C5-Morpholinomethylation of: N 1-sulfonylcytosines by a one-pot microwave assisted Mannich reaction

Article abstract of DOI:10.1039/c8ob00253c

A fast and efficient route for the introduction of a methylene bridged-amine (morpholinomethyl) functionality in the C5 position of the sulfonylated cytosine nucleobase has been developed. First, novel N1-sulfonylcytosine derivatives 3-6 were prepared by the condensation of silylated cytosine with selected sulfonyl chlorides. They were subsequently transformed to 5-morpholinomethyl-N1-sulfonylcytosine derivatives (8, 12-15) using microwave irradiation. As a result of cytosine ring opening in N1-tosylcytosine, depending on the reaction conditions, peculiar tosyl-urea derivative 9 has been isolated, which provided additional insight into the reaction pathway. The influence of the C5-substituent on the antiproliferative activity has been evaluated by performing the MTT test on U251, MCF-7 and MOLT-4 tumor cell-lines.

Full text of DOI:10.1039/c8ob00253c

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C5‐morpholinomethylation of N1‐sulfonylcytosines by one‐pot
microwave assisted Mannich reaction
Josipa Matić,a,b,* Irena Nekola,c Aleksandar Višnjevac,d Renata Kobetić,a Irena Martin‐Kleiner,e
Received 00th January 20xx,
Accepted 00th January 20xx
Marijeta Kralj,e,b and Biserka Žinića,b,
*
DOI: 10.1039/x0xx00000x
A fast and efficient route for introduction of methylene bridged‐amine (morpholinomethyl) functionality in the C5 position
of the sulfonylated cytosine nucleobase has been developed. First, novel N1‐sulfonylcytosine derivatives 3‐6 were prepared
by the condensation of silylated cytosine with selected sulfonyl chlorides. They were subsequently transformed to 5‐
morpholinomethyl‐N1‐sulfonylcytosine derivatives (8, 12‐15) using microwave irradiation. As a result of the cytosine ring
opening in N1‐tosylcytosine, depending on the reaction conditions, peculiar tosyl‐urea derivative 9 has been isolated, which
provided additional insight into reaction pathway. The influence of the C5‐substituent on the antiproliferative activity has
been evaluated by MTT test on U251, MCF‐7 and MOLT‐4 tumor cell‐lines.
www.rsc.org/
promoters of apoptosis in cancer cells through cyclin‐
dependent kinase 9 inhibition.19 In addition, morpholine moiety
is present in many drugs, like analgesic dextromoramide,
Introduction
Over the last few decades, in an extensive pursuit for the
new chemotherapeutics, modified pyrimidine nucleobases and
nucleosides have emerged as promising antitumor agents.1,2,3
Numerous patent applications in the recent years prove that
the interest in these compounds has not decreased.4 On the
other hand, sulfonyl moiety has been recognized as a potent
pharmacophore in carbonic anhydrase and metalloprotease
inhibitors, antifungal, antibacterial, antitumor and other
biologically active compounds.5,6 Since the sulfonylpyrimidines
present an interesting combination of described
pharmacophoric components, they have accordingly drawn
quite attention of our research group.
Sulfonylated derivatives of cytosine, that we have described
earlier, have shown strong antiproliferative activity against
human tumor cell lines in vitro7‐12 and in vivo13. These findings
encouraged us to further investigate modified pyrimidines. It
has been known that different C5‐substituted pyrimidine
nucleosides often display antitumor activity.14‐17 One of the best
known examples is 5‐fluorouracil which is widely used in the
treatment of cancer.18 Moreover, it has been found that C5‐
carbonitrile pyrimidine derivatives modified with heterocyclic
amines, like piperazine, piperidine and morpholine, could act as
antibiotic linezolid or antitumor drug gefitinib.20
We have decided to prepare a series of N1‐sulfonylcytosines
and an analogous series of N1‐sulfonylcytosines with an amine
(morpholine) substituent in the C5‐position. Investigation of
synthetic efforts for introduction of methylene‐bridged
morpholine moiety in the C5 position of N1‐sulfonylcytosine is
described herein. To the best of our knowledge, C5‐
morpholinomethylated derivatives of N1‐sulfonylpyrimidine
have not been reported so far. A secondary goal of the study
was to evaluate the influence of C5‐morpholinomethyl moiety
on the biological activity of N1‐sulfonylcytosines.
Antiproliferative activity on U251, MCF‐7 and MOLT‐4 tumor
cell‐lines was examined by MTT test.
Results and discussion
Chemistry
As
a part of previous sulfonylcyclourea research, N1‐
sulfonylation of cytosine nucleobase was investigated and reaction
conditions optimized throughout the preparation of N1‐
tosylcytosine (2; TsC).21 The reaction was elegantly achieved by
silylation with N,O‐bis(trimethylsilyl)acetamide (BSA) in a dry
acetonitrile and subsequent addition of appropriate sulfonyl chloride
(Scheme 1). Inspired by interesting biological results with
tosylcytosine, we decided to expand investigation to other para‐
substituted phenyl‐derivatives. In this study, several structurally
diverse and commercially available phenylsulfonyl chlorides have
been chosen. All of the selected sulfonyl chlorides contain structural
motifs which have been associated with promising antitumor activity
in the recent literature reports, namely: 4‐chloro‐3‐
nitrobenzenesulfonyl chloride,22‐24 biphenyl‐derivatives (4’‐methyl‐
and 4’‐methoxybiphenyl‐4‐sulfonyl chloride)25‐27 and a fluorophore
a. Laboratory for Biomolecular Interactions and Spectroscopy, Division of Organic
Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička cesta 54, 10000
Zagreb, Croatia.
b. BioZyne Ltd., Bijenička cesta 54, 10000 Zagreb, Croatia.
c. Pliva Croatia TAPI R&D, Prilaz baruna Filipovića 25, 10000 Zagreb, Croatia.
d. Laboratory for chemical and biological crystallography, Division of Physical
Chemistry, Bijenička cesta 54, Ruđer Bošković Institute, 10000 Zagreb, Croatia.
e. Laboratory of Experimental Therapy, Division of Molecular Medicine, Ruđer
Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia.
