Medicinal Chemistry, 2009, 5, 549-557 549
1573-4064/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.
Synthesis, Cytotoxic Evaluation and In Silico Pharmacokinetic Prediction
of Some Benzo[a]Phenazine-5-sulfonic acid Derivatives
N.S. Hari Narayana Moorthy*, C. Karthikeyan and Piyush Trivedi
School of Pharmaceutical Sciences, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Airport Bypass Road, Gandhi Nagar,
Bhopal – 462036, Madhya Pradesh, India
Abstract: Cancer is one of the life threatening diseases and the development of novel anticancer molecules is limited by
many reasons. In the present investigation, some novel benzo[a]phenazine-5-sulfonic acid derivatives as DNA intercalator
was designed with optimized pharmacokinetic features for cancer treatment. The compounds with desired pharmcokinetic
profile were synthesized and structurally characterized. Cytotoxic activity study against HL-60 tumor cell lines shows that
10-dimethyl carboxamido derivative of benzo[a]phenazine-5-sulfonic acid is found to be the most active in the series with
cytotoxic activity (IC50 = 19 μM) comparable to cisplatin (IC50 = 7 μM). The study concluded that the novel
benzo[a]phenazine-5-sulfonic acid derivatives were found to have enhanced DNA binding affinity and exhibited signifi-
cant activity in vitro against HL-60 cell lines. This work will also guide for further development of effective DNA interca-
lators for cancer treatment.
Keywords: Benzo[a]phenazine-5-sulfonic acid, cytotoxic activity, human leukemia, in silico pharmacokinetic.
INTRODUCTION
Cancer is a leading cause of death worldwide: it ac-
counted for 7.9 million deaths (around 13% of all deaths) in
2007. World Health Organization (WHO) estimated that
about 72% of all cancer deaths in 2007 occurred in low- and
middle-income countries and deaths from cancer worldwide
are projected to continue rising, with an estimated 12 million
deaths in 2030 [1-4]. In the past 20 years, there has been a
tremendous increase in our knowledge of the molecular
mechanisms and pathophysiology of cancer. Many of these
mechanisms have been exploited as new target for drug de-
velopment in the hope that they will have greater antitumor
activity with less toxicity to the patients than is seen with
currently used medicines. The development of anticancer
drug molecule must overcome the failure rate of poor absorp-
tion, distribution, metabolism, excretion (ADME) and toxic-
ity (T) properties. Clinical failure of about 60% of the Inves-
tigational New Drug (IND) filing is attributed to their inade-
quate ADMET attributes. Among that 41% of compounds
fail in drug development because of poor ADME and 22% of
compounds fail in drug development because of toxicity
[5,6]. In silico pharmacokinetic and pharmacodynamic mod-
eling, combined with their speed, versatility and cost effec-
tiveness will see them making vital contributions as they are
integrated in to the drug development process [7].
This literature survey shows that over the past many
years, the search for new cytotoxic intercalators has mainly
followed classical approaches: structural modifications to
conventional molecules, new natural products and modifica-
tions of them, potential synthetic compounds and chemical
conjugates.
*Address correspondence to this author at the School of Pharmaceutical
Sciences, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Airport Bypass
Road, Gandhi Nagar, Bhopal – 462036, Madhya Pradesh, India; Tel: 0755-
2678883; Fax: 0755-2742006;
E-mails: nshnm06@yahoo.co.in; nshnm@yahoo.co.in
In the present work, it is attempted to design and synthe-
sis of some anticancer molecules related to some successful
clinical DNA intercalators such as acridine-4-carboxamide
(DACA), Intoplicine, XR11576. The work is based on the
hypothesis that modification of DNA intercalators nucleus
with the qualitative structural analysis of proven cytotoxic
intercalators and synthesis of new compounds based on gen-
eral structural intercalator characteristics may yield better
intercalators with enhanced affinity for DNA and without
losing cytotoxic effect [8-13].
The selection of benzo[a]phenazine-5-sulfonic acid as a
basic DNA intercalating polycyclic pharmacophore is based
on the following rationale.
• Presence of flat aromatic ring systems capable of inser-
tion in between DNA base pairs. Furthermore, presence
of multiple nitrogen atoms imparts greater degree of pla-
narity to the nucleus and reduces the carcinogenicity and
toxicity related problems associated with non nitrogen
polycyclic systems.
