Synthesis and biological evaluation of novel 5(H)-phenanthridin-6-ones, 5(H)-phenanthridin-6-one diketo acid, and polycyclic aromatic diketo acid analogs as new HIV-1 integrase inhibitors

Article abstract of DOI:10.1016/j.bmc.2006.11.026

A new series of phenanthridinone derivatives, and diketo acid analogs, as well as related phenanthrene and anthracene diketo acids have been synthesized and evaluated as HIV integrase (IN) inhibitors. Several new β-diketo acid analogs with the phenanthrid

Full text of DOI:10.1016/j.bmc.2006.11.026

Bioorganic & Medicinal Chemistry 15 (2007) 1212–1228
Synthesis and biological evaluation of novel 5(H)-phenanthridin-6-
ones, 5(H)-phenanthridin-6-one diketo acid, and polycyclic
aromatic diketo acid analogs as new HIV-1
integrase inhibitors
Shivaputra Patil,a Shantaram Kamath,a Tino Sanchez,b Nouri Neamati,b,*
Raymond F. Schinazic and John K. Buolamwinia,*
aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center,
Memphis, TN 38163, USA
bDepartment of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue,
Los Angeles, California 90089, USA
cEmory University School of Medicine/VA Medical Center, Medical Research 151H, 1670 Clairmont Road, Decatur, GA 30033, USA
Received 15 April 2006; revised 4 November 2006; accepted 13 November 2006
Available online 16 November 2006
Abstract—A new series of phenanthridinone derivatives, and diketo acid analogs, as well as related phenanthrene and anthracene
diketo acids have been synthesized and evaluated as HIV integrase (IN) inhibitors. Several new b-diketo acid analogs with the phe-
nanthridinone scaffold replaced by phenanthrene, anthracene or pyrene exhibited the highest IN inhibitory potency. There is a general
selectivity against the integrase strand transfer step. The most potent IN was 2,4-dioxo-4-phenanthren-9-yl-butyric acid (27f) with an
IC50 of 0.38 lM against integrase strand transfer. The phenanthrene diketo acids 27d–f were more potent (IC50 = 2.7–0.38 lM) than
the corresponding phenanthridinone diketo acid 16 (IC50 = 65 lM), suggesting that the polar amide bridge in the phenanthridinone
system decreases inhibitory activity relative to the more lipophilic phenanthrene system. This might have to do with the possible bind-
ing of the aryl group of the compounds binding to a lipophilic pocket at the integrase active site as suggested by the docking simu-
lations. Molecular modeling also suggested that effectiveness of chelation of the active site Mg2+ contributes to IN inhibitory potency.
Finally, some of the potent compounds inhibited HIV-1 replication in human peripheral blood mononuclear cells (PBMC) with EC50
down to 8 lM for phenanthrene-3-(2,4-dioxo)butyric acid (27d), with a selectivity index of 10 against PBMCs.
ꢀ 2006 Published by Elsevier Ltd.
1. Introduction
transcriptase (RT), (2) integrase (IN), and (3) protease
(PR)2 have been the subjects for anti-HIV drug develop-
ment among others. Drugs targeting RT and PR have
been available for over a decade and have shown efficacy
particularly when employed in combination.3–5 Howev-
er, infection still cannot be eradicated completely with
current highly active antiretroviral therapy (HAART)
combination treatments, and toxicity and drug resis-
tance are problems as well.6,7 Thus, there is a need for
new inhibitors that could block the virus at other steps
of its replication cycle. HIV integrase (IN) is an attrac-
tive potential drug target in this regard because it is
responsible for incorporation of HIV provirus into the
host cell genome, is essential for viral replication, and
does not have a direct human counterpart.8–11 The per-
tinent catalytic activity of IN involves: (1) the cleavage
of a dinucleotide fragment from each end of the proviral
Acquired immunodeficiency syndrome (AIDS), a dis-
ease resulting from infection with human immunodefi-
ciency virus (HIV), is one of the world’s most serious
health problems. It is estimated that approximately 39
million people are living with HIV/AIDS worldwide,
with an infection and death rates of around 4 million
and 3 million per year, respectively.1 Three essential en-
zymes are encoded by the HIV pol gene: (1) reverse
Keywords: HIV integrase inhibitors; Phenanthridinones; Phenanthrid-
inone diketo acid; Polycyclic aromatic diketo acids; Inhibition of HIV
replication; Selectivity; Peripheral blood mononuclear cells.
*
Corresponding authors. Tel.: +1 901 448 7533; fax: +1 901 448 6828
(J.K.B.); Tel.: +1 323 442 2341; fax: +1 323 442 1390 (N.N.); e-mail
addresses: neamati@usc.edu; jbuolamwini@utmem.edu
0968-0896/$ - see front matter ꢀ 2006 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2006.11.026

S. Patil et al. / Bioorg. Med. Chem. 15 (2007) 1212–1228
1213
O
O
OH
O
N
N
O
O
N
H
OH
OH
N
O
F
O
O
O
F
N
S
O
N
NH
L-706,908
L-870,810
S-1360
DNA (i.e., 30-end processing) and (2) insertion of this
donor DNA into the host cellular DNA (i.e., strand
transfer).12–14 For almost the past decade and half,
many integrase inhibitors with diverse structural fea-
tures have been identified, designed, synthesized, and
screened for the inhibitory activity, and the results show
R4
R5
R4
R5
+
O
R3
R3
COCl
R3
H
N
R5
R4
R2
R1
NH2
X
R2
R1
R2
R1
N
X
a
+
O
O
X
R4
R5
1
2
3a-l
4a-g
No.
X
Br
Cl
Br
Br
Cl
Cl
Br
Br
Br
Cl
Br
Br
Br
Cl
Br
Cl
Cl
Br
Br
R1
R2
R3
R4
R5
H
3a
3b
3c
3d
3e
3f
CH3
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CN
CN
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
H
CN
H
CF3
CN
CF3
H
H
H
H
3g
3h
3i
CH3
CH3
CH3
H
NO2
H
NO2
H
H
3j
H
H
3k
3l
CF3
H
H
H
COCH3
H
H
H
4a
4b
4c
4d
4e
4f
CH3
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CN
CN
H
H
H
CH3
H
CN
CN
CF3
H
H
H
H
CH3
CH3
NO2
H
4g
NO2
Scheme 1. Reagents and condition: (a) Et3N, CH2Cl2, RT.

1214
S. Patil et al. / Bioorg. Med. Chem. 15 (2007) 1212–1228
R3
ing this enzyme as a bona fide AIDS therapeutic
target.22
R5
R4
H
R3
b
R2
R1
N
O
H
N
R2
R1
O
In our search for new integrase inhibitors, we have dis-
covered a series of new substituted phenanthridinones
and analogous compounds that show inhibitory activity
against the IN catalytic core. The coupling of the phe-
nanthridinone and their biphenyl open ring mimics with
the known IN inhibitory activity conferring diketo acid
moiety enhanced IN inhibitory activity. Further, con-
verting the phenanthridinone heterocyclic ring system
to the more lipophilic phenanthrene or anthracene sys-
tems remarkably elevated IN inhibitory potency. Some
of the phenanthrene compounds also inhibited HIV-1
replication in cell culture. We report herein the synthesis
and biological testing of these diverse new IN inhibitors,
as well as results of molecular modeling conducted to
explore the binding modes of the potent compounds in
the integrase active site.
X
R4
3a-f & 3i-j
5a-j
R5
Comp No. R1
R2
H
H
H
H
H
H
H
H
H
R3
R4
R5
5a
5b
5c
5d
5e
5f
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CN
CN
H
H
H
H
CH3
CH3
H
CN
CF3
CN
CF3
H
H
H
H
H
5g
5h
5i
CH3
H
H
2. Results and discussion
2.1. Chemistry
H
H
CF3
H
H
H
Incorporation of specific substituents on the 5H-phe-
nanthridin-6-one ring system was achieved by using sev-
eral synthetic methods (Schemes 1–5). Among these
approaches the most convenient method was palladi-
um-catalyzed cyclization of substituted benzamides 3a–
l using Na2CO3 in dry dimethylformamide at 165 ꢁC
yielding the substituted 5H-phenanthridin-6-ones 5a–
j.23,24 Substituted benzamides 3a–l were prepared by
reacting various 2-haloanilines 1 with substituted benzo-
yl chlorides 2 in the presence of Et3N in anhydrous
methylene chloride.25,26 N,N-disubstituted benzamide
derivatives 4a–g (2–5%) observed as side products in
case of electron-withdrawing groups were present on
benzoyl chlorides 2 (Schemes 1 and 2).
5j
COCH3
H
H
H
Scheme 2. Reagents and condition: (b) Pd(OAc)2, Na2CO3, DMA,
165 ꢁC.
H
N
H
H
N
O
N
O
O
c
d
NC
X
6
9
7a-b
X= Br, Cl
e
f
In Scheme 3, 2-bromo-5H-phenanthridin-6-ones 7a and
b were prepared according to a reported procedure27 in
good yields (70%). 2-Methoxy-5H-phenanthridin-6-one
8 was synthesized by refluxing 2-bromo-5H-phenanthri-
din-6-one 7a with cuprous iodide and sodium methoxide
in dry DMF.28 Compound 7a was treated with cuprous
cyanide in DMA at 150–155 ꢁC to yield the 2-cyano-5H-
phenanthridin-6-one 9.29 Compound 9 was refluxed in
ethanolic NaOH to obtain 6-oxo-5,6-dihydrophenan-
thridine-2-carboxylic acid 10. A series of N-substitut-
ed-2-amino-5H-phenanthridin-6-ones, 12a–d, were
readily synthesized by stirring 2-amino-5H-phenanthri-
din-6-one, 11, with appropriate acid chlorides (or ethyl
iodide in the case of 12a) and Et3N in dry DMF. Com-
pounds 13a and b and 14a and b were prepared by cou-
pling 2-amino-5H-phenanthridin-6-one 11 with 3- or 4-
bromomethyl benzonitriles by the following three meth-
ods. In method A, 2-amino-5H-phenanthridin-6-one 11
was stirred with 3- or 4-bromomethyl benzonitriles
and Et3N in dry DMF to yield exclusively 13a–b.
Whereas methods B and C, which employed pyridine
and K2CO3 as bases, respectively, gave 13a and b as ma-
H
N
H
N
O
O
HOOC
H3CO
8
10
Scheme 3. Reagents and conditions: (c) NBS/NCS, DMF; (d) CuCN,
DMA, 150–155 ꢁC; (e) CuI, NaOMe, DMF, reflux; (f) NaOH, EtOH,
reflux.
that effective inhibition is possible.15–20 Compounds
incorporating a b-diketo acid (DKA) moiety as present
in L-706,908 or bioisosteric analogs thereof as depicted
by the diketo triazole in S-3160 and the hydroxy-
naphthyridine formyl moiety in L-870,810 have yielded
clinical integrase inhibitor drug candidates.21
Integrase inhibitors can reduce viral load in patients;
at the recent 13th retrovirus conference, Merck and
Gilead reported positive phase II clinical trial results,
respectively, on new integrase inhibitors; thus validat-

