Effect of substitution at N″-position of N′-hydroxy-N-amino guanidines on tumor cell growth

Article abstract of DOI:10.1016/j.bmcl.2012.06.048

Structural modification of one of our earlier reported lead molecule (ABNM13) has been carried out to study the effect of different substituents at the N″-position of N-hydroxy-N′-amino guanidines (HAGs) on their anticancer activity. Compounds with electron donating substituents were found to be less active. In contrast, those with electron withdrawing groups were found favorable for anticancer activity. The obtained results provide significant SAR information that may be useful for further drug designing with HAGs.

Full text of DOI:10.1016/j.bmcl.2012.06.048

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4934–4938
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Effect of substitution at N00-position of N0-hydroxy-N-amino guanidines on
tumor cell growth
Arijit Basu a, , Barij Nayan Sinha a, Philipp Saiko b, Thomas Szekeres b
⇑
a Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi 835215 , India
b Department of Medical and Chemical Laboratory Diagnostics, Vienna General Hospital, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 January 2012
Revised 9 June 2012
Accepted 15 June 2012
Available online 21 June 2012
Structural modification of one of our earlier reported lead molecule (ABNM13) has been carried out to
study the effect of different substituents at the N00-position of N-hydroxy-N0-amino guanidines (HAGs)
on their anticancer activity. Compounds with electron donating substituents were found to be less active.
In contrast, those with electron withdrawing groups were found favorable for anticancer activity. The
obtained results provide significant SAR information that may be useful for further drug designing with
HAGs.
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Hydroxyguanidine
Ribonucleotide reductase
Lead modification
Hydroxyaminoguanidines (HAGs),1–5 have been reported as
anticancer agents, primarily against L1210 murine, and HL60 hu-
man leukemia cell lines. They were found to inhibit DNA synthesis
by inhibiting the R2 subunit of the enzyme Ribonucleotide reduc-
tase (RR).6,7 Earlier, we have identified5,8 an anticancer lead mole-
(b) Reasons for precluding structure based designing:
HAGs inhibit the M2 subunit of the enzyme RR. Mode of action
of most of these analogs have been deduced by cell line assays, dif-
ferent spectroscopic studies, and enzyme assays. No crystallo-
graphic information of the ligand bound M2 subunit has been
reported till date. In absence of these information, structure based
drug designing on this target has been a serious concern, and
therefore cannot be undertaken for the current work.
We have resorted to a classical drug designing approach. A few
analogs with both electron donating and withdrawing substituents
were synthesized, and tested for their anticancer activity. For the
current work, we have used a synthetic modification strategy on
our earlier developed lead molecule (Fig. 1). The synthesis of the
compounds are presented in Schemes 1 and 2.
cule (ABNM13) with IC50 (HL60 cell line) of 11 lM, CC50 >100 lM
that is a selectivity index of more than ten. It is a potent inhibitor
of RR, and an arabinofuranosylcytosine (Ara-C) synergist. In our
earlier studies we have also emphasized the need for its further
structural modifications. ABNM13 has been identified through a
virtual screening (VS) experiment, by screening an in-house ligand
library, on a developed pharmacophore based QSAR model. To fur-
ther expand these studies, we have explored different structural
modifications of ABNM13, on anticancer activity.
Lead modification process often involves the application of
structure or/and ligand based drug designing. We could not apply
either, due to the following reasons.
We report a method for synthesizing N00 substituted HAGs. The
first step involved the synthesis of methyl dithiocarbazinate (1),
which was prepared as per our earlier reported methodology.9–11
Synthesis of compound 2 is straight forward nucleophilic addition
between the amine and the aldehyde, with good yield of around
85%. The third step in the synthesis involved nucleophilic attack
by different amines on –C@S, carbon of compound 2. We used ex-
cess amine to drive the reaction in forward direction. Excess amine,
and the use of high temperature (>120 °C) favors the hydrolysis of
compound 2 to corresponding aldehyde, which was monitored by
TLC co-spotting with anthraldehyde. This unavoidable side reac-
tion makes the purification difficult for compounds 3–10, and leads
to poor yield. We managed to purify the crude product by using
automated flash chromatography (BUCHI Sepacore, Switzerland),
and employing a gradient elution technique.
