Phenazine-1-carboxamides: Structure-cytotoxicity relationships for 9-substituents and changes in the H-bonding pattern of the cationic side chain

Article abstract of DOI:10.1016/j.bmc.2005.09.032

A series of phenazine-1-carboxamides were prepared, including variations in both chromophore substituents and the nature of the cationic side chain. The novel side-chain analogues were prepared from the corresponding phenazine-1-carboxylic acids via Schmi

Full text of DOI:10.1016/j.bmc.2005.09.032

Bioorganic & Medicinal Chemistry 14 (2006) 1160–1168
Phenazine-1-carboxamides: Structure–cytotoxicity relationships
for 9-substituents and changes in the H-bonding
pattern of the cationic side chain
Swarna A. Gamage,a,* Gordon W. Rewcastle,a Bruce C. Baguley,a
Peter A. Charltonb and William A. Dennya
aAuckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland,
Private Bag 92019, Auckland 1020, New Zealand
bXenova Ltd., 957 Buckinghamshire Avenue, Slough, Berkshire SL1 4NL, UK
Received 2 August 2005; revised 14 September 2005; accepted 14 September 2005
Available online 10 October 2005
Abstract—A series of phenazine-1-carboxamides were prepared, including variations in both chromophore substituents and the nat-
ure of the cationic side chain. The novel side-chain analogues were prepared from the corresponding phenazine-1-carboxylic acids
via Schmidt conversion to the 1-amines and from the corresponding 1-halides. Structure–cytotoxicity relationships for these com-
pounds in a panel of tumor cell lines showed that there is very limited scope for variation of the structure of the 1-carboxamide side
chain, consistent with the recent structural model of how tricyclic carboxamides bind to DNA. There was generally little difference in
IC50s between parent and P-glycoprotein expressing cell lines, suggesting that most of the compounds are not affected by the
presence of this efflux pump.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
binary complex information is of considerable value in
drug design.
Polycyclic aromatic chromophores with an attached cat-
ionic side chain have been a successful general design
motif for DNA-intercalating topoisomerase inhibitors.
Established examples include doxorubicin (1)1 and
mitoxantrone (2),2 with other examples (e.g., intoplicine
(3)3 and XR-11576 (4)4) in clinical trial. An important
subset within this general motif are the tricyclic carbox-
amides5,6 including the dual topo I/II inhibitor DACA
(5), which reached Phase II clinical trial.7 It is accepted
that these compounds work through the formation of
ternary complexes between the drug, the topo enzyme,
and DNA.8 Detailed (atomic-level) structural informa-
tion on these ternary complexes is not yet available,
but has been reported9,10 for binary drug/oligonucleo-
tide complexes of anthracyclines such as 1. Because
the intercalating agents bind preferentially to DNA
rather than the topoisomerase enzymes, even such
Recent determinations11,12 of the structures of several
complexes of 9-aminoacridine-4-carboxamides and the
oligonucleotide d(GCATCG)2 have provided a similar
level of information on the binding mode of the tricyclic
carboxamide class of compounds. The data suggest a
fairly robust binding motif, where the chromophore lies
between the GpC step with the long axis oriented paral-
lel with the base pair long axis (for maximum overlap),
and where the cationic side chain lies in the major
groove. The dimethylamino cation accepts an H-bond
from the N7 and O6 atoms of guanine G2, and the car-
boxamide NH forms a water-bridged H-bond to the
phosphate group of the same guanine. This model is
consistent with the specific structure–activity relation-
ships of the acridine-4-carboxamides, and more general-
ly for other tricyclic carboxamides,5 including the
phenazine-1-carboxamides (e.g., 6).13 The latter
compounds showed good in vitro and in vivo activity,
but structure–activity studies were limited to small chro-
mophore substituents and essentially to the standard
CONH(CH2)2NMe2 side chain (Fig. 1).
Keywords: Phenazine-1-carboxamides; DNA-binders; P-glycoprotein;
Polycyclic aromatic carboxamides.
*
Corresponding author. Tel.: +64 9 3737599x86268; fax: +64 9
3737502; e-mail: s.gamage@auckland.ac.nz
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.032

S. A. Gamage et al. / Bioorg. Med. Chem. 14 (2006) 1160–1168
1161
H
N
O
O
OH
OH O HN
OH
OH
CH2OH
OH
MeO
O
Me
OH O
O
NH2
OH O HN
N
H
2
1
HO
Me
H
N
X
N
N
MeO
NMe2
N
R
NMe2
N
O
4
Me
NMe2
HN
O N
N
H
H
3
5: R = H, X = CH
6: R = H, X= N
HO
7: R = 9-Me, X = N
8: R = 8,9-benzo, X=N
Figure 1.
We report here syntheses and further structure–activity
relationship studies in the phenazine-1-carboxamide ser-
ies, including variations in both chromophore substitu-
ents and the nature of the cationic side chain, and
compare the results against the DNA binding model
deduced for the acridine-4-carboxamides.
3'
F
R
2'
4'
N
N
ii
NH
CONH(CH2)2NMe2
R
N
N
13: R =CO2H
14: R =CONH(CH2)2NMe2
i
15: R = H
iii
16: R = 4'-OMe
17: R = 3'-OMe
18: R = 2'-OMe
19: R = 2'-Me
20: R = 2'-Cl
22: R = 4'-OH
23: R = 3'-OH
24: R = 2'-OH
2. Discussion
21: R = 2',3'-benzo
2.1. Chemistry
Scheme 2. Reagents and conditions: (i) CDI/DMF; then
NH2(CH2)2N(CH3)2. (ii) ArNH2/LDA/THF/ꢀ10 to 0 ꢁC/30 min.
(iii) BBr3/CH2Cl2/ꢀ80 to 20 ꢁC/30 min.
The 9-phenoxyphenazine-1-carboxamides (11 and 12)
were prepared from 9 and 10 by activating with thionyl
chloride or 1,1-carbonyldiimidazole followed by react-
ing with N,N-dimethylethylenediamine (Scheme 1).
The 9-fluorophenazine-1-carboxamide (14) was similarly
prepared from (13) (Scheme 2).
O
HN
X
R
R
9
N
N
N
N
ii
8
The 9-(N-phenylamino)phenazine-1-carboxamides (15–
21) (Scheme 2) were prepared from 14 by fluorine dis-
placement with the anions of the appropriate anilines,
which were formed in situ by the reaction with lithium
diisopropylamide. Demethylation of the methoxy deriv-
atives 16–18 with excess boron tribromide gave the
corresponding hydroxy analogues 22–24 in good yields.
25, 26: X = CO2H
27, 28: X = NH2
29, 30
i
O
iii
iv
O
Br
HN
R
R
HN
NR2
N
R
N
N
N
35, 36
31, 32, 33, 34
iv
O
The amide derivatives 31, 32, 37, and 38 (Scheme 3) were
prepared by Schmidt conversion (polyphosphoric acid/
sodium azide) of the phenazinecarboxylic acids 25 and
26 to the corresponding amines 27 and 28. Reaction of
these with either acryloyl chloride or bromoacetyl bro-
mide gave, respectively, the acrylamides 29 and 30,
and the bromoacetates 35 and 36, which were then treat-
ed with the appropriate amines. The (dimethylami-
no)propoxy derivative 42 (Scheme 4) was prepared
NMe2
HN
N
N
R = 9-Me or 8,9 benzo
NR2 = NMe2 or morpholino
37, 38
Scheme 3. Reagents and conditions: (i) PPA/NaN3/100 ꢁC. (ii) Acry-
loyl chloride/CH2Cl2/N-ethyl di-isopropylamine. (iii) Bromoacetyl-
bromide/CH2Cl2/20 ꢁC. (iv) NHR2/EtOH/reflux.
from 9-fluorophenazine-1-carboxylic acid 13, which
was converted (via the imidazolide)16 to the alcohol
39. Reaction of this compound with triphenylphos-
phine/bromine gave the bromide 40, which was reduced
with sodium borohydride in DMSO to give 9-fluoro-1-
methylphenazine (41). Fluoride displacement with the
anion of 3-(dimethylamino)propanol then gave 42. The
amine derivatives 43 and 44 were prepared (Scheme 4)
from the corresponding carboxamides 31 and 32 by
R
CONH(CH2)2NMe2
R
COOH
N
N
N
N
i, ii
9: R = OPh
10: R = NMe2
11: R = OPh
12: R =NMe2
Scheme 1. Reagents: (i) SOCl2/CH2Cl2 or CDI/DMF. (ii)
NH2(CH2)2N(CH3)2.