S. Patil et al. / Bioorg. Med. Chem. 15 (2007) 1212–1228
1215
H
H
H
N
O
N
O
N
O
H
N
i
j
O
R1
N
N
H2N
+
H
H
N
R2
R2
R1
13a-b
12a-d
11
R1
R2
R1
14a-b
Comp No. R1
Comp No. R1
R2
H
12a
12b
12c
12d
C2H5
13a, 14a
13b, 14b
CN
COCH3
COC6H5
SO2C6H5
H
CN
Scheme 4. Reagents and conditions: (i) Acid chlorides (or ethyl iodide in the case of 12a), Et3N, DMF, RT; (j) 3- or 4-bromomethyl-benzonitrile,
K2CO3, EtOH, reflux or 3- or 4-bromomethyl-benzonitrile, Et3N, DMF, RT or 3- or 4-bromomethyl-benzonitrile, pyridine, DMF.
jor products (60%), and 14a and b as minor products (5–
6%).
O
O
H
H
N
N
O
H3C
H3C
l
O
Br
Scheme 5 shows the synthesis of diketo acids 16 and 18.
Treatment of 3l and 5k with diethyl oxalate and sodium
methoxide provided the 2,4-dioxo-4-(6-oxo-5,6-dihydro-
phenanthridin-3-yl)-butyric acid ethyl ester 15 and 4-(3-
benzoylamino-4-bromo-phenyl)-2,4-dioxobutyric acid
methyl ester 17, which were then hydrolyzed to 2,4-di-
oxo-4-(6-oxo-5,6-dihydrophenanthridin-3-yl)-butyric
acid 16 and 4-(3-benzoylamino-4-bromo-phenyl)-2,4-di-
oxo-butyric acid 18, respectively, using methanolic
NaOH.
5k
3l
o
m
O
O
H
O
O
O
N
O
H
H3CO
N
O
O
O
Br
15
17
n
p
Biphenyl diketo acid 21 was synthesized as depicted in
synthetic Scheme 6. Ullmann coupling of 2-iodobenzoic
acid methyl ester 17 with 1-(4-bromo-3-nitro-phen-
yl)ethanone 18 gave the 40-acetyl-20-nitrobiphenyl-2-car-
boxylic acid methyl ester 19. Reduction of compound 19
with Raney nickel and hydrazine hydrate was intended
to yield the cyclic compound 3-acetyl-5H-phenanthri-
din-6-one 23, but surprisingly we obtained 40-acetyl-20-
aminobiphenyl-2-carboxylic acid methyl ester 22.31
Compound 19 was treated with diethyl oxalate in sodi-
um methoxide to obtain diketoester 20, which was
O
O
H
HO
N
O
O
O
H
HO
N
O
O
O
Br
16
18
Scheme 5. Reagents and conditions: (l) Pd(OAc)2, Na2CO3, DMA,
165 ꢁC; (m) diethyl oxalate, KtOBu, dioxane, reflux; (n) NaOH,
methanol; (o) diethyl oxalate, NaOMe, methanol; (p) NaOH,
methanol.
O
I
O
NO2
O
Br
OCH3
OCH3
q
r
OCH3
+
O
O
OCH3
O2N
O2N
O
O
O
17
18
19
20
s
t
O
O
O
O
OCH3
OCH3
NH
NH
O
O
OH
OH
O2N
H2N
O
O
O
O
O
O
23
24
22
21
Scheme 6. Reagents and conditions: (q) Cu powder, 180–200 ꢁC; (r) diethyl oxolate, NaOMe, MeOH; (s) NaOH, methanol; (t) Raney nickel
NH2NH2, MeOH.

1216
S. Patil et al. / Bioorg. Med. Chem. 15 (2007) 1212–1228
O
O
an electron withdrawing group such as CN at the 8-po-
v
u
R
O
R
R
sition (5b) abolished activity. Introduction of the elec-
tron donating methyl group at the 2-position as in
compound 5a improved inhibitory activity against both
integration steps with IC50 values of 300 and 333 lM
against 30-end processing (cleavage) and strand transfer
steps, respectively. These observations imply that substi-
tution at the phenanthridinone 2-position is important
for inhibitory activity. In other examples, the presence
of halogens like Br and Cl at the 2-position (7a and
7b) conferred moderate inhibitory activity, whereas
more hydrophilic groups like CN and COOH at the
same position produced only low inhibitory activity
compounds (9 and 10), but provided compounds with
selectivity toward strand transfer integration step. Elec-
tron-donating groups like NH2 at the 2-position (com-
pound 11) improved the inhibitory activity against
both 30-end processing (206 lM) and strand transfer
(267 lM) reactions. The NH2 group might also be in-
volved in hydrogen bonding at the binding site.
Compound 14b, which had a bulky N,N-bis-(4-cyanob-
enzyl) substituent, selectively inhibited strand transfer
quite well (IC50 62 11 lM) and was the best in phe-
nanthridinone series, whereas its regioisomer, com-
pound 14a with N,N-bis-(3-cyanobenzyl) substituents,
did not show good inhibitory activity. This indicates
that the 2-position tolerates bulky substituents. The fact
that the para-CN substituted compound (14b) exhibited
higher potency than the meta-CN substituted compound
(14b) could mean that there is a positive and/or hydro-
gen-bond acceptor site that is more accessible to the
para-placed CN than the meta-placed CN group. The
strand transfer inhibitory selectivity of the more potent
compounds in this phenanthridinone series puts them
in the same category of IN inhibitory compounds as
the diketo acid inhibitors.30 This observation prompted
us to try to augment this moderate strand transfer selec-
tivity by introducing a diketo acid moiety onto the phe-
O
OH
O
O
O
O
27a-k
26a-k
25a-k
Compound No.
R
N
O
27a
27b
H
3
C
27c
27d
27e
27f
27g
27h
27i
27j
27k
O
O
N
CH3
nanthridinone
scaffold
to
create
a
hybrid
O
O
pharmacophore. It is important to note that, to date,
one of the most promising classes of integrase inhibitors
are the agents with DKA or bioisosteric substitutions
thereof31,32, therefore we went ahead and appended
the b-diketo acid moiety to the phenanthridinone. This
combination, as exemplified by the first compound syn-
thesized for that purpose, 2,4-dioxo-4-(6-oxo-5,6-dihy-
dro-phenanthridin-3-yl)-butyric acid, 16, indeed led to
a moderate increase in inhibitory activity and selectivity
against the strand transfer reaction, relative to the non-
DKA-substituted phenanthridinones, thus supporting
our hypothesis. It is also interesting that the bis-cyan-
ophenylmethylamine substituted compound, 14b, the
most potent phenanthridinone compound without a
DKA moiety, has about the same potency and selectiv-
ity as the DKA-substituted phenanthridinone, 16. The
bis-N,N-substituted moiety in 14b is reminiscent of a
similar moiety, bis-methoxyphenylmethylamine, found
in IN inhibitory benzimidazole derivatives represented
by compound 28.21
H
C
3
N
CH
3
Scheme 7. Reagents and conditions: (u) diethyl oxalate, NaOMe,
MeOH; (v) NaOH, methanol.
further selectively hydrolyzed to 40-(3-carboxy-3-oxo-
propionyl)-20-nitrobiphenyl-2-carboxylic acid methyl
ester 21 by methanolic NaOH. Scheme 7 shows the
preparation of polycyclic aromatic hydrocarbon diketo
acids. Acetyl derivatives 25a–k were treated with diethyl
oxalate to obtain the corresponding diketo esters 26a–k.
These esters were hydrolyzed directly without further
purification to obtain the desired diketo acids 27a–k in
40–90% yields (Table 1).
2.2. HIV-1 integrase inhibition structure–activity
relationship
Electron withdrawing groups like CN and CF3 at the
9-position of the phenanthridinone scaffold (see 5e and
5f) along with a substituent at the 4-position conferred
moderate inhibitory activity, whereas the presence of
For the acquisition of more SAR data, we next turned
our attention to examining the following: (1) opening
of the central lactam ring of the phenanthridinone ring

S. Patil et al. / Bioorg. Med. Chem. 15 (2007) 1212–1228
1217
O
structural modifications turned out to be very interest-
ing with regard to SAR as follows: (1) the benzylphenyl
ketone DKAs (17 and 18) were practically inactive,
whereas the biphenyl-b-diketo ester, 20, and its acid
hydrolysis product 21 displayed better inhibitory activi-
ty against strand transfer with IC50 values of 27 and
7 lM, respectively (Table 2), than the phenanthridinone
DKA, indicating that opening of the central lactam ring
of phenanthridinone scaffold to a biphenyl system can
lead to enhanced inhibitory activity, (2) the morpholinyl
phenyl compound, 27a, also had improved strand trans-
fer inhibitory activity (IC50 = 23 lM, Table 2), and (3)
replacement of the phenanthridinone system with the
more hydrophobic phenanthrene ring system, as well
as other polycarbocyclic aromatics such as anthracene
and pyrene, led to a marked increase in IN inhibitory
activity, with strand transfer inhibitory activity reaching
submicromolar levels. For example, compounds 27f and
27i exhibited IC50 of 0.38 and 0.53 lM, respectively (Ta-
N
NH2
N
H
N
O
28
system to afford benzylphenyl ketones and biphenyl
compounds as mimics of ring open phenanthridinone
analogs, (2) changing one of the phenyl rings of the
resulting biphenyl compounds to a reduced heterocycle,
and (3) bioisosteric replacement of the phenanthridi-
none ring system in DKA compound (16) with a phen-
anthrene, anthracene, other polycyclic aromatic
systems or heteroaromatic systems like azaxanthone,
to obtain diverse aromatic DKAs. The results of these
Table 1. Inhibition of HIV-1 Integrase catalytic activities
R2
H
N
O
R1
R3
R4
Compound
R1
R2
R3
R4
30-end processing IC50 (lM)
ST IC50 (lM)
5a
5b
5c
5d
5e
5f
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CN
CN
H
H
300
>500
333
H
CH3
H
>500
>500
>500
500
CN
CF3
CN
CF3
H
>500
>500
CH3
H
H
H
>500
449 99
1000
H
CH3
H
109 41
>1000
>1000
900
5g
5h
5i
H
H
CF3
H
H
>1000
>1000
225 48
500
H
H
7a
7b
8
Br
Cl
H
H
H
78 37
100
500
H
H
H
OCH3
CN
H
H
H
>500
>500
>500
9
H
H
H
400
203
10
11
12a
12b
12c
12d
COOH
NH2
H
H
H
H
H
H
206 110
>500
>500
267 153
>500
500
NHC2H5
NH–COCH3
NH–COPh
NH–SO2Ph
H
H
H
H
H
H
>500
>500
>500
415
H
H
H
H
H
H
HN
13a
13b
>500
>500
>500
>500
CN
HN
H
H
H
H
H
H
CN
N
NC
14a
>500
500
>500
CN
CN
N
14b
H
H
H
62 11
NC
S1360 (standard)
N/A
N/A
N/A
N/A
11.0
2
0.6 0.1