(a) Reasons for precluding the already developed QSAR model for
further lead design: ABNM13 has been identified by pharma-
cophore based virtual screening of an in-house library. The
training set for developing such a model consisted of differ-
ent HAGs with aromatic substitutions at the N0-position.
Therefore, the developed model revealed designing informa-
tion pertaining only to this position, and nowhere else.
Therefore, our earlier developed model will be out of scope
for predicting newer compounds with substitution at differ-
ent positions (Fig. 1).
⇑
Corresponding author.
E-mail address: arijit4uin@gmail.com (A. Basu).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.048

A. Basu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4934–4938
4935
observed if we compare compounds 12 & 17. It indicates a clear
influence of aromatic substituent towards biological activity. It
might be an interesting strategy in future, to explore different aro-
matic substitutions at this position. All the other compounds with
aliphatic side chain were found to be much less active.
We also report the development of another series of compounds
with electron withdrawing substituents at N00-position. Three com-
pounds 23–25 (Table1) with the desired properties were synthe-
sized. The synthetic procedure is outlined in Scheme 2. The
method of synthesis is different from those for compounds
11–18. The first step involves the formation of Schiff’s base of thi-
osemicarbazide, and 9-anthraldehyde (19). The next step involves
the amide coupling reaction of different carboxylic acids with com-
pound 19, in the presence of DCC/DMAP, to synthesize compounds
23–25. Final two steps involve S-methylation followed by nucleo-
philic substitution with hydroxyl amine. These two steps are sim-
ilar to that for compounds 11–18. Compounds of the second series,
23–25 were found similar in activity to our earlier reported lead
molecule ABNM13. It indicated that we are moving in right
direction.
H
N
H
N
H
N
NH
N
N
R
We pointed out that the pKa values14 of synthesized compounds
have an influence on activity. Lower pKa value (increase in acidity
due to –NHOH group) favors the biological activity (Fig. 2).
We tried to explain this correlation by reviewing a few earlier
studies,15–17 and subsequently framing a plausible hypothesis.
The M2 subunit of RR generates the free radical that reduces ribo-
nucleotides to their corresponding deoxyribonucleotides. This
reduction occurs via a single electron transfer from TYR176. The
free radical on TYR176 is initiated by the radical-generating, diiro-
n(III/IV) state via a single electron transfer, through a water mole-
cule forming a bridge between the hydroxyl oxygen atom of
TYR176, and the iron center. The radical scavengers interact with
TYR176, replacing/disturbing the water bridge. Therefore, more
acidic (lesser pKa) –NHOH containing compounds may disrupt
the water bridge in a better manner than their lesser acidic ana-
logs. However, we need to support this hypothesis with further
experimental support before we come down to any concrete
conclusion(s). All our synthesized compounds in the first series
(11–18) are alkyl derivatives, which are electron donating groups.
Therefore, when these compounds dissociates (–NHOH/@NOH dis-
sociates to corresponding anion –NHOꢀ/@NOꢀ) the anion is less
stable than that of ABNM13 with unsubstituted N00-position. More
stable anion means more ionization, and consequently more acid-
ity (lower pKa).With aromatic substitutions the anion produced is
more stable than their aliphatic counterparts, which counts for
lower pKa values for compounds 15 & 16. On the other hand, the
NHOH
NOH
ABNM13
(Earlier developed lead)
Current synthetic modification
Figure 1. Lead modification strategy for the current work.
The intermediate step to synthesis of compounds 11–18 was
achieved by the reaction of excess methyl iodide with compounds
3–10. Finally, S-Me group was substituted with –NHOH group by
reaction with hydroxylamine to obtain compounds 11–18. Hydro-
lysis to corresponding aldehyde was observed, but not as drastic as
observed during synthesis of compounds 3–10, possibly due to low
reaction temperature. Physicochemical and spectral data for these
compounds are given in Table 1.
The synthesized compounds 11–18, were screened against HL-
60 human promyelocytic leukemia cell line.12,13 Biological activi-
ties of the compounds are presented in Table 2.
