Conformationally restrained carbazolone-containing α,γ-diketo acids as inhibitors of HIV integrase

Article abstract of DOI:10.1016/j.bmc.2005.12.013

Since α,γ-diketo acid (DKA) compounds were identified as potent and selective inhibitors for HIV integrase, numerous structural modification studies have been carried out to search for a clinical candidate as a supplement for the highly active antiretroviral therapy regimen. Due to the lack of structural information on inhibitor-integrase interactions, a comprehensive structure-activity relationship study is necessary. Most of the reported modification studies on the key α,γ-diketo acid pharmacophore focused on substituting the carboxylate moiety with its bioisosteres or other electron-pair bearing heterocycles. We were interested in studying the conformation and geometry of the central diketo moiety. A series of carbazolone-containing α,γ-diketo acids were designed and synthesized by applying conformational restraint onto the open-chain form of the diketo acid. These compounds showed anti-integrase activity in the low micromolar range, and integrase assay results indicated that the geometry of the diketo acid moiety is crucial to potency. Carbazol-1-one containing DKA analogs (7-8) showed a 2- to 3-fold increase in activity compared with those of carbazol-4-one containing DKA analogs (5 and 6). Alkylation of carbazol-4-one DKA nitrogen (6a-c) led to a loss of activity, suggesting this nitrogen atom may directly interact with the active site of integrase. The halogens (7b-d) and para-fluorobenzyl substituents (8a-d) on carbazol-1-one ring had little effect on potency.

Full text of DOI:10.1016/j.bmc.2005.12.013

Bioorganic & Medicinal Chemistry 14 (2006) 2942–2955
Conformationally restrained carbazolone-containing
a,c-diketo acids as inhibitors of HIV integrase
Xingnan Li and Robert Vince*
Department of Medicinal Chemistry, College of Pharmacy, and Center for Drug Design, Academic Health Center,
University of Minnesota, 8-123A WDH, 308 Harvard Street SE, Minneapolis, MN 55455, USA
Received 26 October 2005; revised 4 December 2005; accepted 5 December 2005
Available online 4 January 2006
Abstract—Since a,c-diketo acid (DKA) compounds were identified as potent and selective inhibitors for HIV integrase, numerous
structural modification studies have been carried out to search for a clinical candidate as a supplement for the highly active anti-
retroviral therapy regimen. Due to the lack of structural information on inhibitor–integrase interactions, a comprehensive struc-
ture–activity relationship study is necessary. Most of the reported modification studies on the key a,c-diketo acid
pharmacophore focused on substituting the carboxylate moiety with its bioisosteres or other electron-pair bearing heterocycles.
We were interested in studying the conformation and geometry of the central diketo moiety. A series of carbazolone-containing
a,c-diketo acids were designed and synthesized by applying conformational restraint onto the open-chain form of the diketo acid.
These compounds showed anti-integrase activity in the low micromolar range, and integrase assay results indicated that the geom-
etry of the diketo acid moiety is crucial to potency. Carbazol-1-one containing DKA analogs (7–8) showed a 2- to 3-fold increase in
activity compared with those of carbazol-4-one containing DKA analogs (5 and 6). Alkylation of carbazol-4-one DKA nitrogen
(6a–c) led to a loss of activity, suggesting this nitrogen atom may directly interact with the active site of integrase. The halogens
(7b–d) and para-fluorobenzyl substituents (8a–d) on carbazol-1-one ring had little effect on potency.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
that the a,c-diketo acid moiety chelates with two Mg2+
ions in the integrase active site to form a tertiary DKA–
Mg2+-integrase complex, thus blocking substrate DNA
binding.17,20
HIV integrase is one of the three essential viral enzymes
encoded by HIV pol gene. It catalyzes the integration of
viral DNA onto the host genome-a critical step for viral
replication-in a two-step process, 30-processing and
strand transfer.1 Since integrase has no human counter-
part, it is considered to be a promising target for develop-
ing new anti-HIV drugs that can be used as alternatives to
current therapeutic agents suffering drug resistance. HIV
integrase was first recognized as a novel anti-AIDS target
in the early 1990s (see reviews1–6). Several classes of HIV
integrase inhibitors have since been developed, including
dinucleotides,7 hydroxylated aromatics,8–12 and a,c-dike-
to acids.13–19 Among these inhibitors, only the diketo acid
compounds (DKAs) were biologically validated as target-
ing integrase by specifically inhibiting the strand transfer
step in the integration process.14 The inhibition is depen-
dent on the presence of divalent metal ions.17 It is believed
The first DKA types of inhibitors were independently
reported by Shionogi & Co. and Merck.15,14 In their pat-
ent, authors from Shionogi & Co. revealed that
OH
O
HO
HO
N
O
NH
N
O
Cl
Cl
N
N
H
N
H
2
1
(5-CITEP, Shionogi)
(Shionogi)
O
O
O
OH
N
O
OH
O
OH
O
OH
F
Keywords: HIV integrase inhibitor; a,c-Diketo acids; Carbazolone;
Conformationally restrained.
4
3
(L-708,906, Merck)
(L-731,988, Merck)
*
Corresponding author. Tel.: +1 612 624 9911; fax: +1 612 624
0139; e-mail: vince001@umn.edu
Figure 1. Reported a,c-diketo acids as HIV integrase inhibitors.
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.013

X. Li, R. Vince / Bioorg. Med. Chem. 14 (2006) 2942–2955
2943
indole-containing a,c-diketo acid (Fig. 1) showed good
potency against integrase with an IC50 less than 1 lM.15
They also disclosed the tetrazole-containing 5-CITEP
(2, Fig. 1) as carboxylic acid bioisostere with an IC50 of
2.1 lM.13 Similar a,c-diketo acids (3–4, Fig. 1) with
anti-integrase activities were also reported by Merck. L-
731,988 (3) showed good potency against integrase, par-
ticularly in the strand transfer step with an IC50 of
0.08 lM compared with 6 lM in the 30-processing step.14
form of diketo acid. The effect of b-substitution and
conformational restraint on potency was investigated.
In addition, the geometry between the aromatic substit-
uents was examined since the angle of bisection is be-
lieved to be crucial for potency.16
In our SAR study, two of the reported potent integrase
inhibitors, compounds 1 and 3 (Fig. 1), were used as the
lead. By combining the important features of these two
molecules, we designed the conformationally restrained
b-cyclized DKA analogs by connecting the b carbon
of a,c-diketo acid to the indole ring via a methylene
chain bridge (Fig. 3). This modification afforded a
‘drug-like’ tricyclic carbazolone molecule, which is an
important pharmacophore present in many pharmaceu-
tical agents such as 5HT3 inhibitors and anti-tumor nat-
ural alkaloids.24–27 Three other major modifications on
the carbazolone core were made to further examine
the effects of substitutions on activity. First, we investi-
gated the substitution position of the DKA group to
look for the optimal geometry. The 2-hydroxy-acrylic
acid side chain was substituted either at the 3 position
of carbazol-4-one (5–6, Fig. 3) derived from compound
1 or at the 2 position of carbazol-1-one (7–8, Fig. 3) de-
rived from compound 3. Second, a para-fluorobenzyl
group was introduced to carbazolone core (6 and 8) be-
cause its presence was reported to improve activity.16,18
Last, the effect of halogen substitution on the carbazo-
lone ring was examined. Based on these modifications,
four series of molecules (5–8, Fig. 3) were synthesized
and their biological activities were evaluated.