Electronic Supplementary Information (ESI) available: 1H and 13C NMR spectra of all
products, COSY NMR spectrum of
data for the compound (CCDC 1588667). See DOI: 10.1039/x0xx00000x
8, additional reaction scheme, crystallographic
9
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on, it is well‐known that cytidine reacts with formaldehyde in ethanol
to provide the N‐ethoxymethyl cytidine derivative.33 In our case,
corresponding N‐ethoxymethly derivatives of TsC and cytosine were
isolated from the mixture as well. In addition, as a possible source of
byproducts, formation of 1,4 adducts in the reaction of cytosine with
morpholine and formaldehyde has also been described previously.34
dabsyl
chloride
(4‐(dimethylamino)azobenzene‐4’‐sulfonyl
chloride)28. In addition, due to its specific features, azobenzene
moiety plays a crucial role in a sophisticated drug‐delivery systems,
described in the latest studies.29‐31
Novel N1‐sulfonyl derivatives 3‐6 were prepared in very good
yield (57–80%). On the other hand, synthesis of 5‐
morpholinomethylcytosine 7 was described earlier by Prukała, and it
was prepared by acid catalyzed Mannich reaction of cytosine with
paraformaldehyde and morpholine in ethanol.32
In an attempt to modify this method, we reasoned how to
provide enough energy for a reaction to occur, but, at the same time,
shorten the reaction time in order to prevent the formation of
numerous byproducts. Following this logic, we have turned attention
to microwave irradiation. Reaction conditions were explored with
respect to temperature and time (Table 1). Aminomethylation of TsC
2 was studied as a model reaction. Two equivalents of morpholine
and paraformaldehyde and four equivalents of acetic acid in respect
to starting 2 were used in all entries, as described earlier.32 Unlike
previously described method, all reagents were added to reaction
vial at once. The best yield on the compound 8 (65%), was obtained
when the reaction mixture was heated 30 minutes at 100 C. The
product precipitated spontaneously from the reaction mixture upon
cooling, which eliminated the need for a tedious isolation. Product
was isolated simply by filtration, and purified by recrystallization
from the methanol. Reasonably good results were obtained from
reactions at higher temperature (120 C). Although 8 was isolated in
somewhat reduced yield (32‐41%), the purity of the precipitated
product was much higher and there was no need for recrystallization.
In a quest for new pyrimidine analogs with potential medical
application, we wanted to prepare cytosine derivatives which
contain both N1‐sulfonyl and C5‐morpholinomethyl substituents.
First we attempted the sulfonylation of 7 through several known
methods: 1) activation/silylation of 7 with BSA in CH3CN; 2) activation
of
7 with 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) in N,N‐
dimethylformamide (DMF) and 3) activation of 7 with K2CO3 in DMF,
4) sulfonylation of 7 in pyridine.21
While method 4) provided traces of desired product 8 (Scheme
2), other methods resulted solely in isolation of starting compound 7
as a single product.
NH2
NH2
N
1. BSA/CH3CN
2. R-SO2Cl
N
O
N
H
O
N
S
Table 1. Optimization of reaction conditions for the Mannich
aminomethylation of N1‐sulfonylpyrimidines.
O
O
R
NH2
2-6
1
N
O
AcOH
CH3
2: R =
5: R =
CH3
4: R =
3: R =
Cl
HN
O
O
N
S
72%
80%
80%
EtOH
MW
77%
H
H
O
O
NO2
O
N
N
CH3
6: R =
2
CH3
CH3
57%
N
CH3
NH
Scheme 1. Synthesis of N1‐sulfonylcytosine derivatives.
O
NH
HN
N
O
O
S
O
N
N
N
O
N
S
H
H
O
O
O
NH
H3C
9
NH2
HN
N
8
CH3
O
pyridine
N
N
O
N
S
O
r.t.
O
O
Entry
1
2
3
4
5
6
7
8
9
10
11
Temp.
80 C
80 C
80 C
80 C
Time
Yield of 8 Yield of 9
O
N
H
traces of product
20 min
30 min
40 min
50 min
60 min
20 min
30 min
40 min
20 min
30 min
40 min
23%
27%
48%
49%
49%
33%
65%
56%
41%
34%
32%
45%
30%
22%
17%
16%
2%
traces
traces
0%
7
8
CH3
Scheme 2. N1‐sulfonylation of 5‐morpholinomethylcytosine 7 in
pyridine.
80 C
100 C
100 C
100 C
120 C
120 C
120 C
Since sulfonylation of 7 did not yield satisfactory results, we have
decided to apply reversed synthetic approach. In this attempt, we
planned to introduce a morpholinomethyl moiety to TsC 2. We
applied the Prukała method (4 h reflux), but a mixture of several
byproducts was formed. Again, only traces of desired product 8 were
detected, alongside with a large amount of starting TsC 2. Upon
prolongation of the reaction time, complexity of the byproduct
mixture increased (Supporting information, Figure S14). One of the
reasons lies in the instability of N‐SO2 bond in the given conditions,
namely several hours of heating in the acidic solution. Detection of
cytosine 1 in the product mixture supports this assumption. Further
0%
0%
However, reactions at lower temperatures (80 C) gave the
unexpected results. As a result of cytosine ring opening, tosylurea
derivative 9 was isolated in 45% yield, as a main product after 20
minutes. In addition, yield of 8 dropped significantly (Table 1). The
2 | J. Name., 2012, 00, 1‐3
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NH2
amount of 9 decreased with prolongation of the reaction time, to
disappear almost completely at higher temperatures. At 100 C, 9
was isolated in only 2% yield after 20 minutes in a microwave reactor,
while at longer reaction times merely traces were detected.
Formation of similar acyclic analogues of uracil and cytosine was
N
H
N
3:HCOH:
1:4:4:8
:AcOH/
O
O
N
S
O
O
NO2
Cl
100 °C
30 min
3
NH
EtOH, MW
55%
reported as
a
result of photoreactions in the presence of
H
N
HN
N
ethylamine.35
100 °C
3:HCOH:
:AcOH/
30 min
O
O
1:2:2:4
EtOH, MW
O
N
S
83%
O
O
NO2
NH2
In a separated experiment, we have proven that 9 undergoes ring
closure upon heating. When 9 was heated for 1 hour in a microwave
reactor at 100 C, in pure ethanol, traces of TsC 2 were detected.
Furthermore, heating of 9 in acidic ethanol solution resulted in
almost complete conversion into TsC 2 after one hour. It is
reasonable to assume that higher reaction temperatures promote
formation of 5‐morpholinomethyl‐N1‐tosylcytosine (8), by
cyclization of tosylurea‐derivative 9 into starting TsC 2.
H
N
N
N
12
11:HCOH:
1:2:2:4
:AcOH/
O
O
O
N
S
O
O
100 °C
NO2
N
30 min
EtOH, MW
48%
11
O
Scheme 4. Synthetic routes for the preparation of 5‐
morpholinomethyl derivative 12.
It is important to emphasize the significance of order in which the
reagents were added. When we heated the reaction mixture prior to
addition of acetic acid, as reported in described method, 5‐
morpholinomethylcytosine (7) was isolated as a main product.32 This
is due to the N‐SO2 bond breakage of TsC 2, which takes place in
these conditions. In addition, as a result of the transamination
reaction, an adduct 10 of morpholine and p‐toluenesulfonic acid was
isolated as well (Scheme 3).
However, 30 minute‐reaction at 100 C did not provide adequate
results with compounds 4‐6, bearing sulfonylbiphenyl or azobenzene
substituents at N1‐position (Scheme 5). Major amount of starting
compound (77‐100%) was isolated after the reaction. Also,
corresponding sulfonylurea derivative was isolated from reaction
with methylbiphenyl derivative 4. Nevertheless, 13 and 14 were
prepared in good yields (52 and 49%) when higher temperature (120
C) was applied for 30 minutes (Table 2). Compound 15 was isolated
in somewhat modest yield (30%) after 60 minutes at 120 C.
NH2
1. 100° C, 10 min
2. AcOH, 120°C, 30 min
N
O
HN
O
O
N
S
H
H
EtOH
MW
O
O
NH2
NH
AcOH
EtOH
MW
O
2
N
HN
N
CH3
HN
O
O
H
H
O
O
N
S
O
N
S
O
O
O
NH2
N
S
O
R
R
O
O
N
N
2-6, 11
8, 12-15
O
O
N
H
CH3
2, 8: R =
CH3
CH3
4, 13: R =
10
7
Scheme 3. Sequential addition of reagents leading to N‐SO2 bond
breakage of TsC 2.