1162
S. A. Gamage et al. / Bioorg. Med. Chem. 14 (2006) 1160–1168
CO and NH moieties making specific binding
contributions.
R
F
Me
O(CH2)3NMe2
N
N
N
iv
N
13: R = CO2H
i
42
39: R = CH2OH
40: R = CH2Br
41: R = Me
ii
3. Conclusion
iii
O
The results showed that while small 9-substituents en-
hance cytotoxic potency in phenazine-1-carboxamides,
there is a size limit, with the larger arylamino groups
giving less effective compounds. There were tight SAR
for the C-1 side chain, with only ꢀreversed amideꢁ ana-
logues showing any significant potency. This is consis-
tent with the structural model of how the tricyclic
carboxamide bind to DNA, where the C-1 carboxamide
makes specific binding contributions. Most of the com-
pounds were not affected by P-glycoprotein-mediated
drug resistance.
HN
NMe2
HN
NMe2
R1
R1
N
N
N
N
v
43: R1 = Me
44: R1 = 8,9 benzo
31: R1 =Me
32: R2 = 8,9-benz
H
H
O
N
N
Me
NMe2 Me
NMe2
N
N
v
N
N
45
7
Scheme 4. Reagents and conditions: (i) a—CDI/THF; b—NaBH4/
THF/H2O then concd HCl. (ii) PPh3/Br2/CH3CN. (iii) NaBH4/
DMSO. (iv) NaH/3-N,N-dimethylamino-1-propanol/THF/50 ꢁC. (v)
BH3-SMe2/THF/reflux then THF/HCl.
4. Experimental
Analyses were performed by the Microchemical Labora-
tory, University of Otago, Dunedin, NZ. Melting points
were determined with an Electrothermal Model 9200
digital melting point apparatus and are as read. NMR
spectra were measured on Bruker AC-200 or Bruker
DRX-400 MHz spectrometers, and referenced to Me4Si.
Mass spectra were recorded on a Varian VG 7070
spectrometer. Compounds tested for cytotoxicity
were >98% pure by HPLC.
reduction with BH3-SMe2, followed by acid decomplex-
ation of the resulting boron complexes. Similar reduc-
tion of 7 gave the benzylamine derivative 45.
2.2. Biology
The phenazinecarboxamides, as the highly water-soluble
monohydrochloride salts, were evaluated using a panel
of cell lines in culture. The murine P388 leukaemia cell
line provided comparison with previous compounds,
while the H69 parental human small lung carcinoma cell
line and its derived drug resistant line LX4, which over-
expresses P-glycoprotein, were used to evaluate the
ability of the compounds to overcome P-glycoprotein-
mediated drug resistance.17 Table 1 compares the poten-
cy of these compounds for the variation of substituents
at C-9. Methyl, 8,9-benz and OMe improved the
potency in all three cell lines, whereas substitution with
N-alkyl or N-phenyl did not, and substitution with N-(2-
methoxy) and N-(2-hydroxyphenyl) had only slight
effect. There was generally a little difference in IC50s in
the parent and P-glycoprotein expressing lines, suggest-
ing that most of the compounds are not affected by the
presence of this efflux pump.
4.1. Cytotoxicity assays
They were performed as described previously.18–21 After
drug addition, cells were incubated for 5–6 days before
adding Alamar blue (H69/P, H69/LX4) or for 3 days be-
fore adding [3H]thymidine (P388) to measure cell prolif-
eration as IC50s (concentration required to give 50% cell
kill). Independent assays were performed in duplicate.
The ratio of IC50s in the resistant cell line (LX4) divided
by that in the parental cell line (P) gives an indication of
the degree to which a compound is affected by P-gp and
is termed the resistance factor.
4.2. General procedures for synthesis of compounds 11–45
4.2.1. N-[2-(Dimethylamino)ethyl]-9-phenoxyphenazine-
1-carboxamide (11). A solution of 9-phenoxyphen-
azine-1-carboxylic acid (9)14 (1.0 g, 2.85 mmol) in SOCl2
(30 mL) was refluxed for 30 min, and after removal of
the solvent under vacuum, the residue was treated with
a solution of N,N-dimethylethylenediamine (3 mL) in
CH2Cl2 (200 mL). After 5 min, the solution was washed
twice with water and extracted four times with 0.1 M
HCl. The aqueous layer was basified with concd aque-
ous NH3 and extracted with CH2Cl2. Drying and
removal of the solvent gave crude N-[2-(dimethylami-
no)ethyl]-9-phenoxyphenazine-1-carboxamide (11) (0.8 g,
Table 2 explores the effect of variations in the C-1 side
chain. Previous work with tricyclic and tetracyclic carb-
oxamides has suggested there is tight SAR around this
position, and there has been little exploration of alterna-
tive linker groups for the side-chain amide. The more
extensive study carried out here confirms that there is
limited scope for variation in this position. In compari-
son with the ꢀparentꢁ carboxamides 7 and 8, only the ꢀre-
versed amidesꢁ 31A and 32 showed significant potency,
and even these were 30- to 50-fold less effective than
the parent compounds. All of the compounds with other
side chains, including those with O, NH and CH2NH
linkers (43–45A), were essentially inactive. This is con-
sistent with the recent structural model of how the tricy-
clic carboxamides bind to DNA, with the carboxamide
1
73%); H NMR (CDCl3) d 2.14 [s, 6H, CH2N(CH3)2],
2.52 (t, J = 7.1 Hz, 2H, CH2NMe2), 3.65 (q,
J = 6.3 Hz, 2H, CH2NH), 7.18–7.21 (m, 3H, H-8, H-20
and H-60), 7.24 (t, J = 7.5 Hz, 1H, H-40), 7.43–7.47 (br
t, J = 8.4 Hz, 2H, H-30 and H-50), 7.76 (dd, J = 8.8,

S. A. Gamage et al. / Bioorg. Med. Chem. 14 (2006) 1160–1168
Table 1. 9-Substituted 1-CONH(CH2)2NMe2 phenazines
1163
H
O
N
R
NMe2
N
N
a
IC50 (nM)
Compound
R
P388b
1,100
H69c
X4d
Ratioe
6
7
Hf
>5,000
200
68
>5,000
290
130
Mef
18
37
1.5
1.8
3.3
8
8,9-Benzf
OMef
OPh
9
63
245
820
11
12
15
16
17
18
19
20
21
22
23
24
2,300
16,000
1,600
>20,000
3,400
770
5,000
5,000
2,330
1,840
5,000
330
5,000
5,000
4,070
2,330
5,000
2,530
5,000
2,260
3,470
1,570
2,260
ꢁ1
ꢁ1
1.7
1.3
ꢁ1
7.6
NMe2
NHPh
NH(4-OMePh)
NH(3-OMePh)
NH(2-OMePh)
NH(2-MePh)
NH(2-ClPh)
NH(1-naphth)
NH(4-OHPh)
NH(3-OHPh)
NH(2-OHPh)
15,000
2,100
12,400
>20,000
1,100
690
5,000
2,190
2,620
2,105
2,370
ꢁ1
1.0
1.3
0.74
0.95
a IC50; concentration of drug (nM) to reduce cell number to 50% of control cultures. Number is the average of at least two independent
determinations.
b Murine P388 leukemia cell line.
c H69 parental human small cell lung carcinoma cell line.
d LX4: H69 line over-expressing P-glycoprotein.
e IC50 ratio (X4/H69).
f Ref. 13.