1218
S. Patil et al. / Bioorg. Med. Chem. 15 (2007) 1212–1228
Table 2. Inhibition of HIV-1 integrase catalytic activities of diketo acid analogs
O
O
OR2
R1
O
Compound
R1
R2
30-end processing IC50 (lM)
ST IC50 (lM)
H
N
O
16
H
400 10
65 10
17
18
CH3
H
>500
>500
>500
>500
O
O
Br
Br
NO2
20
CH3
102
3
27 15
H3CO
O
O
NO2
21
H
H
26 10
7
1
9
H3CO
27a
>100
23
N
O
27b
27c
H
H
54 16
7
3
3
2
11
7
H3C
27d
27e
H
H
25
23
7
6
2.7
4.8
2
2
27f
H
9
2
9
0.38 0.11
27g
27h
H
H
32
13
4
54 10
1.7
27i
H
7
2
0.53 0.25

S. Patil et al. / Bioorg. Med. Chem. 15 (2007) 1212–1228
1219
Table 2 (continued)
Compound
27j
R1
R2
H
30-end processing IC50 (lM)
ST IC50 (lM)
35 15
>100
O
O
N
CH3
O
H3C
27k
H
92
5
2
16
8
O
N
CH3
S1360 (standard)
N/A
N/A
11.0
0.6 0.1
ble 2). These compounds turned out to be the most po-
tent and selective compounds synthesized in our study,
with 27f and 27i being 13- and 24-fold selective toward
strand transfer relative to 30-end processing, respective-
ly. The azaxanthone DKAs 27j and 27k also showed
moderate improvement in inhibitory activity over the
phenanthridinone DKA compound 16. Thus, the prefer-
ence of a very hydrophobic polycyclic aromatic moiety
of these new aryl DKAs is clearly indicated by the
results.
for the ability to inhibit HIV replication in primary hu-
man peripheral blood mononuclear cells (PBMC). The
toxicity of the compounds was also evaluated using
three cell types: PBMC, human CEM lymphoblastic leu-
kemia cells, and African green monkey Vero cells. The
results are presented in Table 3 and indicate that some
of the compounds exhibit selective inhibition of HIV
replication in cell culture. The most potent compound,
27d, exhibited an EC50 of 8.0 lM in these initial results,
which is reasonable anti-viral activity when compared to
the anti-viral activity of reported diketo acid IN inhibi-
tors. For example, compound L-706,908 (vide supra)
has been shown to inhibit HIV replication with an
EC50 of 5.7 4.7.33 To put these results in perspective
with regard to AZT’s activity, in the same assay, the
known diketo acid IN inhibitor L-706,90821 exhibited
an EC50 of 5.7 4.7 lM, and AZT exhibited an EC50
of 0.001 lM.33 Thus, the large difference between the
EC50 values of AZT and compound 27d is not unusual
for DKAs. Moreover, this cell culture HIV inhibitory
activity of 27d is comparable to that of 5H-pyrano[2,3-
d:-6,5-d0]dipyrimidines reported recently such as V-165
(EC50 of 14.7 lM).34 Interestingly, compound 27d also
exhibited a reasonable therapeutic index, with a selectiv-
ity index of 10 with reference to PBMC, the cell system
used for the anti-viral assays. One aspect of the biolog-
ical activity that is notable is that, the activity was not
correlated with IN inhibitory activity. Compound 27f
is the most potent IN inhibitor among the new com-
pounds, but it exhibited a higher EC50 (83.7 lM) than
compound 27d (EC50 = 8 lM), which exhibited the
highest activity against HIV replication in PBMC. Com-
pound 27e was the second best inhibitor of HIV replica-
tion, whereas compound 27h was virtually inactive.
The concern with these flat polycyclic aromatics is that
they may be DNA intercalators, and possibly interfering
with donor and/or target in the integration step. This ap-
pears not to be the case because we did not observe DNA
retardation in our gel electrophoresis experiments. An
interesting observation, however, is that the position of
diketo acid substitution on these polycyclic aromatic
compounds is very important in the SAR. Substitutions
that have the diketo acid long axis aligned with the long
axis of the polycyclic compound (as in 27d, 27e, and
27g) are less potent than the isomers in which the diketo
acid long axis is perpendicular or almost perpendicular
to the long axis of the polycyclic system (as in 27f and
27h). This positional effect seems to be contrary to a
DNA intercalation mechanism, as the former compounds
(i.e., 27d, 27e, and 27g) should probably more easily inter-
calate than the latter compounds (i.e., 27f and 27h), and
thus be more potent as well, but that was not the case.
2.3. Inhibition of HIV replication in culture
To assess the potential utility of the new compounds as
anti-HIV agents, we tested selected potent compounds
Table 3. Anti-HIV activity of selected novel potent diketo acids
Compound
Anti-HIV-1 activity in PBMCsa
Slope
R
Cytotoxicity (IC50, lM) in
EC50(lM)b
EC90 (lM)b
PBMCs
CEM
Vero
AZT (control)
27e
0.00073
26.6
83.7
0.019
>100
>100
72.8
0.68 0.06
1.5 0.26
1.1 0.37
1.0 0.19
0.99
0.98
1.0
>100
>100
83.5
81.0
>100
NA
14.3
16.2
73.1
56.4
>100
NA
50.0
10.8
>100
>100
>100
NA
27f
27d
8.0
>100
5.7 4.7c
0.96
27h
L-706,908
>100
NA
NA
NA
a Human peripheral blood mononuclear cells (PBMCs) were infected with HIV-1/LAI.
b EC50 and EC90 are the effective concentrations inhibiting 50% and 90% HIV replication, respectively.
c In the human leukemic T-cell line MOLT-4; in that assay, the EC50 of AZT was 0.001 lM.34

1220
S. Patil et al. / Bioorg. Med. Chem. 15 (2007) 1212–1228
Interestingly, the two least potent inhibitors of HIV rep-
lication, among compounds tested, were compounds 27f
and 27h in which the diketo acid long axis is perpendic-
ular or almost perpendicular to the long axis of the poly-
cyclic ring system, whereas the more potent HIV
inhibitory compounds 27d and 27e have the diketo acid
long axis aligned with the long axis of the polycyclic sys-
tem. However, these examples are too few for any con-
clusions to be drawn. The differences in anti-viral
activity could also be the result of solubility or cell per-
meability differences among the compounds. These fac-
tors will be examined in future testing.
Figure 1. Docking of phenanthridinone diketo acid and polycyclic aromatic diketo acids into HIV integrase catalytic site. Binding modes depicted
are: (a) parent phenanthridinone diketo acid, compound 16, (b) phenanthrene-3-b-diketo acid, compound 27d, (c) phenanthrene-2-b-diketo acid,
compound 27e, (d) phenanthrene-9-b-diketo acid, compound 27f, (e) anthracene-2-b-diketo acid, compound 27g, and (f) anthracene-9-b-diketo acid,
compound 27h. The docked compounds are shown as colored cyan. The protein is represented by gray ribbons, and, active site amino acid residues
within 5 A radius of each docked compound are displayed in green. The Mg2+ ion is colored magenta. Other atoms are colored as follows: O, red; N,
˚
blue; S, yellow. Hydrogen atoms have been omitted to reduce crowding.

S. Patil et al. / Bioorg. Med. Chem. 15 (2007) 1212–1228
1221
2.4. Molecular modeling
rings, while hydrophilic residues within the vicinity of
the Mg2+ ion such as Asp64, Asp116, and Asn155 inter-
acted with the polar diketo acid moiety region of the
molecules. This might have to do with the possible bind-
ing of the aryl group of the compounds binding to a lipo-
philic region of the integrase active site as suggested by
the docking simulations.
To explore the binding modes of the new compounds at
the HIV integrase catalytic site, we conducted binding
simulations using the GOLD molecular docking pro-
gram35 for the phenanthridinone diketo acid and the po-
tent polycyclic diketo acids as presented in Figure 1. We
used the crystal structure of the integrase catalytic core in
which the flexible catalytic loop has been resolved, PDB
ID 1BIS,36 to perform docking simulations. We docked
the phenanthridinone diketo acid compound 16, and sev-
eral potent phenanthrene and anthracene diketo acids.
As can be seen in the panels in Figure 1, the aryl part
of compounds binds into the hydrophobic pocket involv-
ing the flexible catalytic loop region, while the diketo
acid moiety is extended to interact with the Mg2+ ion
as one would expect since it is generally believed that
b-diketo acid moiety chelates Mg,37,38 the most probable
divalent cation in the active site of IN in vivo, although
this has not yet been proven. The degree of interaction
between the DKA moiety and the Mg ion appears to cor-
relate with potency, with the more potent compounds,
such as compounds 27f (Fig. 1d) and 27h (Fig. 1f), hav-
ing two oxygen atoms, the enolized oxygen, and one of
the carboxylate oxygens interacting with the Mg ion.
This is not the case in their less potent counterparts, com-
pounds 16, 27d, 27e, and 27g, shown in Figure 1a–c and
e, respectively, where only one oxygen atom, a carboxyl-
ate oxygen, is interacting with the Mg ion. This implies
that the more potent compounds are able to dock in a
pose (configuration) that allows more effective chelation
of the active site Mg2+ ion. The favorable orientation of
the diketo acid moiety in the compounds such that there
was more effective chelation of the active site Mg2+ is also
suggested by the docking results to influence IN inhibito-
ry activity, with the compounds which involved two oxy-
gen atoms of the diketo acid in interaction with the Mg
ion being more potent than those that involved only
one oxygen in this interaction. In addition to binding
to the active site Mg2+ ion to different extents, the dock-
ing results suggest these inhibitors also interacted with
integrase active site amino acid residues to varying de-
3. Conclusion
In summary, we have successfully synthesized and eval-
uated the HIV-1 integrase inhibitory activities of series
of new phenanthridinones, diketo acid substituted phe-
nanthridinones, and related compounds. Generally, the
compounds were selective against IN strand transfer
step as compared to the 30-end processing step of inte-
gration, similar to the much studied DKA class of IN
inhibitors.21 Introduction of the DKA functionality into
the phenanthridinone system resulted in enhancement of
inhibitory activity, and replacement of the phenanthrid-
inone ring system with more lipophilic polycyclic aro-
matic bioisosteres dramatically increased IN inhibitory
potency. Molecular docking simulations identified a
possible binding region in the IN catalytic site for these
novel potent polycyclic DKAs that could further drug
design. More importantly, some of the new potent phen-
anthrene diketo acid analogs also inhibited HIV replica-
tion in cell culture, with good selective toxicity. To the
best of our knowledge, this is the first report of potent
phenanthrene, anthracene, and pyrene DKA IN inhibi-
tors, and the fact that they possess anti-HIV activity in
cell culture with low toxicity makes them a new lead ser-
ies for further exploration in attempts to discover novel
DKA IN inhibitors. Thus, we have expanded the DKA
IN inhibitor armentarium, and this is timely as clinical
results are beginning to show promise for the use of
DKAs as anti-HIV therapeutics.22
4. Experimental
˚
grees (amino acid residues within 5 A radius of each
All the reagents and solvents were purchased from Al-
drich and used without further purification. Progress
of all reactions were monitored by TLC on silica gel
plates (Analtech, Inc.). Most of the reactions were per-
docked compound are displayed in Fig. 1) as described
in the following. Compound 16, which has the phenanth-
ridinone moiety, interacts extensively with Asp64,
Asp116, and Pro142, and substantially with His114
and Thr115, and to a limited extent with Gln62 and
Ile151. Compound 27d interacts extensively with
Asp116, and also interacts substantially with Thr115,
Gly140, and Ile141. It also interacts with Phe139. Com-
pound 27e interacted extensively with Gln62, His114,
and Pro142, substantially with Thr115 and Ile151, and
limited interaction with Asp116. Compound 27f interact-
ed extensively with His114, Asp116, Pro142, substantial
interaction with Ile151, and limited interaction with
Gln62 and Thr115. Compound 27g interacted extensive-
ly with Asp116, substantially with Gln62, Phe139, and
Pro142, and limited interaction with Gly140. Compound
27h interacted extensively with Asp64 and Asn155, and
substantially with Ile151 and Glu152. Hydrophobic res-
idues in the catalytic loop like Gly140, Pro142, and
Ile151 interact with the lipophilic polycyclic aromatic
1
formed under nitrogen atmosphere. H NMR spectra
were recorded on Brucker AR, 300-MHz spectrometer:
chemical shifts are expressed in d values (ppm) and cou-
pling constants (J) in Hertz. Mass spectral data were
determined on a Brucker-HP Esquire-LC spectrometer
(ESI-MS). Fisher scientific Da visil grade 1740 (170–
400 mesh) silica gel was used for flash column chroma-
tography. Elemental analysis (C, H, N) was performed
by Atlantic Microlab, Inc. (Norcross, GA), and results
were within 0.4% of the theoretical values for the for-
mula given. Yields refer to purified products.
4.1. General procedure for the synthesis of substituted
benzamides (3a–l)
Benzoyl chlorides (2.75 mmol) were added portionwise
to the stirred solution of 2-halo anilines (2.5 mmol)