3: R =
Cl
5, 14: R =
6, 15: R =
O
CH3
NO2
N
CH3
CH3
N
O
11, 12: R =
N
N
Once the optimal conditions for Mannich reaction were
established, we have expanded the reaction scope on the series of
N1‐sulfonyl‐aromatic cytosine derivatives 12‐15 (Schemes 4 and 5,
Table 2). Beside biological activity, we wanted to assess the
robustness of the method regarding diverse N1‐sulfonylcytosine
derivatives. Interestingly, the dominant reaction with the compound
3 was substitution of chlorine with morpholine (Scheme 4).
Compound 11 was isolated in 83% yield. 5‐morpholinomethyl‐
derivative 12 was isolated when the 11 was subsequently subjected
to the established Mannich conditions, or alternatively, when the
compound 3 was treated with double amount of reagents (four
equivalents of morpholine and paraformaldehyde and eight
equivalents of acetic acid).
NO2
Scheme 5. Synthesis of 5‐morpholinomethyl derivatives 12‐15.
Table 2. Reaction conditions for the synthesis of 5‐
morpholinomethyl derivatives 8, 12‐15.
N1‐comp. N1‐C5‐
Temp.
Time
Yield
comp.
2
3
11
4
5
8
30 min
30 min
30 min
30 min
30 min
60 min
65%
55%
48%
52%
49%
30%
100 C
100 C
100 C
120 C
120 C
120 C
12
12
13
14
15
6
Characterization of the products
NMR spectra
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353
Structure of 5‐morpholinomethyl‐N1‐tosylcytosine (8) was
100
80
confirmed by 2d NMR spectroscopy. Absence of characteristic C5‐
proton signal in 1H NMR spectrum, as well as coupling interaction of
C6‐proton with methylene protons in COSY spectrum confirms that
the aminomethylation occurred at the C5‐position of cytosine base
(Supporting information, Figure S13). According to the chemical
shifts of NH‐protons, aminomethylated cytosine base of the
compound 8 occupies keto‐imino tautomeric form.36
A
100
80
60
40
20
0
PI 353
60
40
130 140 150 160 170 180 190
20
m/z
0
200
400
100
100
80
356
B
80
60
40
20
0
Protons of acyclic urea derivative 9 exhibit significant shifts in
NMR spectra compared to TsC 2 and 8, indicating dramatically
different structure. Signals of tosyl‐protons of 9 are shifted upfield
for about 0.2 ppm, and morpholine N‐CH2 protons are 0.9 ppm
shifted downfield, compared to 8. The most pronounced shift of 1.2
ppm displays vinyl CH proton, compared to C5 proton of TsC 2.
PI 356
60
40
20
0
130 140 150 160 170 180 190
m/z
200
400
m/z
The acyclic structure of 9 was not unambiguously determined by
NMR in solution (DMSO‐d6) and therefore, additional studies were
performed in gas‐phase (mass spectrometry) and solid phase (single
crystal diffraction X‐ray).
Figure 2. Full scan ES+ spectrum for compound 9 dissolved in
methanol (A) and deutero methanol (B) at about 10‐6 mol dm‐3.
Inserted spectrum is PI for molecular ion signals m/z for [M+H]+ 353
(CH3OH) and m/z 356 for [M+D]+ (CD3OD).
Mass Spectra
Molecular and crystal structure
H/D exchange experiments were performed to prove open
cytosine structure of 9, with three fast exchangeable protons, in
contrast to N1‐substituted cytosine molecule, having only two
exchangeable protons. The collision‐induced fragmentation (CID)
experiments of 9 showed completely different composition of the
obtained fragments in regards to the open structure. N1‐substituted
cytosine molecular ion increases mass by 2 i.u. while molecular ion
of 9 increased by 3 i.u. Additionally, we observed that 9 has three not
equivalent C‐N bonds in the gas phase (Figure 1). Heterolytic bond
cleavage of 9 molecular ion (m/z for [M+H]+ 353 or [M+D]+ 356)
occurred at three positions. The variation of the collision energy (CE
5‐20 eV) or the ionization mode (ES+ or ES‐) yielded the major
fragment formed upon the a bond cleavage (i.e. the most abundant
signal at m/z 182.1 in ES+). Bond a was the most unstable C‐N bond,
followed by c and finally b (Figures 1 and 2).
Molecular structure of 9 reveals no peculiarities, with the
exception of an unexpected proton migration from N8 to N12 and
formation of a zwitterionic molecule that was discovered by the X‐
ray single crystal diffraction studies. Morpholine unity is in a usual
chair conformation (Figure 3).
O
NH
H+
O
O
a
b c
S
N
N
N
Figure 3. ORTEP drawing of compound 9 with the atom numbering.
H
H
Displacement parameters are scaled to 50 % probability value.
O
H3C
Figure 1. Marked up three C‐N bonds that have similar but not the
equivalent bonds in the gas phase. Bond a is the most unstable C‐N
bond, followed by c and finally b.
Crystal packing of 9 is characterized by intensive networks of
hydrogen bonds in which the co‐crystallized water molecules of O5
and O6 are heavily involved (Table 3, Figures 4 and 5).
Table 3. Hydrogen bonds in the structure of 9.
D‐H ˑˑˑA
D‐H (Å)
H ˑˑˑA (Å)
DˑˑˑA (Å)
D‐H ˑˑˑA (°)
symm op. on A
O5‐H5AˑˑˑN8
0.90 (4)
0.85 (3)
0.75 (3)
0.75 (3)
0.78 (3)
0.89 (2)
0.87 (3)
0.89 (3)
2.10 (4)
2.02 (3)
2.35 (3)
2.49 (4)
2.05 (3)
2.19 (2)
2.01 (3)
2.02 (3)
2.992 (2)
2.855 (2)
3.047 (3)
3.103 (2)
2.830 (2)
3.075 (2)
2.875 (2)
2.657 (2)
174 (3)
168 (3)
155 (3)
139 (3)
174 (4)
171 (2)
172 (2)
128 (2)
1
O5‐H5BˑˑˑO2iii
O6‐H6AˑˑˑO1i
O6‐H6AˑˑˑN8i
O6‐H6BˑˑˑO5
N10‐H10ˑˑˑO5i
N12‐H12AˑˑˑO6ii
N12‐H12BˑˑˑO3
1+x, y, z
1‐x, 1‐y, 1‐z
1‐x, 1‐y, 1‐z
1
1‐x, 1‐y, 1‐z
1‐x, 2‐y, 1‐z
1 (intra)
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N12 effectuates a single intra‐molecular H‐bond in this structure, via
its hydrogen H12B to the carbonyl oxygen O3. With its remaining
hydrogen, H12A, N12 forms the hydrogen bond towards the co‐
crystallized water molecule of O6ii. All these interactions contribute
to a construction of a two dimensional molecular tube of the (roughly
measured) dimensions 4.58 Å x 6.24 Å, stretching parallel to the 100
crystallographic plane (Figure 5).