Table 2. 9-Methylphenazines with varying 1-side chains
Me
R
R
N
N
N
N
B
A
a
IC50 (nM)
Compound
Form
R
P388b
18
H69c
X4d
290
Ratioe
7
31
33
37
42
43
45
8
A
A
A
A
A
A
A
B
B
B
B
B
CONH(CH2)2NMe2
NHCO(CH2)2NMe2
200
1.5
1.2
1.4
2,500
>20,000
>20,000
10,000
11,200
10,900
37
1,930
2,450
>5,000
5,000
2,450
>5,000
70
2,220
3,640
>5,000
2,770
3,420
>5,000
130
NHCO(CH2)2Nmorph
NHCOCH2NMe2
O(CH2)3NMe2
0.55
1.4
NH(CH2)3NMe2
CH2NH(CH2)2NMe2
CONH(CH2)2NMe2
NHCO(CH2)2NMe2
NHCO(CH2)2Nmorph
NHCOCH2NMe2
NH(CH2)3NMe2
1.8
1.2
32
34
38
44
1,040
430
3,830
4,970
495
>5,000
4,770
12,900
>20,000
2,900
>1.3
0.95
a IC50; concentration of drug (nM) to reduce cell number to 50% of control cultures. Number is the average of at least two independent
determinations.
b Murine P388 leukemia cell line.
c H69 parental human small cell lung carcinoma cell line.
d LX4: H69 line over-expressing P-glycoprotein.
e IC50 ratio (X4/H69).
7.6 Hz, 1H, H-7), 7.96–8.01 (m, 2H, H-3 and H-6), 8.37
(dd, J = 8.0, 1.5 Hz, 1H, H-4), 8.99 (dd, J = 7.1, 1.5 Hz,
1H, H-2), 11.10 (br s, 1H, NH). Treatment of a metha-
nolic solution of the crude amide with HCl, followed by
recrystallization from methanol/ethyl acetate, gave
the hydrochloride; mp 239–241 ꢁC; Anal. Calcd for

1164
S. A. Gamage et al. / Bioorg. Med. Chem. 14 (2006) 1160–1168
C23H23ClN4O2: C, 65.3; H, 5.5; N, 13.3; Cl, 8.4; found:
C, 65.2; H, 5.3; N, 13.2; Cl, 8.6%.
2.01 (s, 6H, N(CH3)2), 2.56 (t, J = 5.7 Hz, 2H,
CH2N(CH3)2), 3.78 (q, J = 5.5 Hz, 2H, NHCH2), 7.02
(dd, J = 7.4, 1.1 Hz, 1H, H-8), 7.29 (t, J = 7.3 Hz, 1H,
ArH), 7.43 (d, J = 7.1 Hz, 2H, 2· ArH), 7.49 (t,
J = 7.7 Hz, 2H, 2· ArH), 7.61 (dd, J = 8.7, 1.3 Hz, 1H,
ArH), 7.66 (t, J = 8.1 Hz, 1H, ArH), 7.94 (dd, J = 8.6,
7.2 Hz, 1H, H-3), 8.02 (br s, 1H, NH), 8.37 (dd,
J = 8.4, 1.5 Hz, 1H, H-4), 8.91 (dd, J = 7.1, 1.5 Hz,
1H, H-2), 10.21 (br s, 1H, CONH); HRMS (EI) calcd
for C23H23N5O (M+) m/z: 385.1903, found: 385.1906.
4.2.2. N-[2-(Dimethylamino)ethyl]-9-(dimethylamino)phen-
azine-1-carboxamide (12). A mixture of 9-(dimethylami-
no)phenazine-1-carboxylic acid (10)14 (1.25 g, 4.7 mmol)
and CDI (1.51 g, 9.4 mmol) in DMF (25 mL) was heated
at 50–60 ꢁC for 15 min. N,N-Dimethylethylenediamine
(2.6 mL, 5 equiv) was added, and the mixture was
stirred at room temperature for 15 min. The mixture
was then diluted with water and extracted with CH2Cl2
to give crude N-[2-(dimethylamino)ethyl]-9-(dimethyl-
amino)phenazine-1-carboxamide (12); 1H NMR (CDCl3)
d 2.35 [s, 6H, CH2N(CH3)2], 2.72 (t, J = 7.0 Hz, 2H,
CH2NMe2), 3.16 [s, 6H, 9-N(CH3)2], 3.83 (q,
J = 6.9 Hz, 2H, CH2NH), 7.19 (dd, J = 7.3, 1.2 Hz, 1H,
H-8), 7.83 (dd, J = 8.7, 7.3 Hz, 1H, H-7), 7.83 (dd,
J = 8.7, 1.3 Hz, 1H, H-6), 7.94 (dd, J = 8.6, 7.1 Hz, 1H,
H-3), 8.33 (dd, J = 8.6, 1.5 Hz, 1H, H-4), 8.93 (dd,
J = 7.1, 1.5 Hz, 1H, H-2), 11.03 (br s, 1H, NH). Treat-
ment with HCl in MeOH gave the dihydrochloride
(1.7 g, 89%); mp (MeOH/EtOAc). 220–222 ꢁC; Anal.
Calcd for C19H25Cl2N5O: C, 55.6; H, 6.1; N, 17.1; Cl,
17.3; found: C, 55.7; H, 6.4; N, 17.3; Cl, 17.4%.
4.2.4. N-[(2-Dimethylamino)ethyl]-9-[N-(4-methoxy)phe-
nylamino]phenazine-1-carboxamide (16). Similar reaction
of 4-methoxyaniline with 14 gave 16 (50%): mp
(CH2Cl2/hexane) 165–166.5 ꢁC; 1H NMR (CDCl3) d
2.04 (s, 6H, N(CH3)2), 2.54 (t, J = 5.7 Hz, 2H,
CH2N(CH3)2), 3.79 (q, J = 5.4 Hz, 2H, CH2NH), 6.70
(dd, J = 7.6, 1.2 Hz, 1H, H-8), 7.03 (d, J = 8.7 Hz, 2H,
H-20, 60), 7.34 (d, J = 8.8 Hz, 2H, H-30, 50), 7.55 (dd,
J = 8.7, 1.1 Hz, 1H, H-6), 7.62 (t, J = 8.1 Hz, 1H, H-
7), 7.93 (dd, J = 8.7, 7.2 Hz, 1H, H-3), 7.99 (br s, 1H,
NH), 8.36 (dd, J = 8.7, 1.5 Hz, 1H, H-4), 8.94 (dd,
J = 7.2, 1.5 Hz, 1H, H-2), 10.44 (br s, 1H, CONH);
HRMS (EI) calcd for C24H25N5O2 (M+) m/z 415.200,
found 415.2005.
4.2.3. N-(2-Dimethylaminoethyl)-9-(N-phenylamino)phen-
azine-1-carboxamide (15). A suspension of 9-fluorophen-
azine-1-carboxylic acid (13)15 (1.40 g, 5.78 mmol) and
CDI (1.9 g, 11.6 mmol) in DMF (10 mL) was heated at
40 ꢁC for 10 min to give a clear solution, which was
cooled to 0 ꢁC and treated with N,N-dimethylethylenedi-
amine (3.17 mL 5 equiv) in CH2Cl2 (10 mL). After stir-
ring for 15 min at 0 ꢁC, the mixture was diluted with
water and extracted with CH2Cl2 (3 · 50 mL). Evapora-
tion, and chromatography of the residue on alumina,
eluting with CH2Cl2, gave crude N-[2-(dimethylami-
no)ethyl]-9-fluorophenazine-1-carboxamide (14); 1H
NMR (CDCl3) d 2.37 [s, 6H, CH2N(CH3)2], 2.72 (t,
J = 6.4 Hz, 2H, CH2NMe2), 3.80 (q, J = 6.4 Hz, 2H,
CH2NH), 7.56–7.60 (m, 1H, H-8), 7.81–7.87 (m, 1H, H-
7), 8.01 (dd, J = 8.7, 7.2 Hz, 1H, H-3), 8.11 (br d,
J = 8.9, 1H, H-6), 8.38 (dd, J = 8.7, 1.5 Hz, 1H, H-4),
9.04 (dd, J = 6.4, 1.5 Hz, 1H, H-2), 10.92 (br s, 1H,
NH). Treatment with HCl in MeOH gave the hydrochlo-
ride (1.67 g, 83%); mp (MeOH/EtOAc) 245–247 ꢁC;
Anal. Calcd for C17H18ClFN4OÆ1.1 H2O: C, 55.4; H,
5.5; N, 15.2; Cl, 9.6; found: C, 55.2; H, 5.5; N, 15.3; Cl,
10.1%.