We explored a range of substitutions, primarily aliphatic side
chains, starting from smaller methyl group to bulkier phenyl ethyl
group. The SAR analysis reveals, presence of aromatic ring in the N00
(compounds 16 and 17) position is favorable for biological activity
as compared with only aliphatic side chains. If we compare 11 &
16, compound 11 with IC50 of 32 lM is twice less active than com-
pound 16 with benzyl substitution. Similar correlation can also be
H
S
H
ii.
i.
N
S
O
H2NHN
N
NH2NH2 CS2
SCH3
SCH3
1
2
iii.
H
H
N
N
NHOH
S
N
iv
N
N
NH
R
R
11-18
3-10
Scheme 1. Reagents and conditions: (i) KOH, <10 °C, stirring for 1–1.5 h; CH3I, <10 °C, stirring for 2 h; (ii) dry EtOH, reflux, 2 h; (iii) DMF, K2CO3, aliphatic amines; (iv) in two
steps (a) CH3I, MeOH, reflux, 6 h, (b) hydroxyl amine, MeOH, rt, stirring, 12 h.

4936
A. Basu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4934–4938
iii.
i.
ii.
N
N
H
N
H
N
N
O
HN
O
HN
O
HN
NH2
NOH R
S
R
S
23-25
20-22
19
Scheme 2. Reagents and conditions: (i) thiosemicarbazide, EtOH, reflux 2 h; (ii) RCOOH, DCC, DMAP, stirring rt for 12–14 h; (iii) in two steps (a) CH3I, DMF, reflux, 2 h, (b)
hydroxyl amine, DMF/MeOH, rt, stirring, 12 h.
Table 1
Structures, physicochemical and spectral data (1H NMR and FAB-MS) for compounds 11–18 and 23–25
NH
N
N
NHOH
R
11-18 & 23-25
Code
R
Yield
(%)
Mp
(°C)
Spectral data
11
12
–CH3
–C2H5
65
65
195.1 MS-FAB (m/z): [M+H]+ 294,1H NMR (300 MHz, DMSO-d6) d 11.41 (s, 1H), d 7.5–8.25 (m,9H), d 6.31 (s, 1H), d 2.6 (s, 3H).
196.8 MS-FAB (m/z): [M+H]+ 308, 1H NMR (300 MHz, DMSO-d6) d 11.83 (s, 1H), d 7.5–9.27 (m,10H), d 6.48 (s, 1H), d 6.21 (s, 1H), d
5.76 (s, 1H), d 2.13(m,2H), d 1.27 (t, 3H).
13
14
15
16
17
18
–C3H7
63
70
62
69
73
75
179.2 MS-FAB (m/z): [M+H]+ 306, 1H NMR (300 MHz, DMSO-d6) d 11.8 (s, 1H), d 7.5–8.27 (m, 5H), d 6.89 (s, 1H), d 5.89 (s, 1H), d 1.04
(s, 3H).
–iC3H7
181.9 MS-FAB (m/z): [M+H]+ , 306,1HNMR (300 MHz, DMSO-d6) 11.37 (s, 1H), d 7.91–9.21 (m, 9H), d 9.81 (s, 1H), d 3.57–3.61 (m,
1H), d 1.22 (s, 6H).
Cyclohexyl
–CH2 Ph
–CH2CH2Ph
–C4H10
202.6 MS-FAB (m/z): [M+H]+,361, 1HNMR (300 MHz, CDCl3, DMSO-d6) d 11.76 (s, 1H) 7.54–9.21 (m, 10H), 3.54–3.56 (d, 3H), 1.63–
1.44 (m, 1H), 1.30–1.33 (m, 2H), 1.57(t, 2H) 0.93 (t, J = 7.5 Hz, 1H).
223.9 MS-FAB (m/z): [M+H]+ 319, 1H NMR (300 MHz, DMSO-d6) d 11.68 (s, 1H), 7.56–9.28 (m, 12H), 3.54–3.56 (d, 3H), 1.63–1.44 (m,
1H), 1.30–1.33 (m,2H),1.57(t,2H) 0.93 (t, J = 7.5 Hz, 1H).