In vitro screening of antitumor activity
Antiproliferative activity of 2 (tosyl‐derivative) on various tumor
cell lines was extensively reviewed in previous studies, showing
moderate activity on MCF‐7 cells and no activity towards MOLT‐4
cells.7,11 In addition to in vitro studies, mechanism of action of 2 was
analyzed in an extensive in silico study.11 The results revealed that 2
exhibits uncommon mechanism of cytostatic action, combining
nucleic acid antimetabolite and a mechanism involving the activation
of CYP1A1 and CYP1B1 genes by aryl hydrocarbon receptor, and a
subsequent DNA damage response. Although a direct parallel cannot
be drawn for all sulfonylcytosine derivatives, these assumptions
present a valuable starting point for further analyses.
Figure 4. Hydrogen bonding pattern in the structure of 9.
The activity of new compounds was tested using MTT assay.
Compounds showed diverse effect on proliferation of U251, MCF‐7
and MOLT‐4 tumor cell lines (Table 4).
Among N1‐sulfonylcytosine derivatives, compounds 3 (4‐chloro‐
3‐nitrophenyl‐derivative),
4 (methylbiphenyl‐derivative) and 5
(methoxybiphenyl‐derivative) showed low micromolar IC50 activities,
especially on MCF‐7 and MOLT‐4 cell lines.
Introduction of C5‐morpholinomethyl group produced very
heterogeneous effect on the antitumor activity, strongly depending
on the N1‐substituent. Tosyl‐derivative 8 and azobenzene‐derivative
15 showed very modest activity. Introduction of C5‐
morpholinomethyl moiety completely deteriorated antitumor
activity in 15, compared to its moderately active N1‐sulfonyl
analogue 6. Introduction of two morpholine units also diminished
activity in the compound 12, which showed moderate activity,
compared to very active N1‐sulfonyl analogue 3. However, insertion
of C5‐morpholinomethyl moiety had favorable influence on the
antitumor activity of biphenyl‐derivatives. Compounds 13
(methylbiphenyl‐derivative) and 14 (methoxybiphenyl‐derivative)
exhibited even more pronounced antiproliferative activity toward
U251 cell line than their N1‐sulfonyl analogues 4 and 5. At the same
time, 13 and 14 preserved remarkable potency toward MCF‐7 and
MOLT‐4 cell lines.
Figure 5. The H‐bonded molecular tube a motif of the crystal
packing in the structure of 9.
The water molecule of O5 acts as a hydrogen bonding bridge
between two main molecules (mutually related by the symmetry
operation 1+x, y, z) being H‐bonded to the deprotonated N8 of the
first main molecule by its hydrogen H5A, and to the sulphonyl oxygen
of the second one by its remaining hydrogen, H5B. The remaining
water molecule (of O6) connects through its hydrogen atom H6B to
the water molecule of O5, while its second hydrogen, H6A acts as a
double donor forming two hydrogen bonds, towards the sulfonyl
oxygen O1i and deprotonated nitrogen N8i, of the neighboring main
molecule (related by the symmetry operation 1‐x, 1‐y, 1‐z). Hence,
the deprotonated nitrogen N8 acts as a double acceptor of the
hydrogen bonds, connecting the main molecule to both of the co‐
crystallized water molecules. Finally, the protonated imino‐nitrogen
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Journal Name
Table 4. Antiproliferative activity of the new compounds.
solution was cooled to 0

C and sulfonyl chloride (1 mmol) was
added. After heating under reflux for 60 min a small amount of
ammonia/methanol was added in the reaction mixture which
immediately resulted in precipitation of crystals. Solid was
filtered off and washed with cold methanol. Crude product was
recrystallized from methanol to give analytically pure sample.
IC50a/μM
MCF‐7
4 ± 3
3 ± 0.2
3 ± 1
30 ± 11
≥100
Compound
U251
3 ± 2
22 ± 1
23 ± 2
8 ± 3
≥100
61±1
4±0.6
3±0.2
46±38
7±4
MOLT‐4
2 ± 1
1 ± 0.1
5 ± 2
>100
≥100
3
4
5
6
8
N
1‐(4‐chloro‐3‐nitrophenylsulfonyl)cytosine
(3): Synthesis
12
13
14
15
5‐FUb
28±3
18±2
was performed according to the general procedure with 4‐
2±0.2
2±0.5
>100
2±0.1
2±0.1
>100
chloro‐3‐nitrobenzenesulfonyl chloride to give the product
(453 mg, 77%) as white crystals.
3
M.p. = 222–224 °C; Rf = 0.3 (CH2Cl2:CH3OH/9:1); UV (MeOH):
λmax/nm: 225 and 247; log ε/ dm3 mol–1 cm–1: 4.21 and 4.21; IR
(KBr) νmax/cm–1: 3381 (m), 3099 (m), 1672 (s), 1659 (s), 1593 (w),
1572 (w), 1535 (s), 1491 (m), 1387 (m), 1356 (s), 1283 (m), 1256
(w), 1234 (w), 1188 (s), 1111 (m), 1097 (m), 1058; (w), 1020 (w);
1H NMR (DMSO‐d6) δ/ppm: 8.66 (d, 1H, J = 2.2 Hz, Ar), 8.28 (dd,
1H, J1 = 8.6 Hz, J2 = 2.3 Hz, Ar), 8.13–7.97 (m, 4H, Ar, H‐6, NH2),
6.00 (d, 1H, J5,6 = 7.9 Hz, H‐5); 13C NMR (DMSO‐d6) δ/ppm: 166.1
(Cq), 150.8 (Cq), 147.2 (Cq), 139.2 (CH, C‐6), 137.1 (Cq), 133.6 (CH,
Ar), 132.7 (CH, Ar), 131.4 (Cq), 126.2 (CH, Ar), 98.0 (CH, C‐5). (see
Supporting information Fig. S1) ESI‐MS: calcd. for C10H7ClN4O5S:
330.0; found [M+H]+ at m/z 331.0.
1±0.3
1±0.2
aIC50; the concentration that causes 50% growth inhibition
b5‐FU – 5‐fluorouracil
As a well‐known chemotherapeutic agent with pyrimidine structure,
5‐fluorouracil (5‐FU) was assayed for the comparison of the
antitumor activity with the novel cytosine derivatives presented
herein.18
Considering IC50, 5‐FU showed superior activity than compound 8
(tosyl‐derivative), 12 (4‐morpholino‐3‐nitrophenyl‐derivative) and
compounds 6 and 15 (both azobenzene derivatives). However,
compounds
3
(4‐chloro‐3‐nitrophenyl‐derivative),
13
(methylbiphenyl‐derivative) and 14 (methoxybiphenyl‐derivative)
showed stronger activity on U251 cells, while they retained very
similar, only slightly lower potency against MOLT‐4 and MCF‐7 cell‐
N
1‐(4’‐methylbiphenyl‐4)sulfonylcytosine
(4): Synthesis
was performed according to the general procedure with 4’‐
methylbiphenyl‐4‐sulfonyl chloride to give the product
mg, 72%) as white crystals.
lines. Compounds
4
(methylbiphenyl‐derivative) and
5
4 (217
(methoxybiphenyl‐derivative) also showed somewhat lower activity
on all the tested cell lines, with the exception of 4 being equally
active towards MOLT‐4 cells, as the standard 5‐FU.