4.2.5. N-[(2-Dimethylamino)ethyl]-9-[N-(3-methoxy)phe-
nylamino]phenazine-1-carboxamide (17). Similar reaction
of 3-methoxyaniline with 14 gave 17 (32%): mp
(CH2Cl2/hexane) 142–145 ꢁC; 1H NMR (CDCl3) d
2.04 (s, 6H, N(CH3)2), 2.56 (t, J = 5.7 Hz, 2H,
CH2N(CH3)2), 3.78 (q, J = 5.5 Hz, 2H, CH2NH), 3.85
(s, 3H, OCH3), 6.83 (dd, J = 8.5, 2.2 Hz, 1H, H-8),
6.99 (t, J = 1.7 Hz, 1H, H-20), 7.02 (d, J = 7.6 Hz, 1H,
ArH), 7.10 (dd, J = 7.4, 1.2 Hz, 1H, ArH), 7.39 (t,
J = 8.1 Hz, 1H, H-7), 7.60–7.69 (m, 2H, 2· ArH), 7.93
(dd, J = 8.6, 7.2 Hz, 1H, H-3), 7.96 (br s, 1H, NH),
8.35 (dd, J = 8.6, 1.5 Hz, 1H, H-4), 8.90 (dd, J = 7.1,
1.5 Hz, 1H, H-2), 10.18 (br s, 1H, CONH); Anal. Calcd
for C24H25N5O2: C, 69.4; H, 6.1; N, 16.8; found C, 69.1;
H, 5.8; N, 16.8%.
4.2.6. N-[(2-Dimethylamino)ethyl]-9-[N-(2-methoxy)phe-
nylamino]phenazine-1-carboxamide (18). Similar reaction
of 2-methoxyaniline with 14 gave 18 (98%): mp
(CH2Cl2/hexane) 144–146 ꢁC; 1H NMR (CDCl3) d
2.04 (s, 6H, N(CH3)2), 2.55 (t, J = 6.1 Hz, 2H,
CH2N(CH3)2), 3.78 (q, J = 5.8 Hz, 2H, CH2NH),
7.04–.09 (m,
2
H, 2· ArH), 7.17–7.23 (m, 2H,
Aniline (0.15 g, 1.6 mmol) was dissolved in THF
(10 mL) and cooled in an ice/salt bath (ꢀ10 to 0 ꢁC).
A solution of lithium di-iso-propylamide (5.3 mL,
8 mmol) was added, and the mixture was stirred under
nitrogen at this temperature for 30 min. A solution of
14 (0.044 g, 0.33 mmol) in THF (5 mL) was added,
and the mixture was stirred for a further 30 min at
(ꢀ10- to 0-ꢁC). Water (20 mL) was added and the mix-
ture was extracted with EtOAc (3 · 25 mL). The com-
bined extracts were dried (Na2SO4) and the solvent
was evaporated, The crude product was chromato-
graphed on silica gel (20–40 lm), eluting with a
CH2Cl2/MeOH gradient, to give 15 (0.1 g, 79%): mp
(CH2Cl2/hexane) 169–172 ꢁC; 1H NMR (CDCl3) d
2· ArH), 7.56 (dd, J = 7.7, 1.6 Hz, 1H, ArH), 7.63 (dd,
J = 8.7, 1.2 Hz, 1H, ArH), 7.6–7.72 (m, 1H, ArH),
7.94 (dd, J = 8.6, 7.2 Hz, 1H, H-3), 7.98 (s, 1H, NH),
8.37 (dd, J = 8.7, 1.5 Hz, 1H, H-4), 8.90 (dd, J = 7.1,
1.4 Hz, 1H, H-2), 10.29 (br s, 1H, CONH); Anal. Calcd
for C24H25N5O2: C, 69.4; H, 6.1; N, 16.7; found: C, 69.5;
H, 7.5; N, 16.7%.
4.2.7. N-[(2-Dimethylamino)ethyl]-9-[N-(2-methyl)phe-
nylamino]phenazine-1-carboxamide (19). Similar reaction
of 2-methylaniline with 14 gave 19 (89%) as a foam; H
1
NMR (CDCl3) d 2.04 (s, 6H, N (CH3)2), 2.40 (s, 3H,
CH3), 2.65 (br s, 2H, CH2(CH3)2), 3.82 (q, J = 5.3 Hz,
2H, NHCH2), 6.54 (d, J = 7.2 Hz, 1H, ArH), 7.28–7.43

S. A. Gamage et al. / Bioorg. Med. Chem. 14 (2006) 1160–1168
1165
(m, 4H, 4· ArH), 7.57 (dd, J = 8.7, 1.4 Hz, 1H, ArH),
Similarly were prepared:
7.63 (t, J = 8.0 Hz, 1H, ArH), 7.94 (dd, J = 8.7,
1.2 Hz, 1H, ArH), 8.05 (br s, 1H, NH), 8.38 (dd,
J = 8.6, 1.3 Hz, 1H, ArH), 8.93 (dd, J = 7.1, 1.1 Hz,
1H, H-2), 10.52 (br s, 1H, CONH); Anal. Calcd for
C24H25N5O: C, 72.2; H, 6.3; N, 17.5; found C, 71.9;
H, 6.0; N, 17.4%.
4.2.11. N-[(2-Dimethylamino)ethyl]-9-[N-(3-hydroxy)phe-
nylamino]phenazine-1-carboxamide (23). From 17 (65%);
mp (CH2Cl2/hexane) 172–174 ꢁC; H NMR [(CD3)2SO]
1
d 2.06 (s, 6H, N(CH3)2), 2.49 (CH2N(CH3)2; peak over-
lapped with DMSO peak), 3.56 (q, J = 6.3 Hz, 2H,
NHCH2), 6.49 (d, J = 8.5, 0.7 Hz, 1H, H-8), 6.85 (s,
1H, H-20), 7.39 (d, J = 7.4 Hz, 1H, H-60),7.18 (t,
J = 7.9 Hz, 1H, ArH) 7.51 (dd, J = 7.5, 5.5 Hz, 1H, H-
7), 7.68 (d, J = 8.7 Hz, 1H, ArH), 7.86 (t, J = 8.2 Hz,
1H, ArH), 8.05 (dd, J = 7.3, 8.5 Hz, 1H, H-3), 8.39 (d,
J = 8.7 Hz, 1H, ArH), 8.54 (d, J = 7.4 Hz, 1H, H-2),
8.55 (s, 1H, OH), 9.85 (br s, 1H, NH), 9.78 (br s, 1H,
CONH); HRMS (EI) calcd for C23H23N5O2 (M+) m/z
401.1852, found: 401.1855.