1222
S. Patil et al. / Bioorg. Med. Chem. 15 (2007) 1212–1228
and 452 lL of triethyl amine (3.25 mmol) in dry methy-
lene chloride (5 mL) at 0 ꢁC over a period of 15 min,
then the mixture was stirred at room temperature for
5–11 h. The reaction mixture was extracted with ethyl
acetate, dried, and solvents were removed under reduced
pressure. The crude residue was subjected to flash
chromatography.
7.67 (br s, 1H, NH), 7.31 (s, 1H, ArH), 7.06 (s, 1H,
ArH), 2.33 (s, 3H, CH3), 2.30 (s, 3H, CH3); MS (ESI):
m/z 371.3 [M+Na]+; Anal. Calcd (C15H13BrN2O3) C,
H, N.
4.1.9. N-(2-Bromo-4,6-dimethylphenyl)benzamide (3i).
Yield 66%; 1H NMR (CDCl3): d 7.97 (d, J = 9 Hz,
2H, ArH), 7.63–7.49 (m, 3H, ArH), 7.32 (s, 1H, ArH),
7.07(br s, 1H, NH), 2.33 (s, 6H, CH3); MS (ESI): m/z
327 [M+Na]+; Anal. Calcd (C15H14BrNO) C, H, N.
4.1.1. N-(2-Bromo-4,6-dimethylphenyl)-3-cyanobenzamide
(3a). Yield 61%; 1H NMR (CDCl3): d 8.24 (s, 1H),
8.19 (d, J = 7.8 Hz, 1H), 7.87(d, J = 7.5 Hz, 1H),
7.65(t, J = 7.7 Hz, 1H,) 7.59(br s, 1H, NH), 7.32 (s,
1H), 7.07 (s, 1H), 2.41 (s, 3H, CH3), 2.33 (s, 3H,
CH3); MS (ESI): m/z 351.1 [M+Na]+; Anal. Calcd
(C16H13BrN2O) C, H, N.
4.1.10. N-(2-Chloro-6-methylphenyl)benzamide (3j). Yield
52%; 1H NMR (CDCl3): d 7.95 (d, J = 7.2 Hz, 2H, ArH),
7.68–7.49 (m, 4H, ArH), 7.33–7.16 (m, 3H, ArH), 2.36 (s,
3H, CH3); MS (ESI): m/z 268 [M+Na]+; Anal. Calcd
(C14H12ClNO) C, H, N.
4.1.2. N-(2-Chloro-6-methylphenyl)-3-cyanobenzamide (3b).
1
Yield 72%; H NMR (CDCl3): d 8.29 (s, 1H, ArH), 8.22
4.1.11. N-(2-Bromo-4-trifluoromethylphenyl)benzamide
(3k). Yield 70%; 1H NMR (CDCl3): d 8.77 (d,
J = 8.7 Hz, 1H, ArH), 8.63 (br s, 1H, NH), 7.97 (d,
J = 7.2 Hz, 2H, ArH), 7.87 (s, 1H, ArH), 7.68–7.54
(m, 5H, ArH); MS (ESI): m/z 365.98 [M+Na]+; Anal.
Calcd (C14H9BrF3NO) C, H, N.
(d, J = 7.8 Hz, 1H, ArH), 7.91 (d, J = 7.8 Hz, 1H, ArH),
7.72 (br s, 1H, ArH), 7.70 (t, J = 7.8 Hz, 1H, ArH)
7.39–7.24 (m, 3H, ArH), 2.38 (s, 3H, CH3); MS (ESI):
m/z 293.2 [M+Na]+; Anal. Calcd (C15H11ClN2O) C,H, N.
4.1.3. N-(2-Bromo-4,6-dimethylphenyl)-4-cyanobenzamide
(3c). Yield 52%; 1H NMR (CDCl3): d 8.06 (d, J =
8.4 Hz, 2H, ArH), 7.81 (d, J = 8.4 Hz, 2H, ArH), 7.59
(br s, 1H, NH), 7.33 (s, 1H, ArH), 7.28 (s, 1H, ArH),
2.31 (s, 3H, CH3), 2.34 (s, 3H, CH3); MS (ESI): m/z
351 [M+Na]+; Anal. Calcd (C16H13BrN2O) C, H, N.
4.1.12. N-(5-Acetyl-2-bromo-phenyl)-benzamide (3l).
Yield 61%; 1H NMR (CDCl3): d 9.19 (s, 1H, ArH),
8.54 (br s, 1H, NH) 7.97 (d, J = 6.9 Hz, 2H, ArH),
7.72–7.52 (m, 5H, ArH), 2.67 (s, 3H, CH3); MS (ESI):
m/z 340 [M+Na]+; Anal. Calcd (C15H12BrNO2) C, H, N.
4.1.4. N-(2-Bromo-4,6-dimethylphenyl)4-trifluorometh-
ylbenzamide (3d). Yield 45%; 1H NMR (CDCl3): d
8.06 (d, J = 8.1 Hz, 2H, ArH), 8.77 (d, J = 8.1 Hz, 2H,
ArH), 7.58 (br s, 1H, NH), 7.32 (s, 1H, ArH), 7.07 (s,
1H, ArH), 2.33 (s, 3H, CH3), 2.31 (s, 3H, CH3); MS
4.1.13. N-(2-Bromo-4,6-dimethyl-phenyl)-3-cyano-N-(3-
cyano-benzoyl)- benzamide (4a). Yield 3.4%; 1H NMR
(CDCl3): d 8.06 (s, 2H, ArH), 8.01 (d, J = 7.8 Hz,
2H, ArH), 7.76 (d, J = 7.5 Hz, 2H, ArH), 7.52 (t,
J = 7.8 Hz, 2H, ArH), 7.36 (s, 1H, ArH), 6.95 (s,
1H, ArH), 2.29 (s, 3H, CH3), 2.19 (s, 3H, CH3);
MS (ESI): m/z 458 [M+H]+; Anal. Calcd
(C24H16BrN3O2) C, H, N.
(ESI):
m/z
394.3
(C16H13BrF3NO) C, H, N.
[M+Na]+;
Anal.
Calcd
4.1.5.
N-(2-Chloro-6-methylphenyl)-4-cyanobenzamide
(3e). Yield 52%; 1H NMR (CDCl3): d 8.03 (d,
J = 8.4 Hz, 2H, ArH), 7.77 (d, J = 8.4 Hz, 2H, ArH),
7.72 (br s, 1H, NH), 7.33–7.19 (m, 3H, ArH), 2.31 (s,
3H, CH3); MS (ESI): m/z 293.2 [M+Na]+; Anal. Calcd
(C15H11ClN2O) C, H, N.
4.1.14. N-(2-Chloro-6-methylphenyl)-3-cyano-N-(3-cya-
nobenzoyl)benzamide (4b). Yield 4.0%; 1H NMR
(CDCl3): d 8.03 (s, 2H, ArH), 8.98 (d, J = 8.1 Hz, 2H,
ArH), 7.76 (d, J = 7.8 Hz, 2H, ArH), 7.52 (t,
J = 15.9 Hz, 2H, ArH), 7.36 (d, J = 8.1 Hz, 1H, ArH),
7.24 (t, 15.3 Hz, 1H, ArH), 2.24 (s, 3H, CH3); MS
(ESI): m/z 422.3 [M+Na]+; Anal. Calcd (C23H14ClN3O2)
C, H, N.
4.1.6. N-(2-Chloro-6-methylphenyl)4-trifluoromethylben-
zamide (3f). Yield 75%; H NMR (CDCl3): d 8.04 (d,
1
J = 8.1 Hz, 2H, ArH), 7.75 (d, J = 8.4 Hz, 2H, ArH),
7.32–7.17 (m, 3H, ArH), 2.32 (s, 3H, CH3); MS (ESI):
m/z 336.3 [M+Na]+; Anal. Calcd (C15H11ClF3NO) C,
H, N.
4.1.15. N-(2-Bromo-4,6-dimethylphenyl)-4-cyano-N-(4-
cyanobenzoyl)benzamide (4c). Yield 3.0%; 1H NMR
(CDCl3): d 7.88 (d, J = 8.4 Hz, 4H, ArH), 7.67 (d,
J = 8.4 Hz, 4H, ArH), 7.34 (s, 1H, ArH), 6.94 (s, 1H,
ArH), 2.18 (s, 3H, CH3), 2.28 (s, 3H, CH3); MS (ESI):
m/z 480 [M+Na]+; Anal. Calcd (C24H16BrN3O2) C, H,
N.
4.1.7. N-(2-Bromo-4,6-dimethylphenyl)-3-nitrobenzamide
(3g). Yield 65%; 1H NMR (CDCl3): d 8.79 (s, 1H, ArH),
8.45 (d, J = 8.1 Hz, 1H, ArH), 8.31 (d, J = 7.8 Hz, 1H,
ArH), 7.72 (t, J = 7.8 Hz, 2H, ArH), 7.32 (s, 1H,
ArH), 7.07 (s, 1H, ArH), 2.34 (s, 3H, CH3), 2.31 (s,
3H, CH3); MS (ESI): m/z 371.3 [M+Na]+; Anal. Calcd
(C15H13BrN2O3) C, H, N.
4.1.16. N-(2-Chloro-6-methylphenyl)-4-cyanobenzoyl) be-
1
nzamide (4d). Yield 3.0%; H NMR (CDCl3): d 7.85 (d,
J = 8.4 Hz, 4H, ArH), 7.66 (d, J = 8.4 Hz, 4H, ArH),
7.35 (d, J = 7.8 Hz, 1H, ArH), 7.23 (t, J = 7.8 Hz, 1H,
ArH), 7.12 (d, J = 7.2 Hz, 1H, ArH), 2.28 (s, 3H,
CH3); MS (ESI): m/z 422.1 [M+Na]+; Anal. Calcd
(C23H14ClN3O2) C, H, N.
4.1.8. N-(2-Bromo-4,6-dimethylphenyl)-4-nitrobenzamide
(3h). Yield 68%; 1H NMR (CDCl3): d 8.35 (d,
J = 8.7 Hz, 2H, ArH), 8.09 (d, J = 8.7 Hz, 2H, ArH),