• Experimentally proven superior DNA binding affinity
and structural resemblance to anticancer acridines [11-
14].
The results are described in this communication.
CHEMISTRY
The benzo[a]phenazine-5-sulfonic acid derivatives were
synthesized by reaction between substituted napthylenedione
derivative and o-phenylenediamine derivatives in the pres-
ence of acid at high temperature. The process yielded an
intermediate of corresponding imines. The formed napthyl-
ene imines were converted in to benzo[a]phenazine-5-
sulfonic acid in the presence of acid [13]. The derivatives
were prepared as per the Scheme 1. Nitration of
benzo[a]phenazine-5-sulfonic acid yielded 3 and 4-nitro de-
rivatives in 1:4 ratio, which was separated by column chro-
550 Medicinal Chemistry, 2009, Vol. 5, No. 6 Moorthy et al.
matography. The carboxylic acid derivatives were treated
with dimethylamine in the presence of N, N-carbonyl diimi-
dazole (CDI) gave the amide derivatives.
EXPERIMENTAL
Reagents, starting materials and solvents were purchased
from common commercial suppliers (CDH, S.D.Fine, Loba,
E. Merck, Lancaster, etc). The melting points were deter-
mined in open capillary tubes on a Jindal melting point appa-
ratus and are reported uncorrected. H1 NMR spectra were
recorded on the Bruker Advance DRX-2000 (300 MHz, FT
NMR) spectrometer in DMSO and CDCl3, TMS (tetramethyl
silane) as an internal standard. The FAB mass spectra were
recorded on a JEOL SX 102/DA-6000 Mass Spectrome-
ter/Data System using Argon/Xenon (6KV, 10mA) as the
FAB gas. The accelerating voltage was 10 KV and the spec-
tra were recorded at room temperature. m-nitrobenzyl alco-
hol (NBA) was used as the matrix. IR absorption spectra
were recorded on Jasco FT/IR - 470 PLUS, using KBr by
diffuse reflectance method. UV-absorption studies were done
on Shimadzu Pharmaspec-UV 1700 spectrophotometer to
find out �max of the compounds. The elemental analysis
(CHN analysis) was done on a Perkin Elmer CHN rapid ana-
lyzer. All the compounds gave satisfactory analysis within ±
0.4% of the theoretical values. The purity and the reactions
progress were monitored by thin layer chromatography using
silica gel-G on glass plate as stationary phase and different
polarity of solvents as mobile phase. Visualization was ac-
complished with UV light and/or iodine vapour.
Synthesis of Benzo[a]phenazine-5-Sulfonic Acid (1) [13]
A mixture of 2.38 g (10 mmol) 1,2 napthoquinone-4-
sulfonic acid, 4.32 g (40 mmol) o-phenylenediamine and 25
mL conc. HCl was heated to reflux in 25 mL ethanol for 7 h.
Then the reaction mixture was cooled overnight. The precipi-
tate was collected by filtration and washed with water and
ether to yield reddish brown precipitate. The crude product
obtained was recrystallized with methanol. Thin layer chro-
matography was performed with isopropanol:ethyl acetate
(4:6).
Synthesis of 4 and 3-Nitro Benzo[a]phenazine-5-Sulfonic
Acid (2)
A mixture of 5 mL conc. sulfuric acid and 5 mL conc.
nitric acid were mixed together at 0oC. To this mixture,
benzo[a]phenazine-5-sulfonic acid (10 mmol) was added and
the reaction mixture was allowed to warm slowly till room
temperature for 3 h. After refrigeration for 24 h, the reaction
mixture was poured on to water yielding a yellow precipitate
of 4-nitro and 3-nitro benzo[a]phenazine-5-sulfonic acid
derivatives in the ratio 4:1. The crude product containing the
two isomers was subjected to column chromatography on
silica gel using isopropanol: ethyl acetate (4:6) as an eluting
solvent. Components in collected fractions were analyzed by
TLC and similar fractions containing the compound were
pooled and the solvent was evaporated to give the desired
compound.