4.2.8. N-[(2-Dimethylamino)ethyl]-9-[N-(2-chloro)phe-
nylamino]phenazine-1-carboxamide (20). Similar reaction
1
of 2-chloroaniline with 14 gave 20 (91%) as a foam; H
NMR (CDCl3) d 2.11 (s, 6H, N(CH3)2), 2.65 (t,
J = 5.8 Hz, 2H, CH2N(CH3)2), 3.82 (q, J = 5.8 Hz, 2H,
NHCH2), 7.09 (m, 1H, H-7), 7.20 (dt, J = 7.7, 1.5 Hz,
1H, H-40), 7.39 (dt, J = 7.7, 1.2 Hz, 1H, H-50), 7.56
(dd, J = 8.0, 1.4 Hz, 1H, H-8), 7.64 (dd, J = 8.0,
1.5 Hz, 1H, H-6), 7.71 (d, J = 4.2 Hz, 2H, 2· ArH),
7.95 (dd, J = 8.6, 7.1 Hz, 1H, H-3), 8.09 (br s, 1H,
NH), 8.38 (dd, J = 8.7, 1.5 Hz, 1H, H-4), 8.90 (dd,
J = 7.1,1.5 Hz, 1H, H-2), 10.08 (br s, 1H, CONH); Anal.
Calcd for C23H22ClN5O: C, 65.8; H, 5.3; N,16.7; Cl, 8.4;
found: C, 65.5; H, 5.5; N, 16.6; Cl, 8.6%.
4.2.12. N-[(2-Dimethylamino)ethyl]-9-[N-(2-hydroxy)phe-
nylamino]phenazine-1-carboxamide (24). From 18 (24%):
1
mp (CH2Cl2/hexane) 200–203 ꢁC; H NMR (CDCl3) d
2.29 (s, 6H, N(CH3)2), 2.77 (t, J = 5.4 Hz, 2H,
CH2N(CH3)2), 3.86 (q, J = 5.6 Hz, 2H, NHCH2),
6.97–7.07 (m, 3H, 3· ArH), 7.33 (dd, J = 1.0, 7.6 Hz,
1H, H-8), 7.54 (d, J = 8.1 Hz, 1H, ArH), 7.66 (dd,
J = 1.0, 8.7 Hz, 1H, H-6), 7.77 (dd, J = 8.7, 7.7 Hz,
1H, H-7), 7.91 (dd, J = 8.7, 7.2 Hz, 1H, H-4), 8.35 (dd,
J = 8.7, 1.6 Hz, 1H, H-4), 8.79 (s, 1H, NH), 8.93 (dd,
J = 1.6, 7.2 Hz, 1H, H-2), 10.43 (t, J = 5.4 Hz, 1H,
CONH), OH peak was not observed; HRMS (EI) calcd
for C23H23N5O2 (M+) m/z 401.1852, found: 401.1862.
4.2.9. N-[(2-Dimethylamino)ethyl]-9-(N-1-naphthylami-
no)phenazine-1-carboxamide (21). Similar reaction of 1-
naphthylamine with 14 gave 21 (98%) as a foam; H
1
NMR (CDCl3) d 1.82 (br s, 6H, N(CH3)2), 2.57 (br s,
2H, CH2(CH3)2), 3.78 (q, J = 5.4 Hz, 2H, NHCH2),
6.58 (d,J = 7.1 Hz, 1H, ArH), 7.46 (ddd, J = 8.3, 6.9,
1.3 Hz, 1H, ArH), 7.51–7.62 (m, 4H, 4· ArH), 7.67 (d,
J = 7.1 Hz, 1H, ArH), 7.88 (d, J = 8.2 Hz, 1 H, ArH),
7.94–7.98 (m, 2H, 2· ArH), 8.10 (d, J = 8.4 Hz, 1H,
ArH), 8.40 (dd, J = 8.6, 1.6 Hz, 1H, ArH), 8.48 (br s,
1H, NH), 8.95 (dd, J = 1.0, 7.1 Hz, 1H, H-2), 10.63 (br
s, 1H, CONH); Anal. Calcd for C27H25N5OÆ0.25 H2O:
C, 74.5; H, 5.8; N, 16.1; found: C, 73.7; H, 5.8; N, 15.9%.
4.2.13. (3-Dimethylamino)-N-(9-methylphenazin-1-yl)pro-
panecarboxamide (31). 9-Methylphenazine-1-carboxylic
acid (25)15 (0.53 g, 2.4 mmol) was dissolved in polyphos-
phoric acid (20 g) by warming to 90 ꢁC. The mixture was
cooled to room temperature, sodium azide (1 g) was add-
ed, and the reaction mixture was then stirred at 100 ꢁC
for 4 h and poured into hot water. Insoluble material
was removed by filtration, and the filtrate was basified
with aqueous ammonia and the resulting precipitate
was filtered, dried and chromatographed on alumina,
eluting with CH2Cl2/hexane (1:1), to give 9-methylphen-
azine-1-amine (27) (0.25 g, 49%) as a red solid, mp
4.2.10. N-[(2-Dimethylamino)ethyl]-9-[N-(4-hydroxy)phe-
nylamino]phenazine-1-carboxamide (22). A solution of 16
(0.15 mg, 0.36 mmol) in CH2Cl2 (10 mL) was cooled to
ꢀ80 ꢁC and treated with boron tribromide (4 mL,
4 mmol). The mixture was stirred for 10 min at ꢀ80 ꢁC
and then allowed to warm to room temperature for
30 min. The excess boron tribromide was quenched with
water (30 mL) and the mixture was acidified with conc.
HCl, stirred for 10 min and then made slightly basic
with concd aqueous Na2CO3. The mixture was extracted
with EtOAc (3 · 60 mL). The combined extracts were
dried (Na2SO4) and the solvent was evaporated under
reduced pressure. The residue was chromatographed
on silica gel, eluting with CH2Cl2/MeOH (94:6) to
give 22 (0.11 g, 72%); mp (CH2Cl2/hexane) 228 ꢁC
1
(CH2Cl2/hexane) 185–187 ꢁC; HMR (CDCl3) d 2.89 (s,
3H, CH3), 5.25 (br s, 2H, NH2), 6.92 (dd, J = 7.2,
1.1 Hz, 1H, H-2), 7.55–7.65 (m, 2H, 2· ArH), 7.69 (dd,
J = 8.7, 6.7 Hz, 2H, 2· ArH), 8.04 (d, J = 8.7 Hz, 1H,
ArH); Anal. Calcd for C13H11N3: C, 74.6; H, 5.3; N,
20.1; found: C, 74.5; H, 5.2; N, 20.3%.
A solution of 27 (0.13 g, 0.06 mmol) and N-ethyldi-iso-
propylamine (1 mL, excess) in CH2Cl2 (5 mL) was
cooled in ice/salt and treated with acryloyl chloride
(1 mL, excess). The mixture was stirred for 30 min at be-
low 0 ꢁC, water (20 mL) and dilute AcOH (10 mL) were
added. The reaction mixture was extracted with CH2Cl2
(3 · 50 mL). The combined extracts were dried
(Na2SO4) and evaporated to dryness. The residue was
chromatographed on alumina, eluting with CH2Cl2/hex-
ane (20:1) to give N-(9-methylphenazin-1-yl)acrylamide
(29) (0.13 g, 82%) as a yellow solid, mp (CH2Cl2/hexane)
186–187 ꢁC; 1H NMR (CDCl3) d 2.94 (s, 3H, CH3), 5.92
(dd, J = 8.7, 2.6 Hz, 1H, CH@CH2), 6.48–6.60 (m, 2H,
1
(dec); H NMR [(CD3)2SO] d 1.98 (s, 6H, N(CH3)2),
2.47 (t, J = 6.1 Hz, 2H, CH2N(CH3)2), 3.60 (q,
J = 5.8 Hz, 2H, NHCH2), 6.88 (d, J = 8.6 Hz, 2H, H-
20, 60), 6.90 (d, J = 7.1 Hz, 1H, H-8), 7.28 (d,
J = 8.6 Hz, 2H, H-30,50), 7.51 (d, J = 8.6 Hz, 1H, H-6),
7.75 (t, J = 8.2 Hz, 1H, H-7), 8.04 (dd, J = 8.5, 7.2 Hz,
1H, H-3), 8.23 (s, 1H, NH), 8.37 (dd, J = 8.7, 1.3 Hz,
1H, H-4), 8.57 (dd, J = 6.4, 1.3 Hz, 1H, H-2), 9.48 (s,
1H, OH), 9.84 (t, J = 5.0 Hz, 1H, CONH); Anal. Calcd
for C23H23N5O2: C, 68.8; H, 5.8; N, 17; found: C, 68.5;
H, 5.5; N, 17.4%.