M.p. = 244–245 °C; Rf = 0.5; UV (MeOH): λmax/nm: 284; log ε/
dm3 mol–1 cm–1: 2.58; IR (KBr) νmax/cm–1: 3377 (m), 3107 (m),
3069 (w), 3030 (w), 1676 (s), 1595 (w), 1524 (s), 1483 (s), 1373
(m), 1362 (m), 1283 (m), 1232 (w), 1171 (s), 1148 (w), 1115 (w),
1092 (m), 1024 (w), 1005 (w); 1H NMR (DMSO‐d6) δ/ppm: 8.15
(d, J6,5 = 7.9 Hz, 1H, H‐6), 8.04–7.99 (m, 2H, Ar), 7.93–7.87 (m,
4H, NH2, Ar), 7.65 (d, J = 8.2 Hz, 2H, Ar), 7.33 (d, J = 8.0 Hz, 2H,
Ar), 5.98 (d, J5,6 = 7.9 Hz, 1H, H‐5), 2.36 (s, 3H, CH3); 13C NMR
(DMSO‐d6) δ/ppm: 165.9 (Cq), 150.9 (Cq), 145.8 (Cq), 139.3 (CH,
C‐6), 138.5 (Cq), 135.4 (Cq), 135.2 (Cq), 129.7 (CH, Ar), 129.3 (CH,
Experimental
Materials and apparatus
Solvents were distilled from appropriate drying agents shortly
before use. Microwave assisted reaction was conducted in a
borosilicate glass vials sealed by reusable snap‐cap with PTFE coated
silicone septum. The microwave heating was performed in the Anton
Paar microwave synthesis reactor Monowave 300. After completed
irradiation, the reaction tube was cooled with high‐pressure air until
the temperature had fallen below 55 °C. TLC was carried out on
DCplastikfolien Kieselgel 60 F254 and preparative thin layer (2 mm)
chromatography was done on Merck 60 F254. NMR spectra were
recorded on 600 and 300 MHz spectrometers. Mass spectrometry
was performed on the Agilent 6410 Triple Quad mass spectrometer
(Agilent Technologies). High resolution mass spectra (HRMS) were
obtained using a MALDI‐TOF/TOF mass spectrometer 4800 Plus
MALDI TOF/TOF analyzer (Applied Biosystems Inc., Foster City, CA,
USA). FT‐IR spectra of the samples in KBr pellets were recorded at
resolution of 4 cm‐1 on an ABB Bomem MB102 single‐beam
spectrometer. The electronic absorption spectra of newly prepared
compounds were measured on a Varian Cary 100 Bio spectrometer.
Ar), 127.0 (CH, Ar), 126.8 (CH, Ar), 97.4 (CH, C‐5), 20.7 (CH3). (see
Supporting information Fig. S2) ESI‐MS: calcd. for C17H15N3O3S:
341.1; found [M+H]+ at m/z 342.1.
N
1‐(4'‐methoxybiphenyl‐4)sulfonylcytosine
(5): Synthesis
was performed according to the general procedure with 4’‐
methoxybiphenyl‐4‐sulfonyl chloride to give the product
mg, 80%) as white crystals.
5 (283
M.p. = 227–228 °C; Rf = 0.4; UV (MeOH): λmax/nm: 246; log ε/
dm3 mol–1 cm–1: 4.00; IR (KBr) νmax/cm–1: 3366 (m), 3099 (m),
1670 (s), 16076 (m), 1522 (s), 1487 (s), 1356 (m), 1286 (m), 1250
(m), 1169 (s), 1140 (w), 1115 (m), 1092 (m), 1024 (w); 1H NMR
(DMSO‐d6) δ/ppm: 8.14 (d, J6,5 = 7.9 Hz, 1H, H‐6), 8.02–7.96 (m,
2H, Ar), 7.93–7.86 (m, 4H, Ar, NH2), 7.75–7.68 (m, 2H, Ar), 7.11–
7.04 (m, 2H, Ar), 5.97 (d, J5,6 = 7.9 Hz, 1H, H‐5), 3.82 (s, 3H,
OCH3); 13C NMR (DMSO‐d6) δ/ppm: 165.9 (Cq), 160.0 (Cq), 150.9
(Cq), 145.5 (Cq), 139.4 (CH, C‐6), 134.8 (Cq), 130.3 (Cq), 129.4 (CH,
Ar), 128.5 (CH, Ar), 126.5 (CH, Ar), 114.6 (CH, Ar), 97.4 (CH, C‐5),
55.3 (OCH3). (see Supporting information Fig. S3) ESI‐MS: calcd.
for C17H15N3O4S: 357.1; found [M+H]+ at m/z 358.1.
Synthesis
General procedure for the preparation of
derivatives (3−6)
N1‐sulfonylcytosine
A mixture of cytosine
1 (1 mmol) and BSA (3 mmol) was
heated under reflux in dry acetonitrile (2 mL) for 30 min. The
6 | J. Name., 2012, 00, 1‐3
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Journal Name
ARTICLE
N
1‐(4‐(dimethylamino)azobenzene‐4’‐sulfonylcytosine (6): M.p. = 272–273 °C; Rf = 0.6 (CH2Cl2:CH3OH/9:1); UV (MeOH):
Synthesis was performed according to the general procedure λmax/nm: 258 and 404; log ε/dm3 mol–1 cm–1: 4.58 and 3.72; IR (KBr)
with 4‐(dimethylamino)azobenzene‐4’‐sulfonyl chloride to give νmax/cm–1: 3420 (s), 3105 (m), 2951 (m), 1692 (s), 1662 (s), 1601 (s),
the product
6
(212 mg, 57%) as purple crystals.
1556 (s), 1526 (s), 1478 (m), 1449 (w), 1413 (m), 1379 (m), 1367 (m),
1352 (m), 1340 (m), 1331 (w), 1296 (m), 1267 (w), 1235 (m), 1178 (s),
1118 (s), 1007 (m); 1H NMR (DMSO‐d6) δ/ppm: 8.37 (d, 1H, J = 2.4 Hz,
Ar), 8.23 (brs, 1H, NH), 8.02 (dd, 1H, J1 = 9.1, J2 = 2.4 Hz, Ar), 7.97 (s,
1H, H‐6), 7.68 (brs, 1H, NH), 7.43 (d, 1H, J = 9.2 Hz, Ar), 3.74–3.50 (m,
4H, O‐CH2), 3.60–3.54 (m, 4H, O‐CH2), 3.29 (s, 2H, CH2), 3.26–3.19 (m,
4H, N‐CH2), 2.42–2.34 (m, 4H, N‐CH2); 13C NMR (DMSO‐d6) δ/ppm:
165.7 (Cq), 150.8 (Cq), 148.4 (Cq), 137.5 (CH, C‐6), 137.3 (Cq), 133.7
(CH, Ar), 128.9 (CH, Ar), 125.0 (Cq), 120.0 (CH, Ar), 104.5 (Cq, C‐5),
66.1 (O‐CH2), 65.6 (O‐CH2), 55.8 (CH2), 52.5 (N‐CH2), 50.2 (N‐CH2).
(see Supporting information Fig. S6) HRMS: m/z: calcd for
C19H24N6O7SNa+: 503.1325; found 503.1342 [M+Na]+.