A
H2N
H2N
R 1
O
O
SO3H
R 1
N
NHO3S
1
3 ( R1= 9-NO 2)
6 (R1 =1 0- COOH)
N
NHO3S
O2N
N
NHO3S
NH2
N
NHO3S
C ON(CH3)2
N
NHO3S
H2N
N
NHO3S
O2N
COOH
+
1
2
3
4
5
6 7 8
9
10
1112
1, 2 Napthoquinone
4-Sulf onic Aci d
o-Phenyl enedia mine
or
3, 4-diam inobenzoi c a cid
or
4-Nit ro 1, 2 di amino benze ne
Be nz o[a]phe na zine-5-sulfoni c aci d
de rivat ives
2
4
E
5
7
8
B
C
D E
Scheme 1. Synthesis of benzo[a]phenazine-5-sulfonic acid derivatives.
A : Conc. HCl/formic acid/acetic acid, Ethanol, reflux for 7 hr., B & D: conc. H2SO4, Conc. HNO3, 0
oC, 24 hr at room temperature, C: CDI,
Dry DMF, stir at room temp for 4 hr. DCM, MgSO4, E: Ethanol, tin, NH4Cl, reflux, DCM.
Synthesis, Cytotoxic Evaluation and In Silico Pharmacokinetic Prediction Medicinal Chemistry, 2009, Vol. 5, No. 6 551
Synthesis 9-Nitro Benzo[a]phenazine-5-Sulfonic Acid (3)
A mixture of 1,2 napthoquinone-4-sulfonic acid (10
mmol), 4-nitrobenzene-1,2-diamine (40 mmol) and conc.
HCl (25 mL) was heated to reflux in ethanol (25 mL) for 7 h.
Then the reaction mixture was cooled overnight. The precipi-
tate was collected by filtration and washed with water and
ether to yield pale yellow precipitate. The crude product ob-
tained was recrystallized with methanol. Thin layer chroma-
tography was performed with isopropanol:ethyl acetate (4:6).
Synthesis of 4 and 9-Amino Benzo[a]phenazine-5-
Sulfonic Acid (4 and 5)
To a mixture of 10 mmol (3.5 g) of 4 or 9-nitro
benzo[a]phenazine-5-sulfonic acid, 2 mL of DMF and 5 mL
ethanol, was added ammonium chloride (3 mL) and 50 mg of
tin. The reaction mixture was heated to reflux for 5-7 h, then
cooled and filtered through a bed of celite. The filtrate was
diluted with water in to dichloromethane and dried magne-
sium sulfate. The solvent was removed in vacuum to yield
the compound. The product obtained was recrystallized with
methanol for 4-amino and DMSO for 9-amino benzo[a]
phenazine-5-sulfonic acid derivatives. TLC was monitored
with acetone: methanol (3:7) for 4-amino compound and
DMSO:acetic acid:water (5:2:3).
Synthesis of 10-Carboxy Benzo[a]phenazine-5-Sulfonic
Acid (6)
A mixture of 2.38 g (10 mmol) 1,2 napthoquinone-5-
sulfonic acid, 4.56 g (30 mmol) diamino benzoic acid, 10 mL
acetic acid was heated to reflux in 30 mL ethanol for 8 h. the
precipitate was collected by filtration and washed with dilute
ethanol and ether to yield brown colour crude product. The
obtained product was recrystallized with DMF to yield the
pure product. TLC was prepared with DMSO:methanol
(5:5).
Synthesis of 4-Nitro-10-Carboxy Benzo[a]phenazine-5-
Sulfonic Acid (7)
5 mL conc. sulfuric acid and 5 mL conc. nitric acid were
mixed together at 0oC. To this mixture, 10 mmol (3.10) of 6
was added and the reaction mixture was allowed to warm
slowly to room temperature for 5 h. After 24 h, the reaction
mixture was poured on to water yielding yellow precipitate.
The crude product was washed cold water and recrystallized
with DMSO. The purity of the compound was checked on
TLC using methanol:DMSO (1:1) as mobile phase.
Synthesis of 10-Dimethylamino Benzo[a]phenazine-5-
Sulfonic acid (8)
A mixture of 3.10 g (10 mmol) compound 6 and CDI (N,
N-carbonyl diimidazole) (138 mg) stirred with dry DMF (8
mL) at room temperature for 48 h. To this mixture, the sub-
stituent (10 mmol dimethylamine) was added and the reac-
tion mixture was stirred at room temperature for further 30
min. The volatile components were then removed in vacuum
and the residue was dissolved in dichloromethane (20 mL)
and washed with water (2 X 20 mL) and dried over magne-
sium sulfate. The solvent was removed in vaccuo to provide
crude product and it was recrystallized with methanol. TLC
was developed with methanol:water:DCM (5:3:2).