241.1 MS-FAB (m/z): [M+H]+ 384,1H NMR(300 MHz,DMSO-d6, d 9.22(s,1H), 6.99–8.68 (m,12H), d 3.95 (t, 2H), 3.76 (t, 2H), 3.13 (s,
1H).
178.9 MS-FAB (m/z): [M+H]+, 319, 1H NMR (400 MHz, DMSO-d6) d 11.68 (s, 1H),7.56–9.28 (m,12H) d 3.62–3.51 (m, 2H), 3.08 (s, 1H),
1.62–1.49 (m, 2H), 1.45–1.34 (m, 2H), 1.07 (s, 3H).
23
24
–COCH3
–CO(4-
55
46
184.6 FAB-MS, 321 (M+1), 1H NMR (300 MHz, DMSO) d 8.49 (s, 1H), d 7.55–8.02 (m,9H), d 4.59 (s, 1H), d 4.59 (s, 1H) d 2.16 (s, 3H).
176.8 FAB-MS, 428(M+1), 1H NMR (300 MHz, DMSO) d 10.13 (s, 1H), 8.46–7.40 (m, 14H), 4.46 (s, 1H), 3.05 (s, 1H).
nitro)Ph
–CO(3,5-
dinitro)Ph
25
50
194.5 FAB-MS, 473(M+1), 1H NMR (300 MHz, DMSO) d 9.76 (s, 1H), 8.98 (s, 1H), 8.91 (s, 1H), 8.59 (s, 1H), 8.33–7.17 (m, 10H), 4.55 (s,
1H), 3.03 (s, 1H).
second series compounds (23–25) with electron withdrawing sub-
stituents are significantly more acidic (lower pKa).
2 h with stirring. After addition, stirring was continued for an addi-
tional 1 h. The white precipitate obtained was filtered and washed
with ice cooled water. The crude product was recrystallized from
dichloromethane. Yield: 93%, mp 90.5 °C (lit.9 mp 90–92 °C).
Methyl dithiocarbazinate (0.02 M) was dissolved in 20 mL of
ethanol and an equimolar amount (0.02 M) of anthraldehyde was
added to it. The mixture was refluxed for 2 h on steam bath and
was monitored by TLC for the completion of reaction. The crude
compound is purified by recrystallization from EtOH. Yield: 85 %,
mp 198 °C.
We conclude, electron donating substituent at N00-position is
unfavorable for anticancer activity, and must be avoided in future
designing. On the other hand we found the effect of electron with-
drawing substituents is not detrimental. This significant SAR infor-
mation may serve as a guideline for future inhibitor design on this
target.
The detailed procedure for synthesis, and biological evaluation
of the compounds 11–18 are given as follows.
To an ice cooled solution (<10 °C) of 19.8 g (0.3 M) of potassium
hydroxide in 24 mL of water and 20 mL isopropanol was added
17.1 mL of 80% pure hydrazine hydrate and constantly stirred.
18.2 mL (0.3 M) of ice cooled carbon disulfide was added drop wise
to the stirred solution, maintained at <10 °C for about 1–1.5 h.
The bright yellow mixture formed was stirred for an additional
1 h. Ice cooled iodomethane solution (18.7 mL, 0.3 M) was added
dropwise to the bright yellow mixture formed over a period of
To a solution of compound 2 (0.01 M) in DMF anhydrous potas-
sium carbonate was added (250 mg/mL) and stirred for 15 min.
Different aliphatic amines (0.1 M) were added; the reaction mix-
ture was stirred, and gradually refluxed at 120 °C for 3–6 h, under
nitrogen atmosphere. For amines of low boiling point, we em-
ployed, coiled condenser with ice cold water circulation ꢁ4 °C.
For compounds 3 & 4 reactions occurred at 65 °C, stirring for 6 h.
The reactions were monitored using lead acetate paper, which

A. Basu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4934–4938
4937
Hydroxyl amine (0.1 M) solution in cold MeOH was added drop
wise over a period of 30 min to a methanolic suspension of com-
pounds from previous step while stirring. Stirring was further con-
tinued for 12 h. Completion of reaction was monitored by TLC
using 50% EtOAc:Hexane as solvent system and evolution of
methyl mercaptan on lead acetate paper.