1166
S. A. Gamage et al. / Bioorg. Med. Chem. 14 (2006) 1160–1168
CH2@CH), 7.60 (d, J = 6.7 Hz, 1H, H-8), 7.78 (dd,
J = 8.7, 6.8 Hz, 1H, H-3), 7.86 (dd, J = 8.8, 7.4 Hz,
1H, H-7), 7.95 (dd, J = 8.8, 1.1 Hz, 1H, H-4), 8.12 (dd,
J = 8.7, 1.1 Hz, 1H, H-6), 8.89 (dd, J = 7.5, 1.2 Hz,
1H, H-2), 10.03 (br s, 1H, NH). Anal. Calcd for
C16H13N3O: C, 73.0; H, 5.0; N,16.0; found: C, 72.8;
H, 5.9; N, 15.8%.
9.93 (m, 1H, ArH), 11.52 (br s, 1H, NH); Anal. Calcd
for C21H20N4O: C, 73.2; H, 5.9; N, 16.3; found: C,
73.0; H, 5.6; N, 16.2%.
4.2.16. 3-(Morpholino)-N-(8,9-benzophenazin-1-yl)propane-
carboxamide (34). Similar reaction of 30 with morpholine
gave 34 (77%): mp (CH2Cl2/hexane) 189–190 ꢁC; 1H
NMR (CDCl3) d 2.66 (t, J = 4.4 Hz, 4H, 2· CH2),
2.89–2.93 (m, 2H, CH2), 2.98–3.01 (m, 2H, CH2), 3.73
(t, J = 4.6 Hz, 4H, 2· CH2), 7.81–7.86 (m, 2H,
2· ArH), 7.88 (dd, J = 8.6, 7.7 Hz, 1H, H-3), 7.96–8.01
(m, 2H, 2· ArH), 8.01 (d, J = 9.3 Hz, 1H, ArH), 8.07
(d, J = 9.3 Hz, 1H, ArH), 8.88 (dd, J = 7.6, 1.0 Hz,
1H, ArH), 9.29–9.34 (m, 1H, ArH), 10.25 (br s, 1H,
NH); Anal. Calcd for C23H22N2O2Æ1.5 H2O: C, 66.8;
H, 6.0; N, 13.6; found: C, 66.7; H, 5.3; N, 13.3%.
A solution of 29 (0.11 g, 0.43 mmol) and 40% aqueous
dimethylamine (1 mL) in EtOH (5 mL) was heated un-
der reflux for 10 min. Solvents were removed under re-
duced pressure, and the residue was chromatographed
on alumina, eluting with CH2Cl2/MeOH (trace), to give
31 (0.12 g, 95%) as a yellow solid, mp (CH2Cl2/hexane)
113–114 ꢁC; 1H NMR (CDCl3) d 2.42 (s, 6H, N(CH3)2),
2.75–2.82 (m, 4H, 2· CH2), 2.98 (s, 3H, CH3), 7.69 (td,
J = 6.7, 1.1 Hz, 1H, H-8), 7.76 (dd, J = 8.7, 6.8 Hz, 1H,
H-3), 7.83 (dd, J = 8.6, 7.6 Hz, 1H, H-7), 7.92 (dd,
J = 8.8, 1.1 Hz, 1H, H-4), 8.10 (dd, J = 8.4, 0.9 Hz,
1H, H-6), 8.89 (dd, J = 7.5, 1.2 Hz, 1H, H-2), 10.96
(br s, 1H, NH); Anal. Calcd for C18H20N4O: C, 70.1;
H, 6.5; N, 18.2; found: C, 69.9; H, 6.5; N, 18.1%.
4.2.17. 2-Dimethylamino-N-(9-methylphenazin-1-yl)ethane-
carboxamide (37). Bromoacetyl bromide (0.5 mL) was
added to a solution of 27 (0.1 g, 0.28 mmol) in CH2Cl2
(10 mL) and stirred for 20 min at room temperature.
Water (20 mL) was added and the organic layer was sep-
arated, dried (Na2SO4), and the solvent was evaporated.
The resulting residue was chromatographed on silica gel
(20–40 lm), eluting with CH2Cl2/MeOH (1000:1), to give
N-(9-methylphenazin-1-yl)-2-bromoethanecarboxamide (35)
(0.13 g, 80%) as a yellow solid: mp (CH2Cl2/hexane) 178–
4.2.14. 3-(Morpholino)-N-(9-methylphenazin-1-yl)propane-
carboxamide (33). Similar reaction of 29 with morpholine
gave 33 (95%): mp (CH2Cl2/hexane) 159–160 ꢁC; 1H
NMR (CDCl3) d 2.60 (t, J = 4.5 Hz, 4H, 2· CH2),
2.82–2.86 (m, 2H, CH2), 2.93–2.96 (m, 5H, CH2 and
CH3), 3.73 (t, J = 7.2 Hz, 4H, 2· CH2), 7.71 (dd,
J = 5.8, 0.9 Hz, 1H, H-8), 7.78 (dd, J = 8.7, 6.9 Hz, 1H,
H-3), 7,84 (dd, J = 8.8, 7.8 Hz, 1H, H-7), 7.94 (dd,
J = 8.9, 1.2 Hz, 1H, H-4), 8.12 (d, J = 8.7 Hz, 1H, H-6),
8.88 (dd, J = 7.3, 1.0 Hz, 1H, H-2), 9.98 (br s, 1H,
NH); Anal. Calcd for C20H22N4O2: C,68.6; H, 6.3; N,
16.0; found: C, 68.3; H, 6.3; N, 16.1%.
1
181 ꢁC. H NMR (CDCl3) d 2.96 (s, 3H, CH3), 4.25 (s,
2H, CH2), 7.71 (d, J = 6.7 Hz, 1H, H-8), 7.78 (dd,
J = 8.7, 6.6 Hz, 1H, H-3), 7.85 (dd, J = 8.8, 7.5 Hz, 1H,
H-7), 7.98 (dd, J = 9.0, 1.1 Hz, 1H, H-6), 8.12 (d,
J = 8.7 Hz, 1H, H-4), 8.76 (dd, J = 7.3, 0.8 Hz, 1H, H-2),
11.12 (br s, 1H, NH). Anal. Calcd for C15H12N3OBrÆ0.25-
H2O: C, 53.8; H, 3.8; N, 12.6; found C, 53.8; H, 3.9; N,
11.9%.
4.2.15. 3-Dimethylamino-N-(8,9-benzophenazin-1-yl)pro-
panecarboxamide (32). Similar reaction of 8,9-benzophen-
azine-1-carboxylic acid (26)13 gave 8,9-benzophenazine-1-
amine (28) (79%): mp (CH2Cl2/hexane) 204–208 ꢁC; H
NMR (CDCl3) d 5.32 (br s, 2H, NH2), 7.03 (d,
J = 7.3 Hz, 1H, H-2), 7.61–7.69 (m, 2H, 2· ArH), 7.74–
7.80 (m, 2H, 2· ArH), 7.91–8.02 (m, 3H, 3· ArH), 9.37
(d, J = 8.4 Hz, 1H- H-7); Anal. Calcd for C16H11N3: C,
78.4; H, 4.5; N, 17; found: C, 78.3; H, 4.3; N, 17.1%.