IN SILICO ADME-TOX STUDY
The Pentium IV work station and Pallas 6.1.1 software
[15] were used to calculate and to predict the ADMET prop-
erties of the molecules. Chem draw ultra software was used
to draw the structure of the compounds to be analyzed and
was saved as MDL file. The sketched molecules were un-
dergo calculation of the following properties viz drug likeli-
ness, metabolite, toxicity, etc. [16, 17].
In Vitro Cytotoxicity Assay in Human Tumor Cell Lines
The compounds were screened in HL-60 cell lines by
adopting the well-established MTT colorimetric micro cul-
ture assay method [18]. Briefly, 5 x 103 (HL-60) cells were
exposed in vitro under sterile cell culture conditions to dif-
ferent concentrations (1-300 �M range) of drug solutions and
incubated. After 72 h of incubation, 10 �L of MTT salt solu-
tion was added in each well. After an additional 4 h, during
which insoluble formazan was produced. The cellular reduc-
tion of the colourless tetrazolium salts yields coloured for-
mazan derivatives in proportion to viable cell numbers. 100
�L of 10% sodium dodecylsulphate were added in each well
and another 12 h were allowed for the dissolution of forma-
zan. The optical density of the reduced formazan derivatives
was determined in ELISA reader at 540 nm to get the per-
centage inhibition. The blank-corrected absorbance of the
control wells was taken as 100% and the results were ex-
pressed as a percentage of the control. IC50 values were de-
termined by computationally on online software (BioData fit
program). Data provided in the Table 1 are the mean of du-
plicate values.
DNA Intercalation Studies: Thermal Denaturation
Method
Two of the synthesized compounds (3 and 8) were sub-
jected to thermal denaturation studies with duplex-form calf
thymus DNA (CT-DNA) using an adaptation of a reported
procedure [19-21].
Working solutions in aqueous buffer (10 mM
NaH2PO4/Na2HPO4, 1 mM disodium EDTA, pH (7.00 +
0.01) containing CT-DNA (100 μM in phosphate) and the
compounds (20 μM) were prepared by addition of concen-
trated compound solution in DMSO to obtain a fixed [Com-
pound]/[DNA] molar concentration. The DNA-compound
mixture was incubated at 37oC for 0, 18 h prior to analysis.
Samples were monitored at 260 nm using a Beckman DU-
7400 spectrophotometer fitted with high performance tem-
perature controller and heating was applied at 1oC min-1 in
the 40-95oC range. DNA helix to coil transition temperature
(Tm) were obtained from the maxima in the d(A260)/dT de-
rivative plots. Drug-induced alterations in DNA melting be-
havior are given by: �Tm = Tm (DNA+Compound)-Tm
(DNA alone), where the Tm value for the compound –free
CT-DNA is 69.2 ± 0.01. The fixed [compound]/[DNA] ratio
used did not result in binding saturation of the host DNA
duplex for any compound examined. The transition in ther-
mal melting points of DNA in the presence of the com-
pounds is recorded in Table 6.
RESULTS AND DISCUSSION
The physicochemical properties such as melting point, Rf
value, solubility and �max of the synthesized compounds
552 Medicinal Chemistry, 2009, Vol. 5, No. 6 Moorthy et al.
Table 1. In Vitro Screening Results in HL-60 Cell Line
N
NHO3S
R2
R1
1
2
3
4
5
6 7 8
9
10
1112
Comp. Code R1 R2 IC50 �M
1 H H 91
2 4-NO 2 H 142
3 H 9-NO 2 124
4 4-NH 2 H 85
5 H 9-NH 2 31
6 H 10-COO H 69
7 4-NO 2 10-COO H 59
8 H 10-CON (CH 3) 2 19
Cis p la tin 7
5-FU 266
Table 2. Physicochemical Characterization of the Benzo[a]phenazine-5-Sulfonic Acid Derivatives
Molecular weight Comp.