M.p. = 263–265 °C; Rf = 0.5; UV (MeOH): λmax/nm: 267 and 454;
log ε/ dm3 mol–1 cm–1: 4.12 and 4.31; IR (KBr) νmax/cm–1: 3373
(m), 3115 (m), 2920 (w), 1655 (s), 1605 (s); 1585 (m), 1520 (m),
1483 (m), 1367 (s), 1358 (s), 1313 (w), 1279 (m), 1234 (w), 1182
(m), 1140 (s), 1121 (m), 1084 (m), 1020 (w); 1H NMR (DMSO‐d6)
δ/ppm: 8.15 (d, 1H, J6,5 = 7.9 Hz, H‐6), 8.09 (d, 2H, J = 8.7 Hz, Ar),
7.99–7.88 (m, 4H, Ar, NH2), 7.84 (d, 2H, J = 9.1 Hz, Ar), 6.85 (d,
2H, J = 9.2 Hz, Ar), 5.98 (d, 1H, J5,6 = 7.9 Hz, H‐5), 3.10 (s, 6H,
N(CH3)2); 13C NMR (DMSO‐d6) δ/ppm: 165.9 (Cq), 155.8 (Cq),
153.4 (Cq), 150.9 (Cq), 142.7 (Cq), 139.3 (CH, C‐6), 136.3 (Cq),
130.1 (CH, Ar), 125. 7 (CH, Ar), 121.8 (CH, Ar), 111.6 (CH, Ar),
97.5 (CH, C‐5), 39.8 (N(CH3)2). (see Supporting information Fig.
S4) ESI‐MS: calcd. for C18H18N6O3S: 398.1; found [M+H]+ at m/z
399.1.
5‐Morpholinomethyl‐N1‐(4’‐methylbiphenyl‐
4)sulfonylcytosine (13): N1‐(4’‐methylbiphenyl‐4)‐sulfonylcytosine
(4) (101 mg, 0.30 mmol), paraformaldehyde (19 mg, 0.61 mmol,
97%), morpholine (52 μL, 0.60 mmol) and acetic acid (68 μL, 1.18
mmol) were suspended in absolute ethanol (3 mL). Reaction mixture
was heated 30 minutes at 120 °C in a microwave reactor (stirring
speed: 600 rpm). Product 13 was isolated by thin‐layer
chromatography (eluent: 10% MeOH in CH2Cl2, white crystals, 69 mg,
52%).
5‐Morpholinomethyl‐N1‐sulfonylcytosine derivatives:
5‐Morpholinomethyl‐N1‐tosylcytosine (8): N1‐tosylcytosine (2)
(200 mg; 0.75 mmol), paraformaldehyde (46 mg, 1.50 mmol, 97%),
morpholine (131 μL, 1.50 mmol, 99%) and acetic acid (172 μL, 3.00
mmol) were suspended in absolute ethanol (5 mL). Reaction mixture
was heated 30 minutes at 100 °C in a microwave reactor. Product 8
precipitated upon cooling of the mixture and it was purified by
recrystallization from hot methanol (white crystals, 178 mg, 58%).
M.p. > 300 °C (dec.); Rf = 0.5 (CH2Cl2/CH3OH 9:1); UV (MeOH):
λmax/nm: 285; log ε/dm3 mol–1 cm–1: 4.69; IR (KBr) ν/cm–1: 3440(s),
3097 (m), 1691 (s), 1654 (m), 1590 (w), 1518 (w), 1474 (w), 1365 (w),
1353 (w), 1340 (w), 1298 (w), 1172 (m), 1121 (w), 1089 (w); 1H NMR
(DMSO‐d6) δ/ppm: 8.21 (brs, 1H, NH), 8.07–7.99 (m, 3H, H‐6, Ar),
7.90 (d, 2H, J = 8.6 Hz, Ar), 7.68 (bs, 1H, NH), 7.66 (d, 2H, J = 8.1 Hz,
Ar), 7.33 (d, 2H, J = 8.0 Hz, Ar), 3.58 (s, 4H, O‐CH2), 3.32 (s, 2H, CH2),
2.41 (bs, 4H, N‐CH2), 2.37 (s, 3H, CH3); 13C NMR (DMSO‐d6) δ/ppm:
165.7 (Cq), 150.7 (Cq), 145.8 (Cq), 138.5 (Cq), 137.5 (CH, C‐6), 135.4
(Cq), 135.2 (Cq), 129.7 (CH, Ar), 129.4 (CH, Ar), 127.0 (CH, Ar), 126.8
(CH, Ar), 104.5 (Cq, C‐5), 66.1 (O‐CH2), 55.8 (CH2), 52.5 (N‐CH2), 20.7
(CH3). (see Supporting information Fig. S7) HRMS: m/z: calcd for
C22H25N4O4S+: 441.1597; found 441.1595 [M+H]+.
M.p. = 245–246 °C; Rf = 0.6 (CH2Cl2:CH3OH/9:1); UV (MeOH):
λmax/nm: 247; log ε/dm3 mol–1 cm–1: 3.84; IR (KBr) νmax/cm–1: 3290
(m), 3098 (m), 3067 (m), 2959 (m), 2922 (m), 2841 (m), 1684 (s), 1517
(s), 1476 (m), 1451 (m), 1369 (m), 1353 (s), 1340 (m), 1291 (s), 1268
(w), 1240 (w), 1205 (w), 1188 (w), 1171 (s), 1116 (s), 1088 (s), 1008
1
(m); H NMR (DMSO‐d6) δ/ppm: 8.19 (s, 1H, NH), 8.01 (s, 1H, H‐6),
7.86 (d, J = 8.3 Hz, 2H, Ar), 7.65 (s, 1H, NH), 7.44 (d, J = 8.1 Hz, 2H,
Ar), 3.63–3.53 (m, 4H, O‐CH2), 3.30 (s, 2H, CH2), 2.44–2.32 (m, 7H, N‐
CH2, CH3); 13C NMR (DMSO‐d6) δ/ppm: 165.7 (Cq), 150.6 (Cq), 145.3
(Cq), 137.5 (CH, C‐6), 134.2 (Cq), 129.5 (CH, Ar), 128.8 (CH, Ar), 104.4
(Cq, C‐5), 66.1 (O‐CH2), 55.8 (CH2), 52.5 (N‐CH2), 21.1 (CH3). (see
Supporting information Fig. S5) HRMS: m/z: calcd for
C16H21N4O4S+: 365.1284; found 365.1271 [M+H]+.
5‐Morpholinomethyl‐N1‐(4’‐methoxylbiphenyl‐
4)sulfonylcytosine
(14):
N1‐(4’‐methoxylbiphenyl‐4)‐
sulfonylcytosine (5) (54 mg, 0.15 mmol), paraformaldehyde (10 mg,
0.32 mmol), morpholine (26 μL, 0.30 mmol) and acetic acid (35 μL,
0.59 mmol) were suspended in absolute ethanol (3 mL). Reaction
mixture was heated 30 minutes at 120 °C in a microvawe reactor.
Product 14 was isolated by thin‐layer chromatography (eluent: 10%
MeOH in CH2Cl2, white crystals, 33 mg, 49%).