S. Patil et al. / Bioorg. Med. Chem. 15 (2007) 1212–1228
1223
4.1.17. N-(2-Chloro-6-methylphenyl)-4-trifluoromethyl-
N-(4-trifluoromethylbenzoyl)benzamide (4e). Yield 2.5%;
1H NMR (CDCl3): d 7.90 (d, J = 8.1 Hz, 4H, ArH),
7.62 (d, J = 8.4 Hz, 4H, ArH), 7.35 (d, J = 7.8 Hz, 1H,
ArH), 7.21 (t, J = 7.8 Hz, 1H, ArH), 7.12 (d,
J = 7.6 Hz, 1H, ArH), 2.26 (s, 3H, CH3); MS (ESI): m/
z 508.1 [M+Na]+; Anal. Calcd (C23H14ClF6NO2) C,
H, N.
4.2.4. 2,4-Dimethyl-9-trifluoromethyl-5H-phenanthridin-
6-one (5d). Yield 28%; H NMR (CDCl3): d 8.64 (d,
J = 8.1 Hz, 1H, ArH), 8.54 (s, 1H, ArH), 7.91 (s, 1H,
ArH), 7.81 (d, J = 8.4 Hz, 1H, ArH), 7.23 (s, 1H,
ArH), 2.48 (s, 3H, CH3), 2.47 (s, 6H, CH3); MS (ESI):
m/z 314.1, [M+Na]+; Anal. Calcd (C16H12F3NO) C, H,
N.
1
4.2.5. 4-Methyl-6-oxo-5,6-dihydro-phenanthridine-9-car-
bonitrile (5e). Yield 23%; 1H NMR (DMSO-d6,
CD3OD): d 9.09 (s, 1H, ArH), 8.46 (d, J = 7.8 Hz, 1H,
ArH), 8.37 (d, J = 7.1 Hz, 1H, ArH), 8.00 (d,
J = 8.4 Hz, 1H, ArH), 7.42 (d, J = 7.5 Hz, 1H, ArH),
7.19 (t, J = 7.2 Hz, 1H, ArH), 2.16 (s, 3H, CH3); MS
(ESI): m/z 257.2 [M+Na]+; Anal. Calcd (C15H10N2O)
C, H, N.
4.1.18. N-(2-Bromo-4,6-dimethyl-phenyl)-3-nitro-N-(3-ni-
tro-benzoyl)- benzamide (4f). Yield 2.0%; 1H NMR
(CDCl3)d 8.65 (s, 2H, ArH), 8.35 (d, J = 8.4 Hz, 2H,
ArH), 8.14 (d, J = 7.5 Hz, 2H, ArH), 7.61 (t,
J = 8.1 Hz, 2H, ArH), 7.36 (s, 1H, ArH), 6.96 (s, 1H,
ArH), 2.28 (s, 3H, CH3), 2.31 (s, 3H, CH3); MS (ESI):
m/z 520 [M+Na]+; Anal. Calcd (C22H16BrN3O6) C, H,
N.
4.2.6.
4-Methyl-9-trifluoromethyl-5H-phenanthridin-6-
1
4.1.19. N-(2-Bromo-4,6-dimethylphenyl)-4-nitro-N-(4- ni-
trobenzoyl)benzamide (4g). Yield 2.5%; 1H NMR
(CDCl3)d 8.24 (d, J = 8.7 Hz, 4H, ArH), 7.96 (d,
J = 8.7 Hz, 4H, ArH), 7.35 (s, 1H, ArH), 6.94 (s, 1H,
ArH), 2.28 (s, 3H, CH3), 2.20 (s, 3H, CH3); MS (ESI):
m/z 520 [M+Na]+; Anal. Calcd (C22H16BrN3O6) C, H, N.
one (5f). Yield 38%; H NMR (DMSO-d6, CD3OD): d
8.77 (s, 1H, ArH), 8.59 (d, J = 9.0 Hz, 1H, ArH), 8.32
(d, J = 8.0 Hz, 1H, ArH), 7.91 (d, J = 9.0 Hz, 1H,
ArH), 7.42 (d, J = 7.0 Hz, 1H, ArH), 7.27 (t,
J = 8.0 Hz, 1H, ArH), 2.50 (s, 3H, CH3); MS (ESI): m/
z 300 [M+Na]+; Anal. Calcd (C15H10F3NO) C, H, N.
4.2. General procedure for synthesis of 5H-phenanthridin-
6-ones (5a–j)
4.2.7. 2,4-Dimethyl-5H-phenanthridin-6-one (5g). Yield
30%; H NMR (CDCl3): d 8.81 (br s, 1H, NH), 8.53
1
(d, J = 8.1 Hz, 1H, ArH), 8.30 (d, J = 8.1 Hz, 1H,
ArH), 7.90 (s, 1H, ArH), 7.79 (t, J = 8.1 Hz, 1H,
ArH), 7.60 (t, J = 8.1 Hz, 1H, ArH), 7.17 (s, 1H,
ArH), 2.47 (s, 6H, CH3); MS (ESI): m/z 224.5
[M+H]+; Anal. Calcd (C15H13NO) C, H, N.
Substituted benzamides (1.31 mmol), palladium(II) ace-
tate (29.34 mg, 0.131 mmol), and anhydrous sodium
carbonate (161 mg, 1.52 mmol) in dry N,N-dimethyl
acetamide (6 mL) were heated together at 165–170 ꢁC
under nitrogen for 11–23 h. The resultant mixture was
cooled to room temperature, poured to cold water,
and extracted with ethyl acetate (3· 5 mL). Ethyl acetate
layer was dried over anhydrous Na2SO4, solvents were
evaporated under reduced pressure, and crude residue
was purified by flash chromatography.
4.2.8. 4-Methyl-5H-phenanthridin-6-one (5h). Yield 30%;
1H NMR (CDCl3)d 8.53 (br s, 1H, NH), 8.51 (s, 1H,
ArH), 8.30 (d, J = 8.1 Hz, 1H, ArH), 8.12 (d,
J = 8.1 Hz, 1H, ArH), 7.80 (t, J = 7.2 Hz, 1H, ArH),
7.60 (t, J = 7.5 Hz, 1H, ArH), 7.35 (d, J = 7.9 Hz, 1H,
ArH), 7.21 (t, J = 7.8 Hz, 1H, ArH), 2.48 (s, 6H,
CH3); MS (ESI): m/z 232 [M+Na]+; Anal. Calcd
(C14H11NO) C, H, N.
4.2.1. 2,4-Dimethyl-6-oxo-5,6-phenanthridine-8-carbonit-
rile (5a). Yield 30%; 1H NMR (CDCl3): d 9.62 (br s, 1H,
NH), 8.63 (s, 1H, ArH), 8.24 (d, J = 8.4 Hz, 1H, ArH),
7.81 (d, J = 8.4 Hz, 1H, ArH), 7.28 (s, 1H, ArH), 7.09 (s,
1H, ArH), 2.29 (s, 3H, CH3), 2.34 (s, 6H, CH3); MS
(ESI): m/z 247.3 [MꢀH]ꢀ; Anal. Calcd (C16H12N2O)
C, H, N.
4.2.9. 2-Trifluoromethyl-5H-phenanthridin-6-one (5i).
1
Yield 53%; H NMR (CDCl3): d 11.43 (br s, 1H, NH),
8.39 (d, J = 8.1 Hz, 1H, ArH), 8.32 (s, 1H, ArH), 8.21
(d, J = 8.1 Hz, 1H, ArH), 7.73 (t, J = 7.2 Hz, 1H,
ArH), 7.54 (t, J = 7.5 Hz, 2H, ArH), 7.37 (d,
J = 8.7 Hz, 1H, ArH); MS (ESI): m/z 286 [M+Na]+;
Anal. Calcd (C14H8F3NO) C, H, N.
4.2.2. 4-Methyl-6-oxo-5,6-dihydrophenanthridine-8-car-
1
bonitrile (5b). Yield 44%; H NMR (CDCl3): d 11.01
(br s, 1H, NH), 8.70 (t, J = 8.4 Hz, 1H, ArH), 8.66 (s,
1H, ArH), 8.34 (d, J = 8.1 Hz, 1H, ArH), 8.23 (d,
J = 8.7 Hz, 1H, ArH), 7.45 (d, J = 7.5 Hz, 1H, ArH),
7.23 (t, J = 7.8 Hz, 1H, ArH), 2.48 (s, 3H, CH3); MS
(ESI): m/z 235.2 [M+H]+; Anal. Calcd (C15H10N2O) C,
H, N.
4.2.10. 3-Acetyl-5H-phenanthridin-6-one (5j). Yield 30%;
1H NMR (CD3OD) d 8.53–8.40 (m, 3H, ArH), 7.96–
7.86 (m, 3H, ArH), 7.70 (t, J = 7.2 Hz, 1H, ArH), 2.67
(s, 3H, CH3); MS (ESI): m/z 236 [MꢀH]ꢀ; Anal. Calcd
(C15H11NO2) C, H, N.
4.2.3.
2,4-Dimethyl-6-oxo-5,6-hydrophenanthridine-9-
4.2.11. Synthesis of 2-methoxy-5H-phenanthridin-6-one
(8). 2-Bromo-5H-phenanthridin-6-one (60 mg, 0.216
mmol), cuprous iodide (41 mg, 216 mmol), and sodium
methoxide (prepared freshly by dissolving 80 mg of sodi-
um metal in 5 mL methanol) were refluxed gently in
anhydrous DMF nearly for 5 h. Reaction mixture was
cooled to room temperature and extracted with ethyl
carbonitrile (5c). Yield 24%; 1H NMR (CDCl3): d
10.95 (br s, 1H, NH), 9.1 (s, 1H, ArH), 8.44 (d,
J = 8.1 Hz, 1H, ArH), 8.23 (s, 1H, ArH), 8.00 (d,
J = 8.2 Hz, 1H, ArH), 7.26 (s, 1H, ArH), 2.39 (s, 3H,
CH3), 2.45 (s, 6H, CH3); MS (ESI): m/z271 [M+Na]+;
Anal. Calcd (C16H12N2O) C, H, N.