One obstacle to the efficient and successful development
of integrase inhibitors is due to the lack of inhibitor–in-
tegrase interaction information on molecular basis. One
co-crystal structure of DKA compound 5-CITEP and
integrase was available,13 but its reliability might be
compromised by crystal packing.21 Therefore, a compre-
hensive structure–activity relationship (SAR) needs to
be established. The general structure of DKAs contains
a central a,c-diketo moiety flanked by an aromatic ring
and a carboxylic acid group (Fig. 2). It is believed that
the diketo acid moiety is the key pharmacophore for en-
zyme inhibition,17 and that an aromatic group adjacent
to the diketo acid moiety improves potency and selectiv-
ity.16,18,22 Many SAR studies on DKAs have been
reported and provided very useful information. Most
of them either focused on the carboxylate moiety by
changing to its bioisosteres and other electron-bearing
heterocycles,18,23 or on the aromatic moiety by changing
its substituents or replacing by other heteroaromat-
ics.23,16,18,19 However, direct modifications on the cen-
tral a,c-diketo moiety are very limited. Since the
diketo group chelates to Mg2+ and mediates inhibitor
binding to integrase, it is necessary to explore the struc-
tural features of this moiety. We were interested in
substituting the b-carbon of the a,c-diketo group and
adding conformational restraint onto the open-chain
2. Results and discussion
2.1. Chemistry
The carbazolone-containing diketo acids 5–8 were syn-
thesized by coupling the corresponding carbazolones
with acylating reagents ethyl chlorooxoacetate 9 or
diethyl oxalate 10 in the presence of sodium hydride
or sodium ethoxide.28,29 The 6(7)-halo-carbazol-4-ones
12a–d were prepared via Fischer-indole synthesis from
4(3)-halo-phenylhydrazine hydrochloride 11a–d and
1,3-cyclohexanedione 1330,31 (Scheme 1). We also used
microwave-assisted synthesis and optimized the reaction
O
OH
COOH
Aryl
diketo
Carboxylate or
bioisosteres
Aromatic or
Heteroaromatic
Figure 2. General structure of DKAs.
O
OH
HO
O
Cl
O
4
OH
O
OH
OH
OH
6
X
OH
OH
X
F
O
O
X
N
O
O
N
H
1
7
N
N
H
1
R
5
6
(Shionogi)
Carbazol-4-one
O
β
4
α
6
O
X
OH
X
O
N
O
O
X
OH
1
N
OH
7
N
R
OH
N
OH
O
OH
H
O
OH
O
OH
F
3
7
8
F
Carbazol-1-one
(L-731,988, Merck)
X= H, F, Cl
Figure 3. Design of carbazolone-containing DKA analogs as HIV integrase inhibitors.

2944
X. Li, R. Vince / Bioorg. Med. Chem. 14 (2006) 2942–2955
O
Since tosyl is a good indole-protecting group, we first
O
X1
X2
protected carbazol-4-one 12b with tosyl chloride. The
resulting N-tosyl carbazol-4-one (18, Scheme 2) was
treated with acylating reagents 9 or 10 and base, but
no reaction products were detected. Similarly, when
Boc was used as the protecting group (19), no coupling
reactions occurred. Since in both of the molecules, the
protecting groups are electron withdrawing, we sur-
mised that the loss of reactivity was due to the very sta-
ble and highly conjugated enolate (Fig. 4). To test this
hypothesis, we placed a nonelectron-withdrawing meth-
yl group onto carbazol-4-one (12b, Scheme 2). As
expected, the resulting methylated compound 20 was
readily coupled with compound 9 and gave the desired
C-acylated product. Ester 21 was then hydrolyzed under
acidic conditions to give the carboxylic acid 22 (Scheme
O
13
N
H
a or b
12a X1=X2= H
X1
X2
12b X1= F, X2=H
12c X1= Cl, X2=H
12d X1= H, X2=Cl
NH2.HCl
Method A
O
N
H
11a X1=X2= H
X1
X2
11b X1= F, X2=H
11c X1= Cl, X2=H
11d X1= H, X2=Cl
O
16
a
N
H
O
15b X1= F, X2=H
15c X1= Cl, X2=H
X2=Cl
15d X1= H,
c
N
N
H
H
O
15a
17
2). Considering
a nonelectron-withdrawing group
Scheme 1. Reagents and conditions: (a) i—H2O, rt, overnight; ii—
TFA, 90 °C, overnight; (b) TFA or H2O, 10 min in microwave; (c)
I2O5, THF/H2O, rt, 30 min.
should be used to protect the carbazol-4-ones, we first
chose the easily removable silanyl group. However, the
low yield of the protection step due to the labile N–Si
bond, coupled with the isolation of the O-acylated prod-
uct, led to the selection of an alternate protecting group.
We therefore selected diethoxymethyl (DEM) as the
protecting group, which can be removed under acidic
conditions. The coupling reaction worked best with
diethyl oxalate 10 and sodium ethoxide. The DEM
conditions in one-pot and one-step with satisfactory
yields. Similarly, carbazol-1-ones 15b–d were synthe-
sized via Fischer-indole synthesis from phenylhydrazine
hydrochloride 11b–d and 1,2-cyclohexanedione 16. Car-
bazol-1-one can also be obtained by oxidizing commer-
cially available carbazole 17 with diiodine pentoxide32
(Scheme 1). Interestingly, direct coupling of carbazol-
4-one 12 with acylating reagents 9 or 10 afforded less
than 30% of the C-acylation product because of the mul-
ti-products formed in the reaction mixture, and both N-
acylated and O-acylated products were found. The yield
could not be improved by adding more acylating reagent
and base or by applying a higher temperature and a
longer reaction time. Reasoning that the free NH group
may affect the reaction, we therefore protected the
indole nitrogen before carrying out the coupling step.
O
O
O
F
F
F
B-
N
S
N
S
N
S
O
O
O
O
O
O
H3C
H3C
H3C
conjugated
enolate
18
Figure 4. The highly stable enolate is unreactive toward acylating
reagents.
O
F
b or c
X
TsCl
NO REACTION
N
S
a
O
O
18
CH3
O
Cl
(Boc)2O
d
b or c
X
NO REACTION
N
O
O
O
O
X
19
O
OH
N
H
12
O
OH
OH
F
F
F
OEt
h
b
MeI
O
X= H, F, Cl
O
N
N
CH3
N
CH3
21
e
CH3
22
20
O
OH
O
OH
O
OH
OEt
h
g
(EtO)3CH
f
O
O
N
H
5a
N
N
OEt
OEt
EtO
EtO
26a
25a
Scheme 2. Selection of protecting groups for carbazol-4-ones. Reagents and conditions: (a) NaH, DMF, TsCl, rt, overnight; (b) NaH, THF, 9, 60 °C,
overnight; (c) Na/EtOH, 10, rt, overnight; (d) (Boc)2O, Et3N, DMAP, DMF, rt, overnight; (e) NaH, DMF, CH3I, rt, overnight; (f) (EtO)3CH, DMF,
toluene, CSA (cat), 130–140 °C, 2 d; (g) Na/EtOH, 10, rt, 1 h; (h) 1 N HCl, 1,4-dioxane, 100–110 °C, 4–10 h.