5‐Morpholinomethyl‐N1‐(4‐morpholino‐3‐
nitrophenylsulfonyl)cytosine (12):
a) N1‐(4‐morpholino‐3‐nitrophenylsulfonyl)‐cytosine (11) (37
mg; 0.10 mmol), paraformaldehyde (7 mg, 0.23 mmol), morpholine
(18 μL; 0.20 mmol) and acetic acid (23 μL, 0.40 mmol) were
suspended in absolute ethanol (2 mL). Reaction mixture was heated
30 minutes at 100 °C in a microwave reactor. Product 12 was isolated
by thin‐layer chromatography (eluent: 10% MeOH in CH2Cl2, yellow
crystals, 23 mg, 48%).
M.p. = 211–212 °C; Rf = 0.6 (CH2Cl2/CH3OH 9:1); UV (MeOH): λmax/nm:
299; log ε/dm3 mol–1 cm–1: 4.17; IR (KBr) ν/cm–1: 3430(m), 1688 (s),
1662 (s), 1609 (m), 1592 (m), 1520 (s), 1489 (s), 1397 (w), 1368 (m),
1354 (m), 1340 (m), 1297 (s), 1270 (m), 1252 (m), 1174 (s), 1116 (m),
1090 (m); 1H NMR (DMSO‐d6) δ/ppm: 8.21 (brs, 1H, NH), 8.07–7.97
(m, 3H, Ar, H‐6), 7.88 (d, 2H, J = 8.7 Hz, Ar), 7.72 (d, 2H, J = 8.8 Hz,
Ar), 7.67 (brs, 1H, NH), 7.08 (d, 2H, J = 8.8 Hz, Ar), 3.82 (s, 3H, O‐CH3),
3.58 (s, 4H, O‐CH2), 3.32 (s, 2H, CH2), 2.41 (s, 4H, N‐CH2); 13C NMR
(DMSO‐d6) δ/ppm: 165.7 (Cq), 160.0 (Cq), 150.7 (Cq), 145.5 (Cq), 137.6
(CH, C‐6), 134.8 (Cq), 130.3 (Cq), 129.5 (CH, Ar), 128.5 (CH, Ar), 126.5
(CH‐Ar), 114.6 (CH, Ar), 104.5 (Cq, C‐5), 66.1 (O‐CH2), 55.8 (CH2), 55.3
(OCH3), 52.5 (N‐CH2). (see Supporting information Fig. S8) HRMS:
m/z: calcd for C22H25N4O5S+: 457.1546; found 457.1529 [M+H]+.
b) N1‐(4‐chloro‐3‐nitrophenylsulfonyl)‐cytosine (3) (196 mg, 0.59
mmol), paraformaldehyde 71 mg, 2.36 mmol), morpholine (206 μL,
2.36 mmol) and acetic acid (270 μL, 4.72 mmol) were suspended in
absolute ethanol (5 mL). Reaction mixture was heated 30 minutes at
100 °C in a microwave reactor. Product 12 was isolated by thin‐layer
chromatography (eluent: 10% MeOH in CH2Cl2, yellow crystals, 155
mg, 55%).
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ARTICLE
5‐Morpholinomethyl‐N1‐(4‐(dimethylamino)azobenzene‐4’‐
Journal Name
temperature on an Oxford Diffraction Xcalibur Nova R diffractometer
sulfonylcytosine
(15):
N1‐(4‐(dimethylamino)azobenzene‐4’‐ with the microfocusing Cu tube (λ = 1.54179 Å). Data reduction and
sulfonylcytosine (6) (60 mg, 0.15 mmol), paraformaldehyde (11 mg, cell refinement were carried out using the CRYSALIS PRO software.37
0.35 mmol), morpholine (26 μL, 0.30 mmol) and acetic acid (34 μL, Structure was solved by direct methods with SIR201438 and refined
0.59 mmol) were suspended in absolute ethanol (5 mL). Reaction by a full matrix least‐squares refinement based on F2, with SHELXL39.
mixture was heated 60 minutes at 120 °C in a microwave reactor Molecular illustrations were prepared with ORTEP‐340 and
(stirring speed: 600 rpm). Product 15 was isolated by thin‐layer MERCURY41 included in the WinGX package42. Calculations of
chromatography (eluent: 10% MeOH in CH2Cl2, red crystals, 23 mg, molecular geometries and crystal packing parameters were
30%).
performed with PLATON43. Hydrogen atoms were located in the
Fourier map and refined freely. CCDC 1588667 contains the
supplementary crystallographic data. These data can be obtained
free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html,
or from the Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: (+44) 1223‐336‐033; or e‐mail:
deposit@ccdc.cam.ac.uk.
M.p. > 300 °C (dec.); Rf = 0.6 (CH2Cl2/CH3OH 9:1); UV (MeOH):
λmax/nm: 272 and 455; log ε/dm3 mol–1 cm–1: 4.03 and 4.26; IR (KBr)
ν/cm–1: 3447 (s), 1607 (m), 1520 (w), 1367 (m), 1178 (m), 1142 (m),
1117 (m), 1084 (m); 1H NMR (DMSO‐d6) δ/ppm: 8.22 (brs, 1H, NH),
8.09 (d, 2H, J = 8.7 Hz, Ar), 8.04 (s, 1H, H‐6), 7.91 (d, 2H, J = 8.7 Hz,
Ar), 7.84 (d, 2H, J = 9.2 Hz, Ar), 7.70 (brs, 1H, NH), 6.87 (d, 2H J = 9.2
Hz, Ar), 3.59 (s, 4H, O‐CH2), 3.37 (s, 2H, CH2), 3.10 (s, 6H, N(CH3)2),
2.41 (s, 4H, N‐CH2); 13C NMR (DMSO‐d6) δ/ppm: 165.7 (Cq), 155.8 (Cq),
153.4 (Cq), 142.7 (Cq), 137.4 (CH, C‐6), 136.3 (Cq), 130.2 (CH‐Ar), 125.7
(CH‐Ar), 121.8 (CH‐Ar), 111.6 (CH‐Ar), 104.6 (Cq, C‐5), 66.1 (O‐CH2),
55.7 (CH2), 52.5 (N‐CH2). (see Supporting information Fig. S9)
HRMS: m/z: calcd for C23H28N7O4S+: 498.1923; found 498.1908
[M+H]+.
Table 5. Crystallographic data
Structure
9
Brutto formula
C15H24N4O6S
388.44
Formula weight (gmol‐1)
Crystal color and habit
Crystal dimensions (mm)
Space group
colourless plate
0.04 x 0.06 x 0.2
P‐1
Byproducts:
a (Å)
b (Å)
c (Å)
α (°)
6.5612 (3)
9.2289 (4)
15.5715 (7)
95.807 (4)
94.598 (4)
98.710 (4)
922.86 (7)
2
1‐Imino‐3‐morpholinoallyl‐3‐tosylurea (9)
Rf = 0.4 (CH2Cl2/CH3OH 9:1); 1H NMR (DMSO‐d6) δ/ppm: 9.90 (bs, 1H,
NH), 9.74 (bs, 1H, NH), 8.69 (s, 1H, NH), 7.88 (d, J = 13.4 Hz, 1H, CH),
7.65 (d, J = 8.1 Hz, 2H, Ar), 7.22 (d, J = 8.0 Hz, 2H, Ar), 4.75 (d, J = 13.4
Hz, 1H, CH), 3.75–3.54 (m, 4H, O‐CH2), 3.30–3.18 (m, 4H, N‐CH2), 2.33
(s, 3H, CH3).; 13C NMR (DMSO‐d6) δ/ppm: 162.6 (Cq), 157.0 (Cq), 151.5
(CH), 142.3 (Cq), 139.8 (Cq), 128.1 (CH‐Ar), 126.6 (CH‐Ar), 80.6 (CH),
65.4 (CH2), 20.6 (CH3). (see Supporting information Fig. S10) ESI‐
MS: calcd. for C15H20N4O4S: 352.1; found [M+H]+ at m/z 353.0
and 351.0 at m/z [M‐H]‐.