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S. Patil et al. / Bioorg. Med. Chem. 15 (2007) 1212–1228
acetate, dried, and solvents were removed under reduced
pressure. Crude residue was further purified by flash
chromatography to get 2-methoxy-5H-phenanthridin-
6-one (41 mg, 83% yield).
The
chromatography.
crude
residue was
purified
by
flash
4.3.1. 2-Ethylamino-5H-phenanthridin-6-one (12a). Yield
50%; 1H NMR (DMSO-d6): d 11.46 (br s, 1H, NH), 8.62
(d, J = 8.1 Hz, 1H, ArH), 8.24 (d, J = 8.1 Hz, 1H, ArH),
7.78 (t, J = 6.9 Hz, 1H, ArH), 7.61 (t, J = 7.2 Hz, 1H,
ArH), 7.40–7.26 (m, 2H, ArH), 6.88 (d, J = 8.7 Hz,
1H, ArH), 3.70 (br s, 1H, NH), 3.28 (q, J = 6.9, 7.2,
6.9 Hz, 2H, CH2), 1.34 (t, J = 7.2, Hz, 3H, CH3); MS
(ESI): m/z 261 [M+Na]+; Anal. Calcd (C15H14N2O) C,
H, N.
1H NMR (C6D6O, DMSO-d6): d 11.43 (br s, 1H, NH),
8.52 (d, J = 8.4 Hz, 1H, ArH), 8.41 (dd, J = 1.5,
1.2 Hz, 1H, ArH), 7.88–7.82 (m, 2H, ArH) 7.64 (t,
J = 8.1 Hz, 1H, ArH), 7.38 (d, J = 9.0 Hz, 1H, ArH),
7.14 (dd, J = 2.7, 2.7 Hz, 1H, ArH), 3.92 (s, 3H,
OCH3); MS (ESI): m/z 248 [M+Na]+; Anal. Calcd
(C14H11NO2) C, H, N.
4.2.12. Synthesis of 6-oxo-5,6-dihydro-phenanthridine-2-
carbonitrile
4.3.2. N-(6-Oxo-5,6-dihydro-phenanthridin-2-yl)-acetam-
ide (12b). Yield 50%; H NMR (DMSO-d6): d 11.64 (br
1
(9).
2-Bromo-5H-phenanthridin-6-one
(150 mg, 0.54 mmol) and cuprous cyanide (58 mg,
0.648 mmol) were heated with stirring in N,N-dimethyl
acetamide (4 mL) at 150–155 ꢁC for a day. After cooling
the reaction mixture to room temperature, 5 mL cold
water was added to the reaction flask and separated sol-
id was filtered. Column chromatography of the crude
product gave 6-oxo-5,6-dihydro-phenanthridine-2-car-
bonitrile as white powder (90 mg, 75% yield).
s, 1H, NH), 11.06 (br s, 1H, NH), 8.61 (s, 1H, ArH),
8.32 (d, J = 7.8 Hz, 1H, ArH), 8.22 (d, J = 7.5 Hz, 1H,
ArH), 7.88 (t, J = 7.2 Hz, 1H, ArH), 7.64 (d t, J = 7.8,
8.1 Hz, 2H, ArH), 7.29 (d, J = 8.4 Hz, 1H, ArH), 2.07
(s, 3H, CH3); MS (ESI): m/z 251 [MꢀH]ꢀ; Anal. Calcd
(C15H12N2O2) C, H, N.
4.3.3. N-(6-Oxo-5,6-dihydro-phenanthridin-2-yl)-benzam-
ide (12c). Yield 60%; 1H NMR (DMSO-d6): d 11.71
(br s, 1H, NH), 10.38 (br s, 1H, NH), 8.79 (s, 1H,
ArH), 8.32 (t, J = 8.4 Hz, 2H, ArH), 8.01 (d,
J = 6.6 Hz, 2H, ArH), 7.93–7.86 (m, 2H, ArH),
7.69–7.53 (m, 4H, ArH), 7.35 (d, J = 8.7 Hz, 1H,
ArH); MS (ESI): m/z 337 [M+Na]+; Anal. Calcd
(C20H14N2O2) C, H, N.
1H NMR (DMSO-d6): d 12.00 (br s, 1H, NH), 8.92 (s,
1H, ArH), 8.59 (d, J = 8.1 Hz, 1H, ArH), 8.28 (d,
J = 7.5 Hz, 1H, ArH), 7.85 (t, d, J = 6.6, 8.7 Hz, 2H,
ArH), 7.67 (t, J = 7.8 Hz, 1H, ArH), 7.42 (d,
J = 8.4 Hz, 1H, ArH); MS (ESI): m/z 219 [MꢀH]ꢀ;
Anal. Calcd (C14H8N2O) C, H, N.
4.2.13. Synthesis of 6-oxo-5,6-dihydro-phenanthridine-2-
carboxylic acid (10). To a suspension of 6-oxo-5,6-dihy-
dro-phenanthridine-2-carbonitrile (100 mg, 0.454 mmol)
in ethanol (10 mL), sodium hydroxide (100 mg) was
added and reaction mixture was refluxed for 21 h. The
cooled reaction mixture was added to ice-cold water
and carefully acidified with dilute hydrochloric acid,
separated white precipitate was filtered and washed with
water repeatedly and air-dried to get sufficiently pure 6-
oxo-5,6-dihydro-phenanthridine-2-carboxylic acid in
30% yield.
4.3.4. N-(6-Oxo-5,6-dihydro-phenanthridin-2-yl)-benze-
nesulfonamide (12d). Yield 85%; H NMR (DMSO-d6,
1
CD3OD): d 8.79 (s, 1H, ArH), 8.51 (d, J = 8.4 Hz, 1H,
ArH), 8.14 (d, J = 8.1 Hz, 1H, ArH), 8.05–7.72 (m,
9H, ArH), 7.45 (d, J = 8.7 Hz, 1H, ArH), 7.22 (d,
J = 8.7 Hz, 1H, ArH); MS (ESI): m/z 348.9 [MꢀH]ꢀ;
Anal. Calcd (C19H14N2O3S) C, H, N.
4.4. General procedures for synthesis of compounds 13a–
b, 14a–b
Method A. 3 or 4-Bromomethylbenzonitriles (37.24 mg,
0.19 mmol) were added to the stirred solution of 2-ami-
no-5H-phenanthridin-6-one (40 mg, 0.19 mmol) and tri-
ethyl amine (29 lL, 0.209 mmol) in dry dimethyl
formamide (4 mL). After the addition was complete,
the reaction mixture was stirred at room temperature
for 4 h. Mixture was extracted with ethyl acetate, dried
over anhydrous Na2SO4, and solvents were removed un-
der reduced pressure. Purification of all the crude prod-
ucts was done by column chromatography.
1H NMR (DMSO-d6): d 13.00 (br s, 1H, OH), 11.89 (s,
1H, NH), 8.91 (s, 1H, ArH), 8.59 (d, J = 7.8 Hz, 1H,
ArH), 8.32 (d, J = 8.1 Hz, 1H, ArH), 8.13 (s, 1H,
ArH), 8.01 (dd, J = 0.9, 1.2 Hz, 1H, ArH), 7.91 (t,
J = 7.5 Hz, 1H, ArH), 7.68 (t, J = 7.5 Hz, 1H, ArH),
7.38 (d, J = 8.4 Hz, 1H, ArH); MS (ESI): m/z 238
[MꢀH]ꢀ; Anal. Calcd (C14H9NO3) C, H, N.
4.3. General procedure for synthesis of N-substituted 2-
amino-5H-phenanthridin-6-ones (12a–d)
Method B. 3 or 4-Bromomethylbenzonitriles (37.24 mg,
0.19 mmol) were added to the stirred solution of substi-
Acid chlorides (ethyl iodide in case of 12a) (0.253 mmol)
were added portionwise to the stirred solution of 2-ami-
no-5H-phenanthridin-6-one (0.23 mmol) and triethyl
amine (42 lL, 0.299 mmol) in dry DMF (5 mL) at
0 ꢁC over a period of 15 min. After the addition was
complete, the reaction mixture was stirred at room tem-
perature for 2–5 h (28 h in case of ethyl iodide). Reac-
tion mixture was extracted with ethyl acetate, dried,
and solvents were removed under reduced pressure.
tuted
2-amino-5H-phenanthridin-6-one
(40 mg,
0.19 mmol) and pyridine (17 lL, 0.209 mmol) in dry
dimethyl formamide (5 mL). The reaction mixture was
stirred for 4 h at room temperature. Workup and purifi-
cation of the reaction mixture is the same as above.
Method C. 3 or 4-Bromomethylbenzonitriles (90 mg,
0.46 mmol), 2-amino-5H-phenanthridin-6-one (100 mg,