X. Li, R. Vince / Bioorg. Med. Chem. 14 (2006) 2942–2955
2945
group was removed in the same step with the hydrolysis
of the ester (Scheme 2).
readily react with diethyl oxalate 10 in the presence of
sodium ethoxide to give the coupling products 29a–d,
which were hydrolyzed to give the free acids 7a–d. Similar-
ly, target compounds 8a–d were made by first alkylating
the carbozol-1-ones 15a–d with 4-fluorobenzyl bromide.
Subsequent coupling with diethyl oxalate 10, followed
by ester hydrolysis gave the final free acids (Scheme 4).
With the selection of DEM as the protecting group, tar-
get compounds 5a–c were synthesized (Scheme 3). Car-
bazol-4-ones (12a–c) were first protected with DEM by
refluxing with triethyl orthoformate,33 the resulting
compounds 25a–c subsequently reacted with diethyl
oxalate 10 to give the esters 26a–c. The DEM groups
were removed together with the hydrolysis of the esters
to give the carbazol-4-one-containing DKAs (5a–c). The
benzylated DKAs 6a–d were synthesized from carbazol-
4-ones 12a–d, which were first benzylated with 4-fluo-
robenzyl bromide to give 27a–d. Compounds 27a–d
were then acylated with ethyl chlorooxoacetate 9 in
the presence of sodium hydride to give the coupling
products 28a–d. The esters were hydrolyzed by refluxing
in dilute HCl to give the free acids 6a–d (Scheme 3).
In the course of synthesis of target compounds 5 and 7,
we found that the carbazol-1-one 15 and carbazol-4-one
12 showed different reactivity toward C-acylation in
thermodynamic-controlled conditions. Compound 15
readily reacted with acylating reagent to give the cou-
pling product 29 with 70–80% yield (Scheme 4). Howev-
er, the acylation reactions under similar conditions with
compound 12 only afforded less than 30% product. This
is possibly due to the different conjugation systems in
each molecule resulting in differences in enolate stability.
For the fully conjugated carbazol-4-one (12, Fig. 5),
enolate 33 that is in equilibrium with 32 is more stable
than enolate 34. As a result, C-acylation is not the major
Target molecules 7 and 8 were synthesized from carba-
zole-1-ones 15a–d (Scheme 4). Carbazole-1-ones can
O
O
OH
O
OH
X1
X1
Method C
OH
X1
OEt d
c
a
Method B
O
O
X2
N
X2
N
X2
N
H
OEt
EtO
OEt
EtO
5a X1=X2= H
5b X1= F, X2=H
5c X1= Cl, X2=H
O
25a X1=X2= H
25b X1= F, X2=H
25c X1= Cl, X2=H
26a X1=X2= H
26b X1= F, X2=H
26c X1= Cl, X2=H
X1
X2
N
H
O
O
OH
OH
O
12a X1=X2= H
OEt
X1
OH
X1
Method E
X2
X1
X2
e
d
12b X1= F, X2=H
12c X1= Cl, X2=H
12d X1= H, X2=Cl
Method C
b
O
O
N
N
X2
N
Method
D
F
F
F
6a X1=X2= H
28a X1=X2= H
27a X1=X2= H
6b X1= F, X2=H
6c X1= Cl, X2=H
6d X1= H, X2=Cl
28b X1= F, X2=H
28c X1= Cl, X2=H
28d X1= H, X2=Cl
27b X1= F, X2=H
27c X1= Cl, X2=H
27d X1= H, X2=Cl
Scheme 3. Synthesis of target compounds 5 and 6. Reagents and conditions: (a) (EtO)3CH, DMF, toluene, CSA (cat), 130–140 °C, 2 d; (b) NaH,
DMF, 4-fluorobenzyl bromide, rt, 4 h; (c) Na/EtOH, 10, rt, 1 h; (d) 1 N HCl, 1,4-dioxane, 100–110 °C, 4 h; (e) NaH, THF, 9, 50 °C, overnight.
X1
X2
X1
X2
X1
X2
O
O
c
b
N
OH
N
OEt Method C
Method B
N
H
H
H
O
OH
O
OH
O
29a X1=X2= H
15a X1=X2= H
7a X1=X2= H
29b X1= F, X2=H
15b X1= F, X2=H
15c X1= Cl, X2=H
15d X1= H, X2=Cl
7b X1= F, X2=H
7c X1= Cl, X2=H
7d X1= H, X2=Cl
29c X1= Cl, X2=H
29d X1= H, X2=Cl
a
Method D
X1
X2
X1
X2
X1
X2
O
O
b
c
N
O
OH
N
O
OEt
N
O
OH
OH
F
F
F
8a X1=X2= H
31a X1=X2= H
30a X1=X2= H
8b X1= F, X2=H
8c X1= Cl, X2=H
8d X1= H, X2=Cl
31b X1= F, X2=H
31c X1= Cl, X2=H
31d X1= H, X2=Cl
30b X1= F, X2=H
30c X1= Cl, X2=H
30d X1= H, X2=Cl
Scheme 4. Synthesis of target compounds 7 and 8. Reagents and conditions: (a) NaH, DMF, 4-fluorobenzyl bromide, rt, 4 h; (b) Na/EtOH, 10, rt,
1 h; (c) 1 N HCl, 1,4-dioxane, 100–110 °C, 4 h.

2946
X. Li, R. Vince / Bioorg. Med. Chem. 14 (2006) 2942–2955
O
O
O
O
B-
>
N
H
N
N
N
H
33
32
34
12
fully conjugated
O
B-
+
N
OEt
N
H
N
N
H
O
OH
H
O
O
O
C-Acylation product
15
36
35
cross conjugated
Figure 5. Fully conjugated carbazol-4-one 12 and cross-conjugated carbazol-1-one 15 showed different reactivity toward acylating reagents.
reaction and both N- and O-acylation products were
found. Whereas for the cross-conjugated carbazol-1-
one 15, enolate 36 is stable and the C-acylation product
is the major product under thermodynamic conditions.
to carbazol-1-ones (8a–d) had little impact on activity.
Interestingly, fluorine substitution (8b) was less active
among this series, and changing the chlorine position
from 6 (8c) to 7 (8d) slightly decreased potency.