Table 2
Growth inhibition of human HL-60 promyelocytic leu-
kemia cells after incubation with 11–18, and 23–25 for
72 h
Code
IC50 (lM)
Thiosemicarbazide (0.5 M), and different anthraldehyde (0.5 M)
were refluxed for 3 h to obtain compound 19. The resulting com-
pound was re-crystallized from 95% ethanol, mp 235 °C, yield 90%.
DCC (0.6 M), DMAP (0.5 M), and different carboxylic acids
(0.5 M) were dissolved in dry DCM. To it 0.5 M of compound 19
in dry DCM was added dropwise for 2 h through a pressure equal-
izing dropping funnel while stirring the reaction mixture. The reac-
tion mixture was further stirred for 12–14 h. The progress of the
reaction was monitored on TLC (5% MeOH:DCM). After completion
of the reaction, it was quenched 10% citric acid solution (to remove
dicyclohexyl urea formed). The resulting suspension was extracted
thrice with sufficient amount DCM. The DCM layer was concen-
trated to dryness to get the crude product.
11
12
13
14
15
16
17
18
23
24
25
32.9 1.30
22.6 0.37
24.9 2.17
29.8 2.11
21.1 0.51
19.3 0.38
19.6 1.79
21.1 1.15
20.0 2.74
10.0 0.40
8.0 1.00
34
32
30
28
26
24
22
20
18
16
14
12
10
8
Compounds 20–22, were further purified by flash chromatogra-
phy using different gradients of DCM and methanol.
adj R2 =0.672
For synthesis of S-methyl derivatives, compounds 20–24
(0.01 M) were dissolved in sufficient dry DMF. Approximately,
10 M excess of iodomethane was added. The mixture was stirred,
and gradually refluxed to 60 °C for 2 h. Coiled condenser with ice
cold water circulation ꢁ4 °C was employed. Completion of the
reactions were confirmed by TLC (50% EtOAc:Hexane) till no spots
for the starting compounds were observed. After completion of the
reaction excess iodomethane was distilled off, and the reaction was
quenched with brine solution, and extracted several times with
ethyl acetate. The organic layer was evaporated to dryness, and
the resulting solid was used in the next step without further
purification.
The solid obtained was dissolved in DMF, to it excess hydroxyl
amine hydrochloride (0.1 M), potassium hydroxide (0.1 M) and
5 mL MeOH was added, and stirred vigorously. Stirring was further
continued for 12 h. Completion of reaction was monitored by TLC
using 5% MeOH:DCM as solvent system. Compounds 23–25, were
purified by flash chromatography using different gradient of
MeOH:DCM.
6
6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0
Pka
Figure 2. Plot of biological activity (IC50 in
predicted pKa values.
lM) of compounds 11–18 with
darkened to brown/black color due to evolution of methyl mercap-
tan. The reaction was continued till the evolution of methyl mer-
captan. Completion of the reactions were also confirmed by TLC
(50% EtOAc:Hexane) till no spots for compound 2 were observed.
The reactions were quenched with brine, extracted thrice with
10 mL EtOAc, and dried with anhydrous Na2SO4. For all the cases
the crude product yielded in three spots on TLC, under UV light
254/365 nm and iodine vapors. We employed 10% H2SO4:MeOH
charring to judge the relative percentage of all the three spots.
We found the middle spot was most prominent amongst all, and
targeted the same for further purification. Compounds 3–10 were
purified by employing 100–200 silica gel as stationary phase and
employing different gradient of EtOAc:Hexane starting from 5%
to 20% to obtain the pure compounds. The pure compounds ap-
peared as bright yellow to yellowish brown crystals. Physicochem-
ical and spectral data are given in Supplementary data.