Code
Molecular formula
Calcd. Found*
Melting point** Solubility Rf. Value �max Yield
1 C16H10N2O3S 310.04 310 215-218
oC DMSO, Metha-
nol
0.91 357, 340, 280 85%
2 C 16H 9N 3O 5S 355.33 355 152
o C Ch lo ro fo rm 0.36 280.5, 357 25%
3 C 16H 9N 3O 5S 355.32 355 317-320
o C D MSO 0.70 281, 341, 357 50%
4 C 16H 11N 3O 3S 325.34 325 323-330
o C Me th a n o l 0.68 355, 295 50%
5 C 16H 11N 3O 3S 325.34 325 128-130
o C D MSO 0.62 356, 306.5 60%
6 C 17H 10N 2O 5S 354.34 354 190-192
o C D MSO , Me th a n o l 0.78 263, 356 78%
7 C 17H 9N 3O 7S 399.34 399 230
o C D MSO , Me th a n o l 0.63 281, 357 48%
8 C 19H 15N 3O 4S 381.41 381 168-170
o C D MSO , Me th a n o l 0.84 283, 342 82%
* MW fo un d ou t by Mas s s p ect ro met er, * * MP o p en cap i l l ary met h od and are u n co rrect ed .
Table 3. Analytical (IR, NMR, MASS and Elemental analysis) result of the Synthesized Compounds
Comp.
Code
Analysis Data
1H NMR(300MHz, � /ppm in
DMSOd6)
1.92 (b r s , 1H, SO 3H), 7.16-7.18(d, J = 5.7 Hz , 2H, A r-H ) , 7.69-7.74 (m , 3H, A r-H ), 8.09 (s, 1H, A r-
H ), 8.21-8.23 (d , J = 6.1 Hz , 1H, A r-H ), 8.35-8.38(d, J =9.0 Hz , 2H, A r -H ).
IR (KBr/Cm-1) 3058 (C-H (A r)), 1 644-1531 (C= N ), 1380 (S= O , Su lfo n ic a c id ), 1045 (C-N ).
1
FAB-MS m/z [M]+ 310
Synthesis, Cytotoxic Evaluation and In Silico Pharmacokinetic Prediction Medicinal Chemistry, 2009, Vol. 5, No. 6 553
(Table 3) contd….
Comp.
Code
Analysis Data
Elemental Analysis Calcd. (Found) for C16H10N2O3S: C 61.93 (61.78), H 3.25 (3.06), N 9.03 (8.97).
1H NMR(300MHz, � /ppm in CDCl3) 1.76 (br, s, 1H, SO3H), 7.49-7.51 (d, J=6.01 Hz, 1H, Ar-H), 7.77-7.79 (d, J=5.3 Hz, 1H, Ar-H),
7.90-8.00 (m, 4H, Ar-H), 8.41 (s, 1H, Ar-H), 8.60 (s, 1H, Ar-H).
IR (KBr/Cm-1) 3097 (C-H (Ar)), 1532 (N=O, Nitro), 1524-1422 (C=N), 1348 (S=O, Sulfonic acid), 760 (Nitro).
FAB-MS m/z [M]+ 355.
2
Elemental Analysis Calcd. (Found) for C16H9N3O5S: C 54.08 (53.92), H 2.55 (2.45), N 11.83 (11.72).
1H NMR(300MHz, � /ppm in
DMSOd6)
1.96 (br s, 1H, SO3H), 7.86-7.88 (d, J=6.0 Hz, 1H, Ar-H), 7.98-8.01(d, J=9.3 Hz, 1H, Ar-H), 8.31-
8.39 (m, 4H, Ar-H), 8.42 (s, 1H, Ar-H), 8.09 (s, 1H, Ar-H).
IR (KBr/Cm-1) 3071 (C-H (Ar)), 1532 (N=O, Nitro), 1533-1452 (C=N), 1348 & 1180 (S=O, Sulfonic acid), 763
(Nitro).
FAB-MS m/z [M]+ 355
3
Elemental Analysis Calcd. (Found) for C16H9N3O5S: C 54.08 (53.82), H 2.55 (2.72), N 11.83 (12.15).
