CC HNH), 110.4 (COC CH), 59.5 (CH2C H2 OH), 55.9 (NC H2 CH2),
50.1 (CC H2 N); ESI-MS 230.30 [M+1]+ .
5-((diethylamino)methyl)uracil (6)
Yield 48%; mp 245 ◦ C; UV λmax (nm) (log ε) 218.0 (1.818), 265.2 (1.107); IR νmax (cm−1) 3430, 2931,
1716, 1670, 1446, 1151; 1 H-NMR (CD3 OD) δ 7.40 (s, 1H, CCH NH), 3.36 (s, 2H, CCH2 N), 2.58 (q, 4H,
J =3.2 Hz, NCH2 CH3), 1.08 (t, 6H, J =3.2 Hz, CH2 CH3); 13 C-NMR (CD3 OD) δ 165.7 (NHC OC), 152.8
(NHC ONH), 142.3 (CC HNH), 109.0 (COC CH), 54.8 (CC H2 N), 46.4 (CH2C H3), 10.3 (NC H2 CH3); ESI-MS
198.20 [M+1]+ .
5-((dimethylamino)methyl)uracil (7)
Yield 72%; mp 204 ◦ C (lit. 203-206 ◦ C);37 UV λmax (nm) (log ε) 262 (3.964); IR νmax (cm−1) 3113, 2971-
2947, 1730, 1702, 1674, 1450, 818, 781; 1 H-NMR (CD3 OD) δ 7.22 (s, 1H, CCH NH), 3.01 (s, 2H, CCH2 N),
2.08 (s, 6H, NCH3); 13 C-NMR (CD3 OD) δ 164.9 (NHC OC), 151.8 (NHC ONH), 140.8 (CC HNH), 109.2
(COC CH), 54.3 (CC H2 N), 45.2 (NC H3); ESI-MS 170.20 [M+1]+ .
5-(piperazinomethyl)uracil (8)
Yield 18%; mp 222 ◦ C; UV λmax (nm) (log ε) 207 (3.005), 263 (2.547); IR νmax (cm−1) 3415, 2935,
1705, 1660, 1455, 831; 1 H-NMR (DMSO-d6) δ 7.21 (s, 1H, CCH NH), 4.02 (s, 2H, CCH2 N), 3.67 (t, 2H,
J =4.8 Hz, CH2 CH2 NH), 3.55 (t, 2H, J =4.8 Hz, CH2 CH2 NH), 2.93 (bs, 1H, NH), 2.96 (t, 2H, J =4.8
Hz, NHCH2 CH2), 2.76 (t, 2H, J =4.8 Hz, NHCH2 CH2); 13 C-NMR (DMSO-d6) δ 165.6 (NHC OC), 152.4
(NHC ONH), 141.8 (CC HNH), 108.5 (COC CH), 54.3 (CC H2 N), 52.4 (C H2 NC H2), 51.3 (C H2 NHC H2);
ESI-MS 211.20 [M+1]+ .
Biological activity assays
Antiproliferative effects of the test compounds were measured in vitro on 3 human cell lines: HeLa (cervix
adenocarcinoma), MCF7 (breast adenocarcinoma), and A431 (skin epidermoid carcinoma), by using the MTT
assay.38 Briefly, cancer cells (5000/well) were seeded onto a 96-well microplate and attached to the bottom of
the well overnight. On the second day, 200 μL of new medium containing the test substances was added. After
incubation for 72 h, the living cells were assayed by the addition of 20 μL of 5 mg/mL MTT solution. MTT
was converted by intact mitochondrial reductase and precipitated as blue crystals during a 4 h contact period.
The medium was then removed, and the precipitated crystals were dissolved in 100 μL of DMSO during a 60
min period of shaking. Finally, the reduced MTT was assayed at 545 nm, using a microplate reader; wells with
588

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Synthesis and cytotoxic evaluation of uracil C-Mannich bases, H. ISTANBULLU, et al.
untreated cells were utilized as controls. All in vitro experiments were carried out on 2 microplates with at
least 5 parallel wells. Stock solutions of the tested substances (30 mM) were prepared with DMSO. The highest
DMSO concentration (0.3%) of the medium did not have any significant effect on the cell proliferation. Their
antiproliferative effects were determined at 2 final concentrations (30 and 90 μM). Doxorubicin was used as
reference antiproliferative agent. It caused 50% inhibition of proliferation at 0.15, 0.28, and 0.16 μM on Hela,
MCF7, and A431 cells, respectively.
Incubation of 1 and 7 with 2-mercaptoethanol
2-Mercaptoethanol (0.005 mol) was added to solutions of 1 and 7 (0.005 mol) in phosphate buffer solution
(pH 7.4, 6.9 and 6.4, 10 mL). After incubation at 37 ◦ C for 72 h on a shaking constant-temperature bath the
aqueous phase was extracted with chloroform. The chloroform layer was separated and dried over anhydrous
sodium sulfate. Removal of the organic solvent afforded a product shown by TLC (CHCl3 -MeOH (4:1)) to be
principally unchanged 1 and 7 along with very small quantities of other compounds. The residue was eluted on
a silica gel column with chloroform-methanol gradually. The starting compounds were mainly obtained. TLC
of the aqueous phase also revealed only the presence of the unchanged 1 and 7.
Results and discussion
We synthesized compounds 1-8 by traditional Mannich reaction using uracil, formalin solution, and appropriate
amines. It was noteworthy that all Mannich reactions to prepare nitrogen mustard with uracil (1) did not take
place when ethanol was used as a solvent. The Elderfield method29 was referred to for the preparation of 1
under hard reaction conditions, namely, by using glacial acetic acid as a solvent and heating under reflux for 3
h. In our study compound 1 was prepared under relatively soft conditions via stirring of the starting materials
in tetrahydrofuran for 24 h at room temperature.
Structures of dimethylamino (7), piperidino (3), and morpholino (2) Mannich bases at the 5 position of
uracil were characterized by their elemental and 1 H-NMR properties by Burckhalter et al.30 and Asherson et
al.37 However, Bombardieri et al.39 reported that the reaction with uracil, formalin, and morpholine gave N-
morpholinomethyluracil (N-Mannich base) depending on nitrogen analyses of the compound. This report was in
disagreement with Burckhalter et al.’s and Asherson et al.’s findings. As seen, it is difficult to understand which
structural isomer (C-5 or N-1 and/or N-3 substituted) of the Mannich bases occurred without interpretation of
NMR data. The other compounds derived, diethylamine (6) and piperazine (8), were not reported clearly in
the literature. Moreover, there are no spectral data for compound 4, which is a pyrrolidine Mannich base. The
structural assignment of diethanolamine derivative (5) was only based on UV and elemental analyses in the
literature.35 Compound 1 containing mustard moiety was characterized by UV, IR, and elemental properties
without giving any NMR data. Consequently, especially high resolution NMR findings were necessary for legal
structure elucidation since the uracil ring has 3 convenient positions for aminomethylation. In our study, the
high resolution 1 H-NMR spectra of these compounds exhibited only one aromatic singlet and disappearance
of the C-5 proton of the uracil ring, indicating formation of the C-Mannich bases at the 5 position. UV, IR,
1 H-NMR, 13 C-NMR, and MS spectral data of each compound are given in the experimental section in detail.
589