β (°)
γ (°)
V (Å3)
Ζ
μ (CuKα) (mm‐1)
Absorption correction
F(000)
1.918
multi‐scan
414
75.881
 max ()
No. refl. measured
No. refl. unique
No. refl. observed [I>2σ(I)]
Rint
7824
3769
3369
0.0309
0.0489
280
0.0537
0.1531
4‐Tosylmorpholine (10)
1
Rf = 0.9 (CH2Cl2/CH3OH 9:1); H NMR (DMSO‐d6) δ/ppm: 7.68–7.58
(m, 2H, Ar), 7.53–7.43 (m, 2H, Ar), 3.72–3.54 (m, 4H, O‐CH2), 2.91–
2.78 (m, 4H, N‐CH2), 2.42 (s, 3H, CH3); 13C NMR (DMSO‐d6) δ/ppm:
143.8 (Cq), 131.5 (Cq), 129.8 (CH‐Ar), 127.6 (CH‐Ar), 65.2 (O‐CH2), 45.8
(N‐CH2), 21.0 (CH3). (see Supporting information Fig. S11) ESI‐MS:
calcd. for C11H15NO3S: 241.1; found [M+H]+ at m/z 241.9.
Rσ
Parameters
R1 [I>2σ(I)]
wR2, all
S
1.001
0.65; ‐0.32
ρmax, ρmin (eÅ‐3)
N1‐(4‐Morpholino‐3‐nitrophenylsulfonyl)cytosine (11):
Rf = 0.3 (CH2Cl2/CH3OH 9:1); 1H NMR (DMSO‐d6) δ/ppm: 8.36 (d, J =
2.4 Hz, 1H, Ar), 8.07 (d, J6,5 = 7.9, 1H, H6), 8.01 (dd, J1 = 9.1, J2 = 2.4
Hz, 1H, Ar), 7.93 (s, 2H, NH2), 7.43 (d, J = 9.2 Hz, 1H, Ar), 5.94 (d, J5,6
= 7.9 Hz, 1H, H5), 3.74–3.66 (m, 4H, O‐CH2), 3.27–3.19 (m, 4H, N‐CH2);
13C NMR (DMSO‐d6) δ/ppm: 166.0 (Cq), 151.0 (Cq), 148.4 (Cq), 139.4
(CH), 137.3 (Cq), 133.6 (CH‐C‐6), 128.8 (CH), 125.0 (Cq), 120.0 (CH),
97.3 (CH‐C‐5), 65.6 (O‐CH2), 50.2 (N‐CH2). (see Supporting
information Fig. S12) ESI‐MS: calcd. for C14H15N5O6S: 381.1;
found [M+H]+ at m/z 382.0.
Cell culturing
Human tumor cell lines U251 (glioblastoma) and MCF‐7 (breast
carcinoma) cells were cultured as monolayers, while MOLT‐4 (acute
lymphoblastic leukemia) cell line was maintained in Dulbecco's
modified Eagle medium (DMEM), supplemented with 10% fetal
bovine serum (FBS), 2mM L‐glutamine, 100 U/ml penicillin and 100
μg/ml streptomycin in a humidified atmosphere with 5% CO2 at 37˚C.
Proliferation assays
Crystalographic data
The cells were inoculated onto a series of standard 96‐well
microtiter plates on day 0, at 1×104 to 3×104 cells/ml, depending on
the doubling times of specific cell line. Test agents were then added
Crystal data, data collection and refinement parameters are
summarized in Table 5. Data collection was performed at room
8 | J. Name., 2012, 00, 1‐3
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in five 10‐fold dilutions (10‐8 to 10‐4 M) and incubated for a further
72 hours. Working dilutions were freshly prepared on the day of
testing. After 72 hours of incubation the cell growth rate was
evaluated by performing the MTT assay, as described previously.11,44
Each test point was performed in quadruplicate in at least two
individual experiments. The results were expressed as IC50, a
concentration necessary for 50% of inhibition. Each result is a mean
value from at least two separate experiments.
3
4
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9
Fast and efficient synthetic method for the synthesis of 5‐
morpholinomethyl‐N1‐sulfonylcytosine derivatives has been
established. N1‐sulfonylcytosine derivatives 3–6 were prepared
by the condensation of pyrimidine bases with different sulfonyl
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chlorides, and then transformed to C5‐substituted N1‐
sulfonylcytosines (8, 12‐15) in moderate to very good yield (up
to 65%). Microwave assisted reaction significantly shortened
the reaction time and favored formation of Mannich product
over numerous possible byproducts. Shortened reaction time
adequately addressed the problem of N‐SO2 bond instability.
Moreover, it has been found that temperature controls the
reaction pathway. As a result of the cytosine ring opening,
13 M. Pavlak, R. Stojković, M. Radačić‐Aumiler, J. Kašnar‐
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interesting tosylurea derivative
reaction at lower temperature (80
(100 C and 120 C), undergoes acid catalyzed cyclization to
form starting TsC and participates in the Mannich reaction. In
9
has been isolated from the
Kraljević Pavelić, S. Raić‐Malić, ACS Med. Chem. Lett., 2015,
1150–1155.
6

C). At higher temperatures
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

9
2
this sense, elevated temperatures promote formation of the
Mannich product. Presented method constitutes a robust tool
for introduction of new pharmacophores, such as cyclic amine
morpholine, into biologically active pyrimidines. Two of the
newly synthesized compounds, 13 and 14, showed a very strong
(micromolar) antiproliferative activity on U251, MCF7 and
MOLT‐4 tumor cell lines. In addition, scope of this method could
be expanded to various amine, as well as pyrimidine derivatives,
and this issue will be studied furtherly.
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The authors declare the following competing financial
interest(s): Marijeta Kralj and Biserka Žinić are minority
shareholders in BioZyne Ltd. The other authors declare no
competing interests.
23 A. Andreani, M. Granaiola, A. Leoni, A. Locatelli, R. Morigi, M.
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Acknowledgements
This work was supported by the Croatian Ministry of
Science, Education and Sport through grant no. 098‐0982914‐
2935 and the Rudjer Boskovic Institute's spin‐off company
BioZyne Ltd. Financial support from the Croatian Science
Foundation (grant no. HRZZ‐1477) is gratefully acknowledged.
26 R. Cincinelli, V. Zwick, L. Musso, V. Zuco, M. De Cesare, F.
Zunino, C. Simoes‐Pires, A. Nurisso, G. Giannini, M. Cuendet
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A fast and efficient route for introduction of morpholinomethyl moiety in the C5 position of the
sulfonylated cytosine nucleobase has been developed.