2.2. Structure–activity relationship studies
3. Experimental
All target compounds were tested against HIV integrase
in microtiter and radioactive gel assays. In general, car-
bazolone-containing a,c-diketo acid analogs inhibited
recombinant HIV integrase at low micromolar range
(Table 1). Modification of lead compound 1 to the
conformationally restrained and b-substituted car-
bazol-4-one DKA (5c) reduced potency by 4- to 5-fold,
suggesting that the open-chain form of DKA adopts a
better conformation for binding to integrase. This
reduction in activity might also be attributed to the
steric hindrance introduced by the methylene bridge or
might result from a twisted coplanarity of Mg2+ and
a,c-diketo complex with bulky b-substituents leading
to a decrease in chelating strength.34 Replacing the chlo-
rine atom on carbazol-4-one (5c) with either a hydrogen
(5a) or fluorine (5b) had little effect on potency, with the
fluorine substitution (5b) exhibiting a better activity.
Alkylation of the carbazol-4-one nitrogen with a para-
fluorobenzyl group (6a–c) or a methyl group (22) led
to significant decrease in activity. This alkylation effect
was different from that found in indole DKAs (1), in
which the size of the alkylating group affected potency.22
Our results suggest that the free NH group of carbazol-
4-one possibly hydrogen bonded with Gln 148 in the
active site of integrase, which is similar to the indolic
nitrogen and Gln 148 hydrogen bond found in the
co-crystal structure of CITEP and integrase.13 Interest-
ingly, the decreased activity due to alkylation was recov-
ered by changing the position of chlorine substituent
from 6 (6c) to 7 (6d), which might be attributed to the
increased binding affinity.
3.1. Biological assays
All the synthesized compounds were tested in a dual
microtiter assay and radioactive gel assay35,36 (data pro-
vided by Southern Research Institute, Birmingham,
AL). The results were shown as the combined activity
of both 30-processing and strand transfer steps given
as percentage of inhibition at 10 lM.
3.2. Chemistry
All reactions were performed in septum-sealed flasks
under argon atmosphere. Progress of the reaction was
followed by TLC. Organic solutions were dried over
MgSO4 and evaporated on a rotary evaporator under re-
duced pressure. Melting points were determined on a Mel-
Temp II apparatus and are uncorrected. 1H and 13C
NMR spectra were obtained using a Varian Unity
VAC-300 spectrometer operating at a field strength of
300 MHz. Chemical shifts (d) are reported in parts per
million (ppm) and coupling constants (J) in hertz. Stan-
dard and peak multiplicities are designated as follows: s,
singlet; d, doublet; dd, doublet of doublets; t, triplet; q,
quartet; p, pentet; br s, broad singlet; and m, multiplet.
Mass spectra were determined using Bruker BioTOF II
spectrometer. Elemental analyses were performed by M-
H-W laboratories (Phoenix, AZ) and gave results within
0.4% of the theoretical values. Flash chromatography
was performed using a 200–300 mesh silica gel. All com-
mercial solvents were of reagent grade or better and used
directly as supplied. Yields refer to purified products and
were not optimized.
Our results also indicated that the geometry of the dike-
to acid side chain was crucial for potency; changing the
position of the a,c-diketo acid from carbazol-4-one (5a–
c) to carbazol-1-one (7a–d) increased activity by 2- to 3-
fold. Substituting the hydrogen (7a) with halogen atoms
(7b–d) on carbazol-1-one only slightly increased activity,
with the fluorine substitution (7b) being the most active
one, which is similar to the observation with carbazol-4-
one (5b). In contrast to the significant loss of activity on
carbazol-4-one (6a–c), adding a para-fluorobenzyl group
3.3. Method A: General procedure for the preparation of
6(7)-halo-1,2,3,9-tetrahydrocarbazol-4-ones (12a–d)
The title compound was prepared as described.31 To a
solution of 1,3-cyclohexanedione (13, 10 mmol) in water
(15 mL) was added 4(3)-halo-phenylhydrazine hydro-
chloride salt (11a–d, 10 mmol) portionwise. The mixture
was allowed to stir for one day at room temperature (rt).

X. Li, R. Vince / Bioorg. Med. Chem. 14 (2006) 2942–2955
2947
Table 1. Anti-HIV integrase activities of carbazol-4-one- (5 and 6) and carbazol-1-one- (7 and 8) containing DKAs in microtiter and radioactive gel
assaysb
Compound
Structure
Inhibition at 10 lM (%)
Microtiter assay
Gel assay
54.4
O
OH
Cl
OH
1
83.3
12.9
36.9
16.4a
1.0a
O
N
H
O
OH
OH
5a
5b
5c
22
15.4
31.5
16.6
2.7a
O
N
H
O
OH
F
OH
O
N
H
O
OH
Cl
F
OH
O
N
H
O
OH
OH
O
N
CH3
O
O
OH
OH
OH
OH
OH
O
6a
6b
6c
6d
N
0a
1.8a
0.7a
2.3a
52.4
F
F
OH
O
N
N
N
5.6a
8.0a
51.3
F
O
OH
Cl
O
F
O
OH
O
Cl
F
O
OH
N
OH
O
3
79.0
37.3
55.0
61.4
26.3
F
O
OH
7a
7b
N
OH
H
O
F
O
57.7
OH
N
H
OH
O
(continued on next page)

2948
X. Li, R. Vince / Bioorg. Med. Chem. 14 (2006) 2942–2955
Table 1 (continued)
Compound
Structure
Inhibition at 10 lM (%)
Microtiter assay
Gel assay
26.1
Cl
O
7c
7d
46.4
46.6
OH
N
OH
H
O
O
OH
Cl
46.7
37.0
N
H
OH
O
O
OH
N
OH
O
8a
48.2
F
F
O
OH
N
OH
8b
33.3
24.5
O
F
Cl
O
OH
N
OH
8c
54.7
47.1
54.9
44.2
O
F
O
OH
Cl
N
OH
O
8d
F
a % of inhibition at 25 lM.
b Reactions were done in duplicate.
The solid precipitated was collected by filtration and
washed with water to give the hydrazone. The dried
hydrazone was treated with 9 mL trifluoroacetic acid
at 90 °C. After the mixture was refluxed overnight, the
excess TFA was removed in vacuo. The mixture was
washed with water and saturated sodium bicarbonate.
The resulting solid was recrystallized with dichlorometh-
ane to give the title compound.
d6): d 193.4, 154.4, 133.0, 125.8, 113.4, 112.5, 111.0,
110.7, 106.0, 38.4, 24.1, 23.6.
3.3.3. 6-Chloro-1,2,3,9-tetrahydrocarbazol-4-one (12c).
Yield 69%; mp 282–284 °C (285 °C30); 1H NMR
(CD3OD): d 7.97 (1H, d, J = 1.5 Hz), 7.32 (1H, d,
J = 7.8 Hz), 7.16 (1H, dd, J = 1.8, 9.0 Hz), 3.0 (2H, t,
J = 6 Hz), 2.54 (2H, t, J = 6 Hz), 2.22 (2H, m).
3.3.1. 1,2,3,9-Tetrahydrocarbazol-4-one (12a). Yield 40%;
mp 223–224 °C (218–220 °C30); 1H NMR (DMSO-d6): d
11.75 (1H, br s, NH), 7.90 (1H, m), 7.38 (1H, m), 7.11
(2H, m), 2.93 (2H, t, J = 6 Hz), 2.40 (2H, t, J = 6 Hz),
2.09 (2H, p, J = 6 Hz); 13C NMR (DMSO-d6): d 193.4,
152.9, 136.5, 125.2, 123.1, 122.2, 120.8, 112.4 (2), 38.6,
24.3, 23.6.