The human HL-60 promyelocytic leukemia cell line was pur-
chased from ATCC (American Type Culture Collection, Manassas,
VA, USA). Cells were grown in RPMI 1640 medium supplemented
with 10% heat inactivated fetal calf serum (FCS), 1% L-glutamine,
and 1% penicillin–streptomycin at 37 °C in a humidified atmo-
sphere containing 5% CO2 using a Heraeus cytoperm 2 incubator
(Heraeus, Vienna, Austria). All media and supplements were ob-
tained from Life Technologies (Paisley, Scotland, UK). Cell counts
were determined using a microcellcounter CC-110 (SYSMEX, Kobe,
Japan). Cells growing in the logarithmic phase of growth were used
for the experiment described below.
HL-60 cells (0.1 ꢂ 106/mL) were seeded in 25 cm2 Nunc tissue
culture flasks and incubated with increasing concentrations of
compounds at 37 °C under cell culture conditions. Cell counts
and IC50 values (IC50 = 50% growth inhibition of tumor cells) were
determined after 72 h using a microcellcounter CC-110. Viability
of cells was determined by staining with trypan blue. Results were
calculated as number of viable cells. Data are means SD of at least
three experiments.
Compounds 3–10 (0.01 M) thus obtained were dissolved/sus-
pended in MeOH, and stirred vigorously for 10 min, and gradually
raising the temp to 50 °C. Excess iodomethane (10 M equiv) was
added drop wise for a period of 30 min, while stirring vigorously.
The reaction mixture was refluxed at 60 °C for 6 h using ice-cold
water circulator ꢁ4 °C, employing
a coiled condenser. After
Acknowledgments
2–3 h, precipitate starts appearing. The reaction was monitored
on TLC 50% EtOAc:Hexane. After completion the reaction mixture
was cooled, the precipitate was filtered. Washed with hexane
and finally with cold MeOH. These compounds were used without
further purification in the next step.
We are thankful to University Grants Commission, India for the
financial assistance. We are also thankful to Dr. Ashoke Sharon, Dr.
Venkatesan J. and Mrs. Nibha Mishra for providing important
inputs for the current work. AB is also thankful to his students

4938
A. Basu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4934–4938
8. Saiko, P.; Graser, G.; Giessrigl, B.; Lackner, A.; Grusch, M.; Krupitza, G.; Basu, A.;
Sinha, B. N.; Jayaprakash, V.; Jaeger, W.; Fritzer-Szekeres, M.; Szekeres, T.
Biochem. Pharmacol. 2011, 81, 50.
Jagganath, Saroj, Vibhor and Dheeraj for their help during the syn-
thesis work.
9. Chetan, B.; Bunha, M.; Jagrat, M.; Sinha, B. N.; Saiko, P.; Graser, G.; Szekeres, T.;
Raman, G.; Rajendran, P.; Moorthy, D.; Basu, A.; Jayaprakash, V. Bioorg. Med.
Chem. Lett. 2010, 20, 3906.
Supplementary data
10. Krishnan, K.; Prathiba, K.; Jayaprakash, V.; Basu, A.; Mishra, N.; Zhou, B.; Hu, S.;
Yen, Y. Bioorg. Med. Chem. Lett. 2008, 18, 6248.
11. Kulandaivelu, U.; Padmini, V.; Suneetha, K.; Shireesha, B.; Vidyasagar, J.; Rao, T.
KN, J.; Basu, A.; Jayaprakash, V Arch. Pharm. 2011, 2, 84.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.06.
048.
12. Madlener, S.; Illmer, C.; Horvath, Z.; Saiko, P.; Losert, A.; Herbacek, I.; Grusch,
M.; Elford, H. L.; Krupitza, G.; Bernhaus, A.; Fritzer-Szekeres, M.; Szekeres, T.
Cancer Lett. 2007, 245, 156.
13. Saiko, P.; Ozsvar-Kozma, M.; Bernhaus, A.; Jaschke, M.; Graser, G.; Lackner, A.;
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14. Predicted pKa values were calculated using Epik, version 2.2, Schrödinger, LLC,
New York, NY, 2011. All the structures were set to ionize at pH 7.0 and
estimated the sequential pKa using maestro molecular modeling environment.
15. Himo, F.; Siegbahn, P. E. M. Chem. Rev. 2003, 103, 2421.
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