A mixture of 35 (0.1 g, 0.34 mmol) and dimethylamine
(40%, 5 mL) in EtOH (10 mL) was heated under reflux
for 3 h. The solvents and the excess reagent were re-
moved under reduced pressure, and the residue was
chromatographed on alumina, eluting with CH2Cl2, to
give 37 (0.95 g, 100%): mp (CH2Cl2/hexane) 203–
204 ꢁC; 1H NMR (CDCl3) d 2.39 (s, 6H, N(CH3)2),
2.95 (s, 3H, CH3), 3.30 (s, 2H, CH2), 7.69 (td, J = 6.9,
1.2 Hz, 1H, H-8), 7.76 (dd, J = 8.7, 6.8 Hz, 1H, H-3),
7.84 (dd, J = 8.8, 7.4 Hz, 1H, H-7), 7.93 (dd, J = 8.9,
1.3 Hz, 1H, H-6), 8.11 (d, J = 8.9 Hz, 1H, H-4), 8.79
(dd, J = 7.2, 1.2 Hz, 1H, H-2), 11.75 (br s, 1H, NH);
Anal. Calcd for C17H18N4O: C, 69.4; H, 6.2; N, 19.0;
found: 69.2; H, 6.1; N, 18.9%.
1
Coupling of 28 as above with acryloyl chloride gave
N-(8,9-benzophenazin-1-yl)acrylamide (30) (78%): mp
(CH2Cl2/hexane) 205–207 ꢁC; 1H NMR (CDCl3) d
5.97 (q, J = 3.8 Hz, 1H, CH@CH2), 6.60–6.69 (m, 2H,
CH2@CH), 7.81–7.92 (m, 3H, 3· ArH), 7.96–8.03 (m,
3H, 3· ArH), 8.07 (d, J = 9.3 Hz, 1H, ArH), 8.98 (dd,
J = 7.6, 0.9 Hz, 1H, ArH), 9.30 (dd, J = 8.0, 2.4 Hz,
1H, ArH), 10.09 (br s, 1H, NH);. Anal. Calcd for
C19H13N3O: C, 76.2; H, 4.4; N, 14.0; Found: C, 76.0;
H, 4.3; N, 14.1%.
4.2.18. 2-Dimethylamino-N-(8,9-benzophenazin-1-yl)eth-
anecarboxamide (38). Similar reaction of 28 with bromo-
acetyl bromide gave N-(8,9-benzophenazin-1-yl)-2-
bromoethanecarboxamide (36) (72%) as a yellow solid:
1
mp (CH2Cl2/hexane) 210–213 ꢁC; H NMR (CDCl3) d
4.31 (s, 2H, CH2), 7.80–7.87 (m, 3H, 3· ArH), 7.89
(dd, J = 8.6, 7.7 Hz, 1H, H-3). 7.91–8.05 (m, 2H,
2· ArH), 8.08 (d, J = 9.3 Hz, 1H, ArH), 8.83 (dd,
J = 7.6, 1.0 Hz, 1H, ArH), 9.37–9.42 (m, 1H, ArH),
11.18 (br s, 1H, NH); Anal. Calcd for C18H12N3O-
BrÆ0.25H2O: C, 58.3; H, 3.4; N, 11.3; found: C, 58.2;
H, 3.0; N, 11.2%.
Similar reaction of 30 with dimethylamine gave 32
(85%): mp (CH2Cl2/hexane) 146–148 ꢁC; 1H NMR
(CDCl3) d 2.55 (s, 6H, N(CH3)2), 2.79–2.85 (m, 4H,
2· CH2), 7.78–7.85 (m, 2H, 2· ArH), 7.87 (dd,
J = 8.6, 7.9 Hz, 1H, H-3), 7.94–8.06 (m, 4H,
4· ArH), 8.97 (dd, J = 7.6, 1.3 Hz, 1H, ArH), 9.45–

S. A. Gamage et al. / Bioorg. Med. Chem. 14 (2006) 1160–1168
1167
Similar reaction of 36 with dimethylamine gave 38 (99%)
as a yellow solid: mp (CH2Cl2/hexane) 212–213 ꢁC; H
(40 mL), and the mixture was heated at 50 ꢁC for
30 min. A solution of 41 (0.08 g, 0.36 mmol) in THF
(2 mL) was then added, and the mixture was heated to
100 ꢁC and stirred at this temperature for 2 h. The
cooled solution was made slightly acidic with aqueous
HCl and extracted with EtOAc. The aqueous layer
was basified with aqueous NaHCO3 and extracted with
EtOAc (4 · 50 mL). These combined extracts were dried
(Na2SO4) and the solvent was evaporated. The residue
was chromatographed on silica gel, eluting with CH2Cl2,
1
NMR (CDCl3) d 2.66 (s, 6H, N(CH3)2), 3.35 (s, 2H,
CH2), 7.80–7.84 (m, 2H, 2· ArH), 7.88 (dd,J = 8.6,
7.8 Hz, 1H, ArH), 7.95–8.00 (m, 2H, 2· ArH), 8.01 (d,
J = 9.5 Hz, 1H, ArH), 8.06 (d, J = 9.4 Hz, 1H, ArH),
8.84 (dd, J = 7.6, 1.0 Hz, 1H, ArH), 9.36–9.40 (m, 1H,
ArH), 11.88 (br s, 1H, NH); Anal. Calcd for
C20H18N4O: C, 72.7; H, 5.5; N, 17.0; found: C, 72.5;
H, 5.6; 17.0%.
1
to give 42 (0.11 g, 100%) as a yellow foam; H NMR
4.2.19. 9-Methyl-1-[(3-dimethylamino)propoxy]phenazine
(42). A mixture of 13 (0.3 g, 1.2 mmol) and CDI (0.58 g,
3.6 mmol) in THF (25 mL) was stirred overnight at room
temperature and then slowly added to a solution of
NaBH4 in H2O (20 mL). After stirring for 15 min, the
mixture was neutralized by dropwise addition of concd
HCl and then extracted with EtOAc. The organic layer
was washed with water, dried (Na2SO4), and evaporated.
The resulting crude product was chromatographed on sil-
ica gel, eluting with CH2Cl2/MeOH (300:1), to give 1-hy-
droxymethyl-9-fluorophenazine (39) (0.23 g, 85%): mp
(CDCl3) d 2.50 (quin, J = 6.8 Hz, 2H, CH2CH2CH2),
2.37 (s, 6H, N(CH3)2), 2.74 (t, J = 7.3 Hz, 2H,
CH2N(CH3)2), 2.96 (s, 3H, CH3), 4.36 (t, J = 6.4 Hz,
2H, OCH2), 7.10 (dd, J = 7.6, 0.8 Hz, 1H, H-2), 7.65
(dd, J = 6.7, 1.5 Hz, 1H, ArH), 7.70–7.75 (m, 2H,
2· ArH), 7.81 (dd, J = 9.0, 1.1 Hz, 1H, ArH), 8.05 (d,
J = 8.7 Hz, 1H, ArH); Anal. Calcd for C18H21N3O: C,
73.2; H, 7.4; N, 14.2; found: C, 73.2; H, 7.4; 13.9%.