3.3.4. 7-Chloro-1,2,3,9-tetrahydrocarbazol-4-one (12d).
Yield 22%; mp 225–229 °C (140 °C30); 1H NMR
(DMSO-d6): d 12.00 (1H, s, NH), 7.91 (1H, d, J = 9 Hz),
7.44 (1H, d, J = 2 Hz), 7.15 (1H, dd, J = 1.5, 9 Hz), 2.96
(2H, t, J = 6 Hz), 2.43 (2H, t, J = 6 Hz), 2.12 (2H, p,
J = 6 Hz); 13C NMR (CD3OD): d 192.0, 154.2, 138.4,
126.1, 124.0, 123.6, 122.3, 121.9, 111.7, 110.6, 24.0, 23.3.
3.3.2. 6-Fluoro-1,2,3,9-tetrahydrocarbazol-4-one (12b).
3.4. General procedure for the synthesis of diethoxym-
ethyl (DEM) protected 1,2,3,9-tetrahydrocarbazol-4-ones
(25a–c)33
1
Yield 75%; mp 253 °C (254 °C30); H NMR (DMSO-
d6): d 12.0 (1H, s, NH), 7.56 (1H, dd, J = 2.4, 9.6 Hz),
7.37 (1H, dd, J = 4.5, 9.0 Hz), 6.98 (1H, dt, J = 2.4,
9.0 Hz), 2.93 (2H, t, J = 6.3 Hz), 2.40 (2H, t, J =
6.9 Hz), 2.08 (2H, p, J = 6.3 Hz); 13C NMR (DMSO-
A solution of 1,2,3,9-tetrahydrocarbazol-4-one (12a–c,
1 mmol) in 1 mL dry DMF was treated with triethyl

X. Li, R. Vince / Bioorg. Med. Chem. 14 (2006) 2942–2955
2949
orthoformate (10 mmol), camphor sulfonic acid
(16 mg), and 2.5 mL benzene. The mixture was heated
to reflux for two days. After cooling down, the reaction
mixture was concentrated and purified by flash column
(EtOAc/hexanes, 1/4) to give the target compound.
151.2, 136.6, 125.0, 124.2, 123.5, 121.8, 113.9, 111.7,
110.6, 102.5, 63.1, 62.1, 23.6, 22.8, 15.2, 14.6; MS (ESI):
m/z 388.2 [M+H]+, 410.2 [M+Na]+, 426.1 [M+K]+.
3.5.2. (9-Diethoxymethyl-6-fluoro-4-oxo-1,2,4,9-tetrahy-
drocarbazol-3-ylidene)-hydroxyacetic acid ethyl ester (26b).
Yield 53%; mp 91–92 °C; 1H NMR (CDCl3): d 8.26 (1H,
dd, J = 2.7, 9.3 Hz), 7.97 (1H, dd, J = 4.2, 9.0 Hz), 7.38
(1H, dt, J = 2.4, 9.0 Hz), 6.61 (1H, s), 4.76 (2H, q,
J = 6.9 Hz), 4.08 (2H, m), 3.88 (2H, m), 3.64 (2H, t,
J = 6.6 Hz), 3.51 (2H, t, J = 6.6 Hz), 1.79 (3H, t,
J = 7.2 Hz), 1.62 (6H, t, J = 7.2 Hz); 13C NMR (CDCl3):
d 189.3, 163.6, 161.6, 158.4, 157.3, 151.9, 132.8, 125.9,
113.6, 113.1, 112.3, 112.0, 110.4, 107.7, 107.4, 102.6, 63.0,
62.2, 23.4, 22.7, 15.1, 14.6; MS (ESI): m/z 428.2 [M+Na]+;
Anal. (C21H24FNO6) C, H, N.
3.4.1. 9-Diethoxymethyl-1,2,3,9-tetrahydrocarbazol-4-one
1
(25a). Yield 71%; mp 85–87 °C; H NMR (CDCl3): d
8.26 (1H, m), 7.60 (1H, m), 7.25 (2H, m), 6.29 (1H, s),
3.70 (2H, m), 3.50 (2H, m), 3.13 (2H, t, J = 6.0 Hz), 2.58
(2H, t, J = 6.0 Hz), 2.24 (2H, p, J = 6.0 Hz), 1.24 (6H, t,
J = 6.9 Hz); 13C NMR (CDCl3): d 194.6, 150.9, 136.0,
125.2, 123.6, 123.0, 121.8, 114.4, 111.3, 102.5, 62.9, 38.4,
23.9, 23.6, 15.2; MS (ESI): m/z 288.2 [M+H]+, 310.1
[M+Na]+.
3.4.2. 6-Fluoro-9-diethoxymethyl-1,2,3,9-tetrahydrocar-
1
bazol-4-one (25b). Yield 78%; mp 130–133 °C; H NMR
3.5.3.
(9-Diethoxymethyl-6-chloro-4-oxo-1,2,4,9-tetra-
(CDCl3): d 7.91 (1H, dd, J = 2.4, 9.0 Hz), 7.56 (1H, dd,
J = 4.5, 9.0 Hz), 7.25 (1H, dt, J = 2.7, 9.0 Hz), 6.21 (1H,
s), 3.68 (2H, m), 3.51 (2H, m), 3.08 (2H, t, J = 6.0 Hz),
2.56 (2H, t, J = 6.0 Hz), 2.24 (2H, p, J = 6.0 Hz), 1.24
(6H, t, J = 6.9 Hz); 13C NMR (CDCl3): d 194.3, 161.4,
158.2, 151.8, 132.2, 126.1, 125.9, 112.7, 112.6, 111.8,
111.4, 107.5, 107.2, 102.6, 62.9, 38.2, 23.8, 23.5, 15.1; MS
(ESI): m/z 328 [M+Na]+.
hydrocarbazol-3-ylidene)-hydroxyacetic acid ethyl ester
(26c). Yield 66%; 1H NMR (CDCl3): d 8.58 (1H, d,
J = 1.8 Hz), 7.97 (1H, d, J = 9.0 Hz), 7.61 (1H, dd,
J = 2.1, 9.0 Hz), 6.63 (1H, s), 4.76 (2H, q, J = 7.5 Hz),
4.10 (2H, m), 3.91 (2H, m), 3.65 (2H, t, J = 6.6 Hz), 3.53
(2H, t, J = 6.6 Hz), 1.80 (3H, t, J = 6.9 Hz), 1.64 (6H, t,
J = 6.9 Hz); 13C NMR (CDCl3): d 189.2, 163.5, 157.4,
151.8, 134.8, 129.3, 126.1, 124.4, 121.4, 113.2, 110.3,
102.6, 63.1, 62.2, 23.4, 22.6, 15.1, 14.6; MS (ESI): m/z
444.1 [M+Na]+.