S. Patil et al. / Bioorg. Med. Chem. 15 (2007) 1212–1228
1225
0.46 mmol), and potassium carbonate (126 mg,
0.92 mmol) were refluxed for 4 h in dry ethanol
(10 mL). Reaction mixture was filtered hot and solvents
removed under reduced pressure. Workup and purifica-
tion of the reaction mixture is the same as above.
ture was stirred for 3–5 h at room temperature. Reac-
tion mixture was poured to cold water and carefully
acidified with dilute HCl. The mixture was extracted
with ethyl acetate, dried over anhydrous Na2SO4,
and solvents were removed under reduced pressure.
The crude product was used without further purifica-
tion in next step.
4.4.1. 3-[(6-Oxo-5,6-dihydro-phenanthridin-2-ylamino)-
methyl]-benzonitrile (13a). Yield 60%; 1H NMR
(DMSO-d6):
d
11.23 (br s, 1H, NH), 8.29 (d,
4.5.1. 4-(3-Benzoylamino-4-bromo-phenyl)-2,4-dioxo-bu-
tyric acid methyl ester (17). Yield 36%; 1H NMR
(DMSO-d6): d 9.92(br s, 1H, NH) 7.90–8.20 (m, 4H,
ArH), 7.80-7.40 (m, 6H, ArH, C@CH), 6.55 (br s, 1H,
OH/or C@CH), 3.73 (s, 3H, OCH3); MS (ESI): m/z
402 [MꢀH]ꢀ; Anal. Calcd (C18H14BrNO5) C, H, N.
J = 7.5 Hz, 1H, ArH), 8.09 (d, J = 8.1 Hz, 1H, ArH),
7.75–7.65 (m, 3H, ArH), 7.53-7.42 (m, 3H, ArH), 7.28
(s, 1H, ArH), 7.12 (d, J = 9.0 Hz, 1H, ArH), 6.78 (dd,
J = 2.4, 2.4 Hz, 1H, ArH), 5.97 (t, J = 6.0 Hz, 1H,
NH), 4.41 (d, J = 1.9 Hz, 2H, CH2); MS (ESI): m/z
348 [M+Na]+; Anal. Calcd (C21H15N3O) C, H, N.
4.5.2. 40-(3-Methoxycarbonyl-3-oxo-propionyl)-20-nitro-
biphenyl-2-carboxylic acid methyl ester (20). Yield 47%;
1H NMR (CDCl3): d 8.70 (s, 1H, ArH), 8.24 (dd,
J = 1.8, 1.5 Hz, 1H, ArH), 8.13 (d, J = 7.5 Hz, 1H,
ArH), 7.63 (t, J = 7.5 Hz, 1H, ArH), 7.53 (t,
J = 7.8 Hz, 1H, ArH), 7.44 (d, J = 8.1 Hz, 1H, ArH),
7.26 (s, 1H, C@CH), 7.23 (d, J = 7.5 Hz, 1H, ArH),
7.15 (br s, 1H, OH/or C@CH), 3.98 (s, 3H, OCH3),
3.70 (s, 3H, OCH3).
4.4.2. 4-[(6-Oxo-5,6-dihydro-phenanthridin-2-ylamino)-
methyl]-benzonitrile (13b). Yield 60%; 1H NMR
(DMSO-d6):
d 11.30 (br s, 1H, NH), 8.22 (t,
J = 8.4 Hz, 2H, ArH), 7.74 (sd, J = 8.1 Hz, 3H, ArH),
7.55 (m, 4H, ArH), 7.34 (s, 1H, ArH), 7.08 (d,
J = 8.7 Hz, 1H, ArH), 6.83 (dd, J = 2.1, 2.4 Hz, 1H,
ArH), 6.30 (t, J = 6.0 Hz, 1H, NH), 4.5 (d, J = 1.9 Hz,
2H, CH2); MS (ESI): m/z 348 [M+Na]+; Anal. Calcd
(C21H15N3O) C, H, N.
13C NMR (CDCl3): d ppm 187.13, 169.98, 166.00,
161.65, 148.00, 141.84, 138.49, 134.45, 131.96, 131.70,
130.67, 130.22, 128.95, 128.12, 127.63, 122.82, 97.46,
52.85, 51.64; MS (ESI): m/z 408 [M+Na]+; Anal. Calcd
(C19H15NO8) C, H, N.
4.4.3. N-[3-(Cyano phenyl methyl)]3-[(6-oxo-5,6-dihydro-
phenanthridin-2-yl amino)methyl]benzonitrile (14a). Yield
1
5%; H NMR (DMSO-d6): d 9.82 (br s, 1H, NH), 8.54
(d, J = 8.1 Hz, 1H, ArH), 7.96 (d, J = 7.8 Hz, 1H,
ArH), 7.74 (t, J = 7.2 Hz, 1H, ArH), 7.407.65–7.45 (m,
10H, ArH), 7.17 (d, J = 8.7 Hz, 1H, ArH), 6.96 (d,
J = 7.5 Hz, 1H, ArH), 4.74 (s, 4H, CH2); MS (ESI): m/
z 463 [M+Na]+; Anal. Calcd (C29H20N4O) C, H, N.
4.6. General procedure for compounds 16, 18, 21, and
27a–k
Diketo ester derivative (0.1 mmol) was stirred with
80 mg of sodium hydroxide in 20 mL of methanol for
3–5 h at room temperature. Poured to ice-cold water
and acidified with dilute HCl. Precipitate was extracted
with ethyl acetate, dried, and evaporated to get the dike-
to acids with good purity.
4.4.4. N-[4-(Cyano phenyl methyl)]4-[(6-oxo-5,6-dihydro-
phenanthridin-2-yl amino)methyl]benzonitrile (14b). Yield
6%; H NMR (DMSO-d6): d 11.31 (br s, 1H, NH), 8.27
(d, J = 7.8 Hz, 1H, ArH), 8.02 (sd, J = 8.1 Hz, 3H,
ArH), 7.64 (d, J = 8.1 Hz, 4H, ArH), 7.55–7.39 (m,
6H, ArH), 7.15 (d, J = 8.7 Hz, 1H, ArH), 6.85 (dd,
J = 2.1, 2.4 Hz, 1H, ArH), 4.78 (s, 4H, CH2); MS
(ESI): m/z 463 [M+Na]+; Anal. Calcd (C29H20N4O) C,
H, N.
1
4.6.1. 2,4-Dioxo-4-(6-oxo-5,6-dihydro-phenanthridin-3-
yl)-butyric acid (16). Yield 72%; H NMR (DMSO-d6):
1
d 11.82 (br s, 1H, NH), 8.61 (d, J = 9.0 Hz, 1H, ArH),
8.56 (d, J = 9.0 Hz, 1H, ArH), 8.36 (d, J = 9.0 Hz, 1H,
ArH), 8.04 (s, 1H, ArH), 7.96–7.86 (m, 3H, ArH,
C@CH), 7.78–7.68 (m, 2H, ArH), 7.00 (br s, 1H, OH);
MS (ESI): m/z 307.8 [MꢀH]ꢀ; Anal. Calcd
(C17H11NO5) C, H, N.
4.4.5. 2,4-Dioxo-4-(6-oxo-5,6-dihydro-phenanthridin-3-
yl)-butyric acid ethyl ester (15). To a solution of 3-ace-
tyl-5H-phenanthridin-6-one (80 mg, 0.337 mmol) in
dry dioxane, potassium tert-butoxide (378 mg,
3.37 mmol) and diethyl oxalate (59.5 lL, 0.438 mmol)
were added with stirring. The reaction mixture was gent-
ly refluxed overnight, cooled to room temperature and
carefully acidified with dilute HCl. Extracted with ethyl
acetate, dried over anhydrous Na2SO4 and solvents were
removed under reduced pressure. The crude product was
used with out further purification in next step.
4.6.2. 4-(3-Benzoylamino-4-bromo-phenyl)-2,4-dioxo-bu-
tyric acid (18). Yield 50%; 1H NMR (CDCl3): d 9.22 (br
s, 1H, NH), 8.54 (br s, 1H, ArH) 7.96 (d, J = 7.2 Hz, 2H,
ArH), 7.77–7.50 (m, 5H, ArH, C@CH), 7.26 (s, 1H,
ArH), 7.20 (s, 1H, OH/or C@CH); MS (ESI): m/z 412
[M+Na]+; Anal. Calcd (C17H12BrNO5) C, H, N.
4.5. General procedure for compounds 17, 20, and 26a–f
4.6.3. 40-(3-Carboxy-3-oxo-propionyl)-20-nitro-biphenyl-
2-carboxylic acid methyl ester (21). Yield 40%; 1H
NMR (CDCl3, DMSO-d6): d 8.71 (s, 1H, ArH), 8.16
(d, J = 7.8 Hz, 1H, ArH), 8.03 (d, J = 7.8 Hz, 1H,
ArH), 7.60–7.34 (m, 5H, ArH, C@CH), 7.16 (d,
J = 9.0 Hz, 1H, ArH), 7.08 (br s, 1H, OH/or C@CH),
A
freshly prepared sodium methoxide (231 mg,
4.278 mmol) in methanol (8 mL) was added to the
mixture of acetyl derivatives (1.337 mmol) and diethyl
oxalate (236 lL, 1.738 mmol), and the reaction mix-