4.2.20. N-[(3-Dimethylamino)propyl]-9-methylphenazin-1-
amine (43). A solution of 31 (0.014 g, 0.045 mmol) in
dry THF (2 mL) was treated with BH3-SMe2 (0.5 mL,
1 mmol) and heated under reflux for 10 min. The excess
reagent was quenched by addition of H2O (100 mL)
and then the reaction mixture was extracted with CH2Cl2
to give a crude boron complex of the product. This was
filtered through silica gel in CH2Cl2/MeOH (500:1) to re-
move baseline material, and the purified complex (0.01 g)
was dissolved in THF (2 mL) and 2 N HCl (4 mL) and
heated under reflux for 20 min. The cooled mixture was
basified with aqueous ammonia and extracted with
EtOAc. The organic layer was dried (Na2SO4), evaporat-
ed, and the residue was suction chromatographed on sil-
ica gel (20–40 lm), eluting with CH2Cl2/MeOH (95:5), to
give N-[(3-dimethylamino)propyl]-9-methylphenazin-1-
amine (43) (0.01, 50%) as a red foam; 1H NMR (CDCl3)
d 2.00 (quin, J = 6.7 Hz, 2H, CH2CH2CH2), 2.32 (s, 6H,
N(CH3)2), 2.52 (t, J = 6.7 Hz, 2H, CH2N(CH3)2), 2.92 (s,
3H, CH3), 3.48 (q, J = 6.2 Hz, 2H, NHCH2), 6.36 (d,
J = 7.6 Hz, 1H, H-2), 6.95 (br s, 1H, NH), 7.42 (dd,
J = 8.2, 0.9 Hz, 1H, ArH), 7.59 (dt, J = 6.8, 1.1 Hz, 1H,
ArH), 7.65–7.71 (m, 2H, ArH), 8.04 (d, J = 8.7 Hz, 1H,
ArH); Anal. Calcd for C18H23N4: C, 73.4; H, 7.5; N,
19.0; found: C, 73.5; H, 7.8; N; 18.7%.
1
(CH2Cl2/hexane) 180–182 ꢁC; H NMR (CDCl3) d 4.30
(t, J = 6.8 Hz, 1H, OH), 5.36 (d, J = 6.8 Hz, 2H, CH2),
7.49–7.54 (m, 1H, H-8), 7.76–7.85 (m, 3H, ArH), 8.07
(dd, J = 10.1, 1.0 Hz, 1H, ArH), 8.18 (dd, J = 8.6,
1.5 Hz, 1H, ArH); HRMS (EI) calcd for C13H9N2FO
(M+) m/z 228.068, found: 228.0694.
Triphenylphosphine (0.71 g, 2.7 mmol) was dissolved in
dry MeCN (15 mL) and bromine (139 lL, 2.7 mmol)
was added dropwise to give a slightly warm pale yellow
solution. A solution of 39 (0.44 g, 1.93 mmol) in MeCN
(10 mL) was then added, giving an immediate orange pre-
cipitate. After stirring at room temperature for 1 h, the
solvent was evaporated and the residue was chromato-
graphed on silica gel, eluting with CH2Cl2/hexane (1:1),
to give (9-fluorophenazin-1-yl)methyl bromide (40)
(0.57 g, 100%); mp (CH2Cl2/hexane) 151–152 ꢁC; 1H
NMR (CDCl3) d 5.37 (s, 2H, CH2), 7.54 (ddd, J = 9.7,
7.7, 1.0 Hz, 1H, H-8), 7.76–7.83 (m, 1H, ArH), 7.86 (dd,
J = 8.8, 6.9 Hz, 1H, H-3), 8.02 (d, J = 6.0 Hz, 1H, ArH),
8.07 (d, J = 8.8 Hz, 1H, ArH), 8.22 (dd, J = 8.8, 1.2 Hz,
1H, ArH); HRMS (EI) (M+) m/z calcd for C14H11N279Br
286.0106, found: 286.0082 and for C14H11N281Br
288.0085, found: 288.0084.
4.2.21. N-[(3-Dimethylamino)propyl]-8,9-benzophenazine-
1-amine (44). A solution of 32 (0.073 g, 0.2 mmol) in dry
THF (5 mL) was treated with BH3-SMe2 (1 mL, 2 mmol)
and heated under reflux for 1 h. The excess reagent was
quenched by addition of H2O (20 mL) and then the reac-
tion mixture was extracted with EtOAc to give a boron
complex of the product. This was filtered through silica
gel in CH2Cl2/MeOH (500:1) to remove baseline material,
and the purified complex (0.06 g) was dissolved in THF
(5 mL) and 2 N HCl (8 mL) and heated under reflux over-
night. The cooled mixture was basified with aqueous
ammonia and extracted with EtOAc. The organic layer
was dried (Na2SO4), evaporated, and the residue was chro-
matographed on silica gel, eluting with CH2Cl2/MeOH
(500:1), followed by preparative HPLC, to give 44
(0.02 g, 29%)asaredfoam; 1H NMR (CDCl3) d2.05(quin,
J = 6.6 Hz, 2H, CH2CH2CH2), 2.37 (s, 6H, N(CH3)2), 2.57
A stirred solution of 40 (0.13 g, 0.44 mmol) in DMSO
(2 mL) was treated with NaBH4 (0.065 g, 1.8 mmol) at
room temperature for 30 min. The reaction mixture
was then diluted with water and extracted with EtOAc
to give a crude product which was chromatographed
on silica gel, eluting with CH2Cl2/hexane (9:1), to give
1-fluoro-9-methylphenazine (41) (0.079 g, 85%): mp:
(CH2Cl2/hexane) 156–158 ꢁC; 1H NMR (CDCl3) d
2.96 (s, 3H, CH3), 7.49 (ddd, J = 10.6, 8.3, 1.1 Hz, 1H,
H-8), 7.70 (dt, J = 6.7, 1.0 Hz, 1H, ArH), 7.74–7.80
(m, 2H, ArH), 8.07 (t, J = 9.2 Hz, 1H, ArH); HRMS
(EI) calcd for C13H9FN2 (M+) m/z 212.0750, found:
212.0747.
Sodium hydride (60%, 0.32 g) was added to a solution of
3-dimethylamino-1-propanol (0.82 g, 80 mmol) in THF

1168
S. A. Gamage et al. / Bioorg. Med. Chem. 14 (2006) 1160–1168
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1994, 22, 2274–2281.
(t, J = 6.6 Hz, CH2N(CH3)2), 3.52 (t, J = 6.6 Hz, 2H,
NHCH2), 6.72 (d, J = 6.6 Hz, 1H, H-2), 7.16 (br s, 1H,
NH), 7.48 (dd, J = 8.7, 0.9 Hz, 1H, ArH), 7.71 (dd,
J = 8.6, 7.7 Hz, 1H, H-3), 7.73–7.81 (m, 2H, ArH), 7.92
(dd, J = 8.1, 1.8 Hz, 1H, ArH), 7.96 (d, J = 9.3 Hz, 1H,
H-6 or H-7), 8.01 (d,J = 9.3 Hz, 1H, H-6 or H-7), 7.37
(dd, J = 7.7, 1.8 Hz, 1H, ArH). HRMS (EI) calcd for
C21H22N4 (M+) m/z 330.18445, found: 330.18376.
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4.2.22. N-[(2-Dimethylamino)ethyl]-9-methylphenazine
methyl amine (45). From reduction of 7 with BH3-
SMe2, and chromatography of the crude product on alu-
1
mina (30%) as a red foam; H NMR (CDCl3) d 2.20 (s,
6H, N(CH3)2), 2.46 (t, J = 6.2 Hz, 2H, CH2N(CH3)2),
2.80 (t, J = 6.2 Hz, 2H, NHCH2), 2.94 (s, 3H, CH3),
4.53 (s, 2H, ArCH2) 7.66 (dt, J = 6.7, 1.2 Hz, 1H,
ArH), 7.66–7.80 (m, 3H, ArH), 8.07 (d, J = 8.7 Hz,
1H, ArH), 8.11–8.15 (m, 1H, ArH). HRMS (EI) calcd
for C18H22N4 (M+) m/z 294.1845, found: 294.1839.
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Acknowledgments
16. Sharma, R.; Voynov, G. H.; Ovaska, T. V.; Marquez, V.
E. Synlett 1995, 839–840.
This work was supported by Xenova plc and by the
Auckland Division ofthe Cancer Societyof New Zealand.
17. Vicker, N.; Burgess, L.; Chuckowree, I. S.; Dodd, R.;
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Charlton, P. A.; Dangerfield, W.; Liddle, C.; Mistry,
P.; Stewart, A. J.; Denny, W. A. J. Med. Chem. 2002,
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