3.4.3. 6-Chloro-9-diethoxymethyl-1,2,3,9-tetrahydrocar-
1
bazol-4-one (25c). Yield 84%; mp 109–110 °C; H NMR
(CDCl3): d 8.24 (1H, d, J = 2.1 Hz), 7.55 (1H, d,
J = 6.0 Hz), 7.19 (1H, dd, J = 2.4, 6.0 Hz), 6.22 (1H, s),
3.70 (2H, m), 3.49 (2H, m), 3.09 (2H, t, J = 6.0 Hz), 2.57
(2H, t, J = 6.0 Hz), 2.24 (2H, p, J = 6.0 Hz), 1.24 (6H, t,
J = 6.9 Hz); 13C NMR (CDCl3): d 194.2, 151.6, 134.2,
128.9, 126.3, 123.8, 121.3, 113.8, 112.8, 102.6, 62.9, 38.2,
23.8, 23.5, 15.1; MS (ESI): m/z 322 [M+H]+, 344 [M+Na]+.
3.6. Method C: General procedure for the synthesis of
(4-oxo-1,2,4,9-tetrahydrocarbazol-3-ylidene)-hydroxy-
acetic acids (5a–c)
(9-Diethoxymethyl-4-oxo-1,2,4,9-tetrahydrocarbazol-3-
ylidene)-hydroxyacetic acid ethyl ester (26a–c, 1 mmol)
was suspended in 4 mL of 1 N HCl and 5 mL of 1,4-di-
oxane. The mixture was heated to reflux for 4 h. After
cooling down, the solid precipitated out was filtered
and washed with water, diethyl ether, and methanol.
The resulting solid was recrystallized with methanol to
give the title compound.
3.5. Method B37: General procedure for the synthesis of
(9-diethoxymethyl-4-oxo-1,2,4,9-tetrahydrocarbazol-3-yl-
idene)-hydroxyacetic acid ethyl esters (26a–c)
9-(Diethoxymethyl)-1,2,3,9-tetrahydrocarbazol-4-one
(25a–c, 1 mmol) was suspended in 1 mL absolute etha-
nol, to which sodium (30 mg, 1.3 equiv) was added por-
tionwise. The reaction mixture was stirred at room
temperature under argon until all the sodium was con-
sumed. To the mixture was then added diethyl oxalate
(10, 200 lL, 1.5 equiv) dropwise and stirring was contin-
ued at room temperature for 1 h. The reaction was
quenched by pouring into 10 mL ice water and the pH
was adjusted to 6 with 1 N HCl. The aqueous layer
was extracted with ethyl acetate and the organic extracts
were combined and dried over MgSO4. After concen-
trated, the crude residue was purified by flash chroma-
tography (EtOAc/hexanes, 1/10–1/5) to give the title
compound.
3.6.1. (4-Oxo-1,2,4,9-tetrahydrocarbazol-3-ylidene)-hydroxy-
acetic acid (5a). Yield 91%; mp 217–219 °C; H NMR
1
(DMSO-d6): (enol form:ketone form = 4:1) enol form d
12.46 (1H, s), 8.15 (1H, d, J = 6 Hz), 7.65–7.60 (1H, m),
7.45–7.35 (2H, m), 3.23 (4H, m); ketone form d 12.25 (1H,
s), 8.07 (1H, d, J = 6 Hz), 4.65 (1H, dd, J = 4.8, 12 Hz),
2.60 (1H, m), 2.48 (1H, m); 13C NMR (DMSO-d6): d
189.0, 165.2, 163.3, 159.0, 153.8, 137.2, 125.0, 123.9, 123.5,
123.0, 122.6, 121.1, 112.8, 111.7, 108.9, 55.8, 26.1, 23.7,
22.7, 22.4; HRMS (ESI): m/z 280.0575 [M+Na]+; Anal.
(C14H11NO4) C, H, N.
3.6.2. (6-Fluoro-4-oxo-1,2,4,9-tetrahydrocarbazol-3-yli-
dene)-hydroxyacetic acid (5b). Yield 91%; mp 223–
225 °C; 1H NMR (DMSO-d6): (enol form:ketone
form = 2.7:1) enol form d 12.35 (1H, s), 7.61 (1H, dd,
J = 2.1, 9 Hz), 7.47–7.39 (1H, m), 7.10–6.99 (1H, m),
3.01 (4H, m); ketone form d 12.16 (1H, s), 7.52 (1H, dd,
J = 2.4, 9 Hz), 4.45 (1H, dd, J = 4.8, 12 Hz), 2.40 (1H,
m), 2.26 (1H, m); 13C NMR (DMSO-d6): d 197.3, 190.1,
189.0, 165.1, 163.2, 159.0, 155.1, 133.8, 133.3, 125.6,
3.5.1.
(9-Diethoxymethyl-4-oxo-1,2,4,9-tetrahydrocar-
bazol-3-ylidene)-hydroxyacetic acid ethyl ester (26a). Yield
66%; mp 68–70 °C; 1H NMR (CDCl3): d 8.47 (1H, m), 7.88
(1H, m), 7.54(2H, m), 6.54(1H, m), 4.62(2H, m), 3.96(2H,
m), 3.77 (2H, m), 3.49 (2H, m), 3.42 (2H, m), 1.65 (3H, m),
1.50 (6H, m); 13C NMR (CDCl3): d 189.5, 163.6, 157.2,

2950
X. Li, R. Vince / Bioorg. Med. Chem. 14 (2006) 2942–2955
114.1, 111.8, 111.5, 108.8, 106.4, 106.1, 55.7, 25.9, 23.6,
22.7; MS (ESI): m/z 274.1 [MꢀH]ꢀ; HRMS (ESI): Calcd
for (C14H10FNO4)H+: 276.0667. Found: 276.0650.
3.7.4.
7-Chloro-9-(4-fluorobenzyl)-1,2,3,9-tetrahydro-
carbazol-4-one (27d). Yield 82%; mp 122–125 °C; 1H
NMR (CDCl3): d 8.1 (1H, d, J = 8.1 Hz), 7.25–6.93
(6H, m), 5.25 (2H, d, J = 16.2 Hz), 2.83 (2H, t,
J = 6 Hz), 2.58 (2H, q, J = 6.6 Hz), 2.19 (2H, m,
J = 6.6 Hz); 13C NMR (CDCl3): d 193.9, 191.5, 164.1,
152.7, 138.9, 131.4, 129.4, 127.9, 124 (4), 116 (2), 113,
109, 108, 46, 39, 38, 23; MS (ESI): m/z 350.1 [M+Na]+.
3.6.3.