1226
S. Patil et al. / Bioorg. Med. Chem. 15 (2007) 1212–1228
3.60 (s, 3H, OCH3); 13C NMR (CDCl3): d ppm 187.07,
171.04, 165.62, 162.62, 147.72, 141.34, 138.32, 134.55,
131.85, 131.61, 130.65, 129.97, 128.89, 127.93, 127.42,
122.49, 97.24, 51.45; MS (ESI): m/z 370 [MꢀH]ꢀ; Anal.
Calcd (C18H13NO8) C, H, N.
4.6.11. 2,4-Dioxo-4-phenanthren-9-yl-butyric acid (27g).
Yield 70%; H NMR (C3D6O): d 8.94 (d, J = 8.1 Hz,
1H, ArH), 8.89 (d, J = 8.1 Hz, 1H, ArH), 8.67 (d,
J = 7.8 Hz, 1H, ArH), 8.49 (s, 1H, ArH), 8.23 (d,
J = 7.8 Hz, 1H, ArH), 7.87–7.72 (m, 4H, ArH), 7.16
(s, 1H, CH); MS (ESI): m/z 315 [M+Na]+; Anal. Calcd
(C18H12O4) C, H, N.
1
4.6.4. 40-Acetyl-20-amino-biphenyl-2-carboxylic acid
methyl ester (22). 40-Acetyl-20-nitro-biphenyl-2-carbox-
ylic acid methyl ester 26 (50 mg, 0.167 mmol), hydrazine
hydrate (20 lL, 0.334 mmol), and Raney nickel (8 mg)
were stirred in methanol for 20 h. Mixture was filtered
through Celite and solvents were removed under re-
duced pressure. The crude residue was purified by flash
chromatography.
4.6.12. 2,4-Dioxo-4-phenanthren-3-yl-butyric acid (27h).
9.02 (d,
Yield 62%; 1H NMR (C3D6O):
d
J = 8.7 Hz, 1H, ArH), 8.95–8.92 (m, 1H, ArH),
8.86 (d, J = 2.1 Hz, 1H, ArH), 8.37 (dd, J = 1.8,
2.1 Hz, 1H, ArH), 8.10–7.97 (m, 3H, ArH), 7.80–
7.76 (m, 2H, ArH), 7.40 (s, 1H, CH); MS (ESI):
m/z 315.1 [M+Na]+; Anal. Calcd (C18H12O4) C,
H, N.
1
Yield 60%; H NMR (CDCl3): d 8.40 (d, J = 1.8 Hz,
1H, ArH), 8.09 (dd, J = 1.2, 1.2 Hz, 1H, ArH), 7.95
(dd, J = 1.8, 1.8 Hz, 1H, ArH), 7.60 (t, J = 7.5 Hz,
1H, ArH), 7.49 (t, J = 7.8 Hz, 1H, ArH), 7.25 (d,
J = 8.1 Hz, 2H, ArH), 5.57 (br s, 2H, NH2), 3.68
(s, 3H, OCH3), 2.19 (s, 3H, COCH3); MS (ESI):
m/z 392 [M+Na]+; Anal. Calcd (C16H15NO3) C, H,
N.
4.6.13. 2,4-Dioxo-4-phenanthren-2-yl-butyric acid (27i).
Yield 55%; H NMR (C3D6O, DMSO-d6): d 9.36 (s,
1H, ArH), 8.99 (d, J = 7.2 Hz, 1H, ArH), 8.14–8.02
(m, 5H, ArH), 7.76–7.66 (m, 2H, ArH); MS (ESI):
m/z 315.1 [M+Na]+; Anal. Calcd (C18H12O4) C, H,
N.
1
4.6.5. 4-(4-Morpholin-4-yl-phenyl)-2,4-dioxo-butyric acid
(27a). Yield 60%; 1H NMR (C3D6O): d 8.02 (d,
J = 9.3 Hz, 2H, ArH), 7.07 (s, d, J = 9.0 Hz, 3H, CH,
ArH), 3.81 (t, J = 4.8, 5.1 Hz, 4H, CH2), 3.43 (t,
J = 5.1, 4.8 Hz, 4H, CH2); MS (ESI): m/z 300
[M+Na]+; Anal. Calcd (C14H15NO5) C, H, N.
4.6.14. 4-(2-Methyl-10-oxo-10,10a-dihydro-8aH-9-oxa-1-
aza-anthracen-3-yl)-2,4- dioxo-butyric acid (27j). Yield
50%; H NMR (DMSO-d6): d 8.75 (s, 1H, ArH), 8.23
(d, J = 8.1 Hz, 1H, ArH), 7.96 (t, J = 8.7 Hz, 1H,
ArH), 7.75 (d, J = 8.7 Hz, 1H, ArH), 7.56 (t,
J = 7.8 Hz, 1H, ArH), 6.88 (s, 1H, CH), 2.83 (s, 3H,
CH3); MS (ESI): m/z 324 [MꢀH]ꢀ; Anal. Calcd
(C17H13NO6) C, H, N.
1
4.6.6. 4-Naphthalen-2-yl-2,4-dioxo-butyric acid (27b).
1
Yield 40%; H NMR (C3D6O): d 8.84 (s, 1H, ArH),
8.20–8.01 (m, 4H, ArH), 7.73–7.64 (m, 2H, ArH), 7.36
(s, 1H, CH); MS (ESI): m/z 265 [M+Na]+; Anal. Calcd
(C14H10O4) C, H, N.
4.6.15. 4-(2,6-Dimethyl-10-oxo-10,10a-dihydro-8aH-9-
oxa-1-aza- anthracen-3-yl)-2,4-dioxo-butyric acid (27k).
Yield 45%; H NMR (C3D6O, DMSO-d6): d 9.31 (s,
1
1H, ArH), 8.48 (s, 1H, ArH), 8.23 (d, J = 8.4 Hz,
1H, ArH), 8.08 (d, J = 8.4 Hz, 1H, ArH), 7.42 (s,
1H, CH), 3.30 (s, 3H, CH3), 2.95 (s, 3H, CH3); MS
(ESI): m/z 338 [MꢀH]ꢀ; Anal. Calcd (C18H15NO6)
C, H, N.
4.6.7. 4-(6-Methyl-naphthalen-2-yl)-2,4-dioxo-butyric acid
(27c). Yield 56%; 1H NMR (C3D6O): d 8.76 (s, 1H,
ArH), 8.11-8.07 (m, 2H, ArH), 7.96 (d, J = 8.7 Hz,
1H, ArH), 7.79 (s, 1H, CH), 7.50 (dd, J = 1.5, 1.5 Hz,
1H, CH), 7.33 (s, 1H, CH), 2.55 (s, 3H, CH3); MS
(ESI): m/z 279 [M+Na]+; Anal. Calcd (C15H12O4) C,
H, N.
4.7. Biological assays
4.7.1. Materials, chemicals, and enzymes. All compounds
were dissolved in DMSO and the stock solutions were
stored at ꢀ20 ꢁC. The c[32P]-ATP was purchased from
either Amersham Biosciences or ICN. The expression
systems for wild-type IN were a generous gift from
Dr. Robert Craigie, Laboratory of Molecular Biology,
NIDDK, NIH, Bethesda, MD. Cell lines were obtained
from American Tissue Type Culture (Rockville, MD).
4.6.8. 4-Anthracen-2-yl-2,4-dioxo-butyric acid (27d).
Yield 92%; H NMR (C3D6O): d 9.06 (s, 1H, ArH),
8.89 (s, 1H, ArH), 8.66 (s, 1H, ArH), 8.26–8.06 (m,
5H, ArH), 7.64–7.58 (m, 2H, ArH), 7.42 (s, 1H, CH);
MS (ESI): m/z 290.8 [MꢀH]ꢀ; Anal. Calcd (C18H12O4)
C, H, N.
1
4.6.9. 4-Anthracen-9-yl-2,4-dioxo-butyric acid (27e).
1
Yield 77%; H NMR (C3D6O): d 8.79 (s, 1H, ArH),
4.7.2. Preparation of oligonucleotide substrates. The oli-
gonucleotides 21top, 50-GTGTGGAAAATCTCTAG-
CAGT-30, and 21bot, 50-ACTGCTAGAGATTTTCC
ACAC-30, were purchased from the Norris Cancer Cen-
ter Microsequencing Core Facility (University of South-
ern California) and purified by UV shadowing on
polyacrylamide gel. To analyze the extent of 30-end pro-
cessing and strand transfer using 50-end labeled
substrates, 21top was 50-end labeled using T4 polynucle-
otide kinase (Epicentre, Madison, WI) and c[32P]-ATP
8.21–8.13 (m, 4H, ArH), 7.64–7.59 (m, 5H, ArH), 6.82
(s, 1H, CH); MS (ESI): m/z 315.1 [M+Na]+; Anal. Calcd
(C18H12O4) C, H, N.
4.6.10. 2,4-Dioxo-4-pyren-1-yl-butyric acid (27f). Yield
60%; 1H NMR (C3D6O): d 8.97 (d, J = 9.3 Hz, 1H,
ArH), 8.53–8.15 (m, 9H, ArH), 7.23 (s, 1H, CH); MS
(ESI): m/z 339 [M+Na]+; Anal. Calcd (C20H12O4) C,
H, N.

S. Patil et al. / Bioorg. Med. Chem. 15 (2007) 1212–1228
1227
(Amersham Biosciences or ICN). The kinase was heat-
inactivated and 21-bot was added in 1.5 M excess. The
mixture was heated at 95 ꢁC, allowed to cool slowly at
room temperature, and run through a G25 mini spin
column (USA Scientific) to separate double-double
stranded oligonucleotide from unincorporated material.
days, and toxicity was evaluated by viable cell counting
using a hemacytometer as described previously.41
4.8. Molecular docking
Monomer B of the crystal structure of HIV-integrase
with PDB ID of 1BIS36 was used for docking, since it
has the flexible catalytic loop resolved, which is missing
in the IN core structure in complex with the inhibitor 5-
CITEP (PDB code 1QS4).42 The Mg2+ ion was placed in
the active site between the carboxylate groups of catalyt-
ic amino acid residues Asp64 and Asp116 using the
geometry of the Mg2+ ion present in subunit A of the
1QS4 structure42 as reference. Phenanthridinones were
constructed using the Biopolymer module in the Sybyl
program (version 7.0, Tripos Associates Inc, St. Louis,
MO). The b-diketo moiety was constructed as the
keto-enol tautomer for docking. The program GOLD
(v2.0)35 from Cambridge Crystallographic Data Center,
UK, was used to dock the ligands into the active site.
GOLD is an automated ligand-docking program that
uses a genetic algorithm to explore ligand conformation-
al flexibility. It also performs limited exploration of
receptor flexibility by optimizing torsion angles of serine
and threonine hydroxyls, and lysine NH3+ groups. The
4.7.3. Integrase assays. To determine the extent of 30-
processing and strand transfer, wild-type IN was prein-
cubated at a final concentration of 200 nM with the
inhibitor in reaction buffer (50 mM NaCl, 1 mM
HEPES, pH 7.5, 50 lM EDTA, 50 lM dithiothreitol,
10% glycerol (w/v), 7.5 mM MnCl2, 0.1 mg/mL bovine
serum albumin, 10 mM 2-mercaptoethanol, 10%
dimethylsulfoxide, and 25 mM MOPS, pH 7.2) at
30 ꢁC for 30 min. An aliquot (5 lL) was electrophoresed
on a denaturing 20% polyacrylamide gel (0.09 M Tris–
borate, pH 8.3, 2 mM EDTA, 20% acrylamide, and
8 M urea). Gels were dried, exposed in a PhosphorImag-
er cassette, and analyzed using a Typhoon 8610 Variable
Mode Imager (Amersham Biosciences) and quantitated
using ImageQuant 5.2. Percent inhibition (%I) was cal-
culated using the following equation:
%I ¼ 100 ꢁ ½1 ꢀ ðD ꢀ CÞ=ðN ꢀ CÞꢂ
˚
active site was defined as residues lying within 15 A radi-
where C, N, and D are the fractions of 21-mer substrate
converted to 19-mer (30-end processing product) or
strand transfer products for DNA alone, DNA plus
IN, and IN plus drug, respectively. The IC50 values were
determined by plotting the logarithm of drug concentra-
tion versus percent inhibition to obtain concentration
that produced 50% inhibition.
us of the active Mg2+ ion. Ten different docking solu-
tions were obtained for each molecule, the docking
being terminated if the top ten solutions were within
an RMSD of 61.5.
Acknowledgments
4.7.4. Anti-HIV-1 activity assay. The testing of the abil-
ity of potent compounds to inhibit HIV replication in
cell culture was done according to a previously report-
ed procedure.38 PBMC (107 cells/T25flask) were stimu-
lated with phytohemagglutinin for 3 days and infected
with a wild-type HIV-1 strain (strain LAI) at 100 50%
tissue culture infective doses, as described previously.38
The cultures were kept for 5 days in the presence of
test compounds at serial 1-log dilutions. Subsequently,
human PBMC were removed from the culture
supernatant by centrifugation (400g, 10 min, 4 ꢁC).
This clarified supernatant was tested by a reverse
transcriptase assay.
Support from funds and resources provided by the
Department of Pharmaceutical Sciences and College of
Pharmacy for the work of J.K.B. is gratefully acknowl-
edged. We would like to also acknowledge the Atlanta
Center for AIDS Research (NIH grant 5P30-AI-
50409) and the Department of Veterans Affairs for sup-
porting the work of R.F.S.
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