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Synthesis and cytotoxic evaluation of uracil C-Mannich bases, H. ISTANBULLU, et al.
In summary, compounds 6 and 8 are reported for first time in the literature and compounds 1, 4, and 5 are
reported for first time with detailed spectral data herein. At pH values of 7.4 and 6.9 representative compounds
1 and 7 were stable and hence the deamination process envisaged in Scheme 2 appears unlikely. No reaction
occurred with a model nucleophile 2-mercaptoethanol and test compounds at pH 7.4 and 6.9. The data in the
Table indicate that the cytotoxic activity was more remarkable in the mustard derivative (1) among the cell lines
covered in our study. As also seen in the Table, this compound yielded a percent inhibition of 34.38 μM, which
was comparable to the effect manifested by the control compound, Etoposide (31.64%), on breast carcinoma cell
lines in 30 μM concentration. This compound was followed by diethanolamine derivative 5 on breast carcinoma
cells with a percent inhibition of 10.75% at the same concentration. The effect of diethanolamine derivative was
comparable to that of diethyl 6 and dimethylamine 7 derivatives on skin dermoid carcinoma cells. The action
mechanism of diethanolamine and mustard derivatives might share similarities. However, the highest inhibitor
activity was obtained with mustard derivative 1 on all test carcinoma cells, while mustard derivative 1 was
followed by diethanolamine derivative 5 on skin dermoid carcinoma cells. This finding might suggest that Cl
rather than OH was required for activity. There was no relation between pKa values and inhibitor activities
of the compounds tested in this study. Stability test results do not appear to support our hypothesis based
on the elimination-nucleophilic addition mechanism under biomimetic test conditions (37 ◦ C and pH 7.4) for
alkylation.
Table 1. Antiproliferative activity of compounds.
Inhibition (%) SEM
Compound
Hela
MCF7
A431
30 μM 19.54 1.19 34.38 1.79 47.82 0.69
90 μM 28.70 1.82 44.64 1.34 63.35 1.22
30 μM 12.28 1.13 5.12 1.76 15.59 2.84
1
2
3
4
5
6
7
8
90 μM 3.49 2.70
30 μM 8.11 3.57
2.64 1.69
4.05 2.57
3.81 1.60 10.23 2.78
90 μM 4.70 2.57 11.54 1.53 3.23 2.87
30 μM 2.05 2.62 –1.00 0.85 15.23 2.78
90 μM –3.84 1.30 0.98 1.18 20.64 + 2.47
30 μM 21.01 + 1.05 10.75 0.84 21.79 2.09
90 μM 15.61 1.68 7.46 1.43 11.88 1.93
30 μM 7.88 0.69
90 μM 9.65 2.23
30 μM 9.61 2.43
5.81 0.47 19.13 0.91
4.78 2.68 24.54 1.12
3.47 1.57 28.40 1.19
90 μM 17.06 1.95 6.09 0.28 19.08 2.72
30 μM 9.33 1.87
90 μM 6.64 3.76
2.81 1.52 15.80 0.86
1.29 3.31 6.30 1.21
Etoposide 30 μM 95.01 0.73 31.64 2.02 93.85 0.59
It is conceivable therefore that the bioactivities of the compounds are due to the molecules per se or that
activity does not involve thiols. Thus future molecular modifications should be aimed at producing N-substituted
590

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Synthesis and cytotoxic evaluation of uracil C-Mannich bases, H. ISTANBULLU, et al.
Mannich bases rather than C-substituted Mannich bases. Such derivatives may have marked cytotoxicity and
value as candidate antineoplastic agents.
Taken together, the results that emerged from this investigation will guide the future expansion of these
series of compounds.
Acknowledgements
We thank the Research Foundation of Ege University for its financial support (project no: 05/ECZ/008) and
Prof. Dr. Ulrike Holzgrabe from the University of Wurzburg (Germany) for providing some chemicals.
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