(6-Chloro-4-oxo-1,2,4,9-tetrahydrocarbazol-3-yli-
dene)-hydroxyacetic acid (5c). Yield 91%; mp >240 °C
(dec.); 1H NMR (DMSO-d6): (enol form:ketone
form = 2:1) enol form d 12.83 (1H, s), 8.30 (1H, d,
J = 1.5 Hz), 7.85 (1H, t, J = 7.5 Hz), 7.65–7.59 (1H, m),
3.42 (4H, m); ketone form d 12.64 (1H, s), 8.22 (1H, d,
J = 2 Hz), 4.85 (1H, dd, J = 4.8, 12 Hz), 2.80 (1H, m),
2.55 (1H, m); 13C NMR (DMSO-d6) d 197.3, 190.2, 189.0,
165.0, 163.3, 159.2, 155.0, 154.8, 135.8, 135.3, 127.5,
127.2, 126.2, 126.0, 123.8, 123.5, 120.1, 119.8, 114.4,
114.1, 111.4, 108.8, 55.7, 25.9, 23.6, 22.7, 22.4; MS (ESI):
m/z 313.9 [M+Ma]+; Anal. (C14H10ClNO4) C, H, N.
3.8. Method E: General procedure for the synthesis of [9-
(4-fluorobenzyl)-4-oxo-1,2,4,9-tetrahydrocarbazol-3-yli-
dene]-hydroxyacetic acid ethyl esters (28a–d)
To
a
solution of 9-(4-fluorobenzyl)-1,2,3,9-tetra-
hydrocarbazol-4-one (27a–d, 1 mmol) in 5 mL anhy-
drous THF was added sodium hydride (2 mmol) at
45 °C. The mixture was stirred at the same temperature
for 20 min, followed by the addition of ethyl chloro-
oxoacetate (9, 2 mmol). The resulting mixture was
heated at 45–50 °C for 4 h, and an additional 1 mmol
of 9 was added. The reaction was complete after heat-
ing overnight. After cooling down, the crude mixture
was poured into 30 mL cold sodium bicarbonate solu-
tion. The aqueous part was extracted with ethyl ace-
tate. The organic layers were combined, washed with
saturated sodium chloride, and dried over MgSO4.
The concentrated crude product was purified by flash
chromatography (EtOAc/hexanes, 1/10–1/4) to give
the title compound.
3.7. Method D: General procedure for the synthesis of 9-(4-
fluorobenzyl)-1,2,3,9-tetrahydrocarbazol-4-ones (27a–d)
To a solution of 1,2,3,9-tetrahydrocarbazol-4-one (12a–
d, 1 mmol) in anhydrous DMF (3 mL) was added sodi-
um hydride (1.2 equiv) portionwise under ice bath. After
20 min of stirring at 0 °C, the ice bath was removed and
4-fluorobenzyl bromide (1.05 equiv) was added to the
reaction mixture dropwise. The resulting mixture was
stirred at room temperature for 4 h. After the reaction
was complete, the mixture was poured into 30 mL satu-
rated sodium bicarbonate and extracted with dichloro-
methane. The combined organic layers were dried over
MgSO4. After concentrated, the crude product was puri-
fied by flash chromatography (EtOAc/hexanes, 1/4) to
give the title compound.
3.8.1.
[9-(4-Fluorobenzyl)-4-oxo-1,2,4,9-tetrahydrocar-
bazol-3-ylidene]-hydroxyacetic acid ethyl ester (28a). Yield
74%; mp 184–186 °C; 1H NMR (CDCl3): d 8.17 (1H, d),
7.25 (3H, m), 6.99 (4H, m), 5.31 (2H, s), 4.35 (2H, q,
J = 6.9 Hz), 3.26 (2H, t, J = 6.9 Hz), 2.90 (2H, t,
J = 6.9 Hz), 1.4 (3H, t, J = 6.6 Hz); 13C NMR (CDCl3): d
185.6, 166.8, 164.1, 160.8, 145.5, 143.9, 137.9, 131.5,
128.1, 125.3, 123.4, 122.7, 121.2, 121.1, 119.1, 116.5,
116.2, 111.2, 110.6, 62.6, 46.8, 24.7, 21.0, 14.2; MS (EI):
m/z 393.2 [M+]; Anal. (C23H20FNO4) C, H, N.
3.7.1. 9-(4-Fluorobenzyl)-1,2,3,9-tetrahydrocarbazol-4-one
1
(27a). Yield 83%; mp 148–150 °C; H NMR (CDCl3): d
8.29 (1H, d, J = 7.2 Hz), 7.25–7.22 (3H, m), 6.99 (4H, d,
J = 7.2 Hz), 5.30 (2H, s), 2.87 (2H, t, J = 6.3 Hz), 2.60
(2H, t, J = 6.0 Hz), 2.37 (2H, p, J = 6.3 Hz); 13C NMR
(CDCl3): d 194.0, 151.7, 137, 128.0, 123.6 (2), 122.1,
116.4 (2), 109.7, 46.8, 38.2, 23.8, 22.7; MS (EI): m/z 293.1
(M+), 265, 236, 157, 156, 128, 109; Anal. (C19H16NOF)
C, H, N, F.
3.8.2. [6-Fluoro-9-(4-fluorobenzyl)-4-oxo-1,2,4,9-tetrahy-
drocarbazol-3-ylidene]-hydroxyacetic acid ethyl ester
(28b). Yield 68%; mp 176–177 °C; H NMR (CDCl3):
d 7.93 (1H, dd, J = 2.7, 9.0 Hz), 7.17 (2H, dd, J = 3.9,
9.0 Hz), 7.0 (5H, m), 5.29 (2H, s), 4.35 (2H, q,
J = 7.2 Hz), 3.25 (2H, t, J = 7.2 Hz), 2.90 (2H, t,
J = 7.2 Hz), 1.4 (3H, t, J = 7.2 Hz); 13C NMR (CDCl3):
d 188.8, 163.6, 157.3, 152.8, 131.1, 128.0 (2), 116.6 (2),
112.3, 112.0, 111.1, 110.1, 108.0, 107.7, 62.2, 47.1,
23.2, 21.9, 14.5; MS (FAB): m/z 412.0 [M+H]+; Anal.
(C23H19ClFNO4) C, H, N, F.
1
3.7.2. 6-Fluoro-9-(4-fluorobenzyl)-1,2,3,9-tetrahydrocar-
1
bazol-4-one (27b). Yield 71%; mp 181–183 °C; H NMR
(CDCl3): d 7.92 (1H, m), 7.12 (1H, dd, J = 3.9, 8.7 Hz),
7.0–6.9 (5H, m), 5.26 (2H, s), 2.85 (2H, t, J = 6.0 Hz),
2.56 (2H, t, J = 6.3 Hz), 2.22 (2H, p, J = 6 Hz); 13C
NMR (CDCl3): d 193.8, 164.1, 161.5, 160.8, 158.3, 152.9,
133.6, 131.6, 128.0, 125, 116.5 (2), 111.7, 111.4, 107.7,
101.6, 46.9, 38.0, 23.6, 22.8; HRMS (EI): Calcd for
(C19H15F2NO)+: 311.1122. Found: 311.1114 [M+].