1H NMR(300MHz, � /ppm in CDCl3) 1.91 (br s, 1H, SO3H), 4.4 (s, 2H, NH2), 7.46-7.49 (d, J=6.3 Hz, 1H, Ar-H), 7.75-7.77 (d, J= 5.6 Hz,
1H, Ar-H), 7.85-7.95 (m, 4H, Ar-H), 8.08 (s, 1H, Ar-H); 8.39 (s, 1H, Ar-H).
IR (KBr/Cm-1) 3233 (N-H), 3122 (C-H (Ar)), 1514-1463 (C=N), 1356 (S=O, Sulfonic acid), 1045 (C-N).
FAB-MS m/z [M]+ 325
4
Elemental Analysis Calcd. (Found) for C16H11N3O3S: C 59.07 (58.97), H 3.41 (3.30), N 12.92 (13.05).
5
1H NMR(300MHz, � /ppm in
DMSOd6)
1.94 (br, s, 1H, SO3H), 3.23 (br, s, 2H, NH2), 6.89-6.91 (d, J= 6.0Hz, 1H, Ar-H), 7.44-7.47 (d, J=
9.0Hz, 1H, Ar-H), 7.72-7.98 (m, 4H, Ar-H), 8.09 (s, 1H, Ar-H), 8.20-8.22 (d, J= 6.6 Hz, 1H, Ar-H).
IR (KBr/Cm-1) 3467 (N-H Poly nuclear), 3057 (C-H (Ar)), 1364 (S=O, Sulfonic acid).
FAB-MS m/z [M]+ 325
Elemental Analysis Calcd. (Found) for C16H11N3O3S: C 59.07 (58.89), H 3.41 (3.34), N 12.92 (13.06).
1H NMR(300MHz, � /ppm in
DMSOd6)
1.94 (br, s, 1H, SO3H), 6.81-6.84 (d, J=6.3 Hz, 1H, Ar-H), 7.60-7.62 (d, J=6.3 Hz, 1H, Ar-H), 7.72
(s, 1H, Ar-H), 7.77-7.86 (m, 4H, Ar-H), 8.02 (s, 1H, Ar-H),12.12 (br, s, 1H, COOH).
IR (KBr/Cm-1) 3003 (C-H (Ar)), 1736 (C=O Carboxilic), 1531-1470 (C=N), 1464 (C-O-H Carboxilic), 1372 (S=O,
Sulfonic acid).
FAB-MS m/z [M]+ 354
6
Elemental Analysis Calcd. (Found) for C17H10N2O5S: C 57.62 (57.92), H 2.84 (2.75), N 7.91 (7.63).
1H NMR(300MHz, � /ppm in CDCl3) 1.90 (br, s, 1H, SO3H), 7.74-7.76 (d, J=5.42Hz, 1H, Ar-H), 7.93-7.96 (d, J=8.5 Hz, 1H, Ar-H), 8.24-
8.27 (d, J=8.2 Hz, 1H, Ar-H), 8.31-8.35 (d, J= 9.4 Hz, 1H, Ar-H), 8.41-8.44 (d, J=9.5Hz, 1H, Ar-H),
8.53 (s, 1H, Ar-H), 8.61 (s, 1H, Ar-H), 12.67 (br, s, 1H, COOH).
IR (KBr/Cm-1) 3122 (C-H (Ar)), 1706 (C=O Carboxilic), 1532 (N=O, Nitro), 1514-1432 (C=N), 1427 (C-O-H
Carboxilic), 1365 (S=O, Sulfonic acid), 763 (Nitro).
FAB-MS m/z [M]+ 399
7
Elemental Analysis Calcd. (Found) for C17H9N3O7S: C 51.13 (51.00), H 2.27 (2.22), N 10.52 (10.44).
1H NMR(300MHz, � /ppm in CDCl3) 1.89 (br, s, 1H, SO3H), 3.09 (s, 6H, CON(CH3)2), 6.74-6.77 (d, J= 6.0 Hz, 1H, Ar-H), 7.50-8.32 (m,
6H, Ar-H), 8.97 (s, 1H, Ar-H).
IR (KBr/Cm-1) 3122 (C-H (Ar)), 2980 (C-H), 1625 (C=O Amide), 1330 (S=O, Sulfonic acid).
FAB-MS m/z [M]+ 381
8
Elemental Analysis Calcd. (Found) for C19H15N3O4S: C 59.83 (59.77), H 3.96 (3.76), N 11.02 (10.95).