3.8.3. [6-Chloro-9-(4-fluorobenzyl)-4-oxo-1,2,4,9-tetra-
hydrocarbazol-3-ylidene]-hydroxyacetic acid ethyl ester
(28c). Yield 90%; mp 123–124 °C; H NMR (CDCl3): d
3.7.3. 6-Chloro-9-(4-fluorobenzyl)-1,2,3,9-tetrahydrocar-
bazol-4-one (27c). Yield 90%; mp 204–205 °C; H NMR
1
1
(CDCl3): d 8.26 (1H, d, J = 1.5Hz), 7.14 (2H, m), 6.99
(4H, m), 5.26 (2H, s), 2.85 (2H, t, J = 6.0 Hz), 2.56 (2H,
t, J = 6Hz), 2.23 (2H, p, J = 6 Hz); 13C NMR (CDCl3): d
193.7, 164.1, 152.6, 135.5, 131.6, 128.8, 127.9, 126.1,
123.7, 121.6, 116.5, 116.2, 113.0, 110.8, 46.9, 38.1, 23.6,
22.7; HRMS (EI): Calcd for (C19H15ClFNO)+: 327.0826.
Found: 327.0818 [M+].
8.12 (1H, s), 7.16 (2H, s), 6.97 (5H, m), 5.28 (2H, s), 4.4
(2H, q, J = 7.2 Hz), 2.9 (4H, m), 1.41 (3H, t, J = 7.5 Hz);
13C NMR (CDCl3): d 185.7, 166.5, 164.2, 160.9, 146.2,
142.9, 136.3, 131.2, 128.5, 128.0, 126.2, 123.6, 120.7,
119.6, 116.6, 116.3, 111.6, 110.7, 62.6, 47.1, 24.7, 20.9,
14.3; MS (FAB): m/z 428.0 [M+H]+; Anal. (C20H19
ClFNO4) C, H, N.

X. Li, R. Vince / Bioorg. Med. Chem. 14 (2006) 2942–2955
2951
3.8.4. [7-Chloro-9-(4-fluorobenzyl)-4-oxo-1,2,4,9-tetra-
hydrocarbazol-3-ylidene]-hydroxyacetic acid ethyl ester
(28d). Yield 78%; mp 214–217 °C; H NMR (CDCl3): d
8.15 (1H, d, J = 8.4 Hz), 7.26 (2H, m), 7.0 (4H, m), 5.27
(2H, s), 4.35 (2H, q, J = 6.9 Hz), 3.26 (2H, t, J = 6.9 Hz),
2.89 (2H, t, J = 6.6 Hz), 1.40 (3H, t, J = 6.9 Hz); 13C
NMR (CDCl3): d 201, 152.4, 130.9, 128.0, 124.1,
123.0, 116.7, 116.4, 110.4, 62, 46.9, 23.3, 21.9, 14.5;
Anal. (C20H19ClFNO4) C, H, N, Cl.
3.10. 2,3,4,9-Tetrahydrocarbazol-1-one (15a)32
1
To a stirred solution of 1,2,3,4-tetrahydrocarbazole (17,
5.0 g, 29.2 mmol) in 300 mL THF and 75 mL water was
added diiodine pentoxide (11.7 g, 35.1 mmol). After stir-
ring for 20 min at room temperature, the solvent was re-
moved in vacuo at room temperature. The residue was
extracted with ethyl acetate. The organic portion was
washed with water, 5% Na2S2O3, saturated sodium bicar-
bonate and brine, and dried over MgSO4. After removing
the solvent, the crude product was purified by flash chro-
matography (EtOAc/hexanes, 1/20–1/15) to give 3.35 g
white solid as a title compound. Yield 55%; mp 164–
3.9. [9-(4-Fluorobenzyl)-4-oxo-1,2,4,9-tetrahydrocarbazol-
3-ylidene]-hydroxyacetic acids (6a–d)
1
The title compounds were prepared from [9-(4-Fluoroben-
zyl)-4-oxo-1,2,4,9-tetrahydrocarbazol-3-ylidene]-hydroxy-
acetic acid ethyl ester (28a–d) according to Method C. The
crude product was recrystallized with EtOAc/hexanes.
165 °C (165–168 °C32); H NMR (CDCl3): d 8.98 (1H,
br s, NH), 7.66 (1H, d, J = 9.0 Hz), 7.44–7.34 (2H, m),
7.15 (1H, t, J = 6.0 Hz), 3.02 (2H, t, J = 6.0 Hz), 2.67
(2H, t, J = 6.0 Hz), 2.27 (2H, p, J = 6.0 Hz); 13C NMR
(CDCl3): d 191.5, 129.7, 127.2, 121.6, 120.6, 112.7, 38.6,
25.4, 21.8; MS (ESI): m/z 208.1 [M+Na]+.
3.9.1. [9-(4-Fluorobenzyl)-4-oxo-1,2,4,9-tetrahydrocar-
bazol-3-ylidene]-hydroxyacetic acid (6a). Yield 54%; mp
177–179 °C; 1H NMR (CDCl3): (enol form:ketone
form = 6.3:1) enol form d 8.29 (1H, d, J = 6 Hz), 7.39–
7.28 (2H, m), 7.07–7.00 (2H, m), 5.35 (2H, s), 3.23 (2H,
t, J = 6.9 Hz), 3.01 (2H, t, J = 6.9 Hz); ketone form d
8.22 (1H, d), 5.33 (2H, s), 3.50 (2H, t, J = 7.5 Hz), 2.94
(2H, t, J = 6.9 Hz); 13C NMR (CDCl3): d 164.6, 152.6,
128.2, 125.4, 125.1, 124.5, 122.8, 116.7, 116.4, 114.5,
110, 47.2, 25.0, 21.5; MS (FAB): m/z 366.0 [M+H]+; Anal.
(C21H16FNO4) C, H, N, F.
3.11. Synthesis of 6-halo-2,3,4,9-tetrahydrocarbazol-1-
ones (15b–d)
The title compounds were prepared from 1,2-cyclohex-
anedione (16) and 4(3)-halo-phenylhydrazine hydro-
chloride salt (11b–d) according to Method A.
3.12. 6-Fluoro-2,3,4,9-tetrahydrocarbazol-1-one (15b)
1
Yield 75%; mp 202–206 °C; H NMR (CDCl3): d 9.42
3.9.2. [6-Fluoro-9-(4-fluorobenzyl)-4-oxo-1,2,4,9-tetrahy-
drocarbazol-3-ylidene]-hydroxyacetic acid (6b). Yield
19% (not optimized); mp 199–200 °C; 1H NMR
(CDCl3): (enol form:ketone form = 2:1) enol form d
7.96 (1H, dd, J = 2.4, 8.7 Hz), 7.23–7.17 (2H, m),
7.07–7.00 (4H, m), 5.34 (2H, s), 3.25 (2H, t, J = 6 Hz),
3.02 (2H, t, J = 6 Hz); ketone form d 7.88 (1H, dd,
J = 2.4, 9 Hz), 5.31 (2H, s), 3.52 (2H, t, J = 6.9 Hz),
2.95 (2H, t, J = 6.6 Hz); 13C NMR (CDCl3): d 184.3,
164.4, 161.0, 158.9, 157.8, 155.6, 152.7, 130.4, 128.2
(2), 116.8 (2), 114.3, 113.2, 112.9, 111.6, 108.9 (2),
47.5, 24.9, 21.5; MS (ESI): m/z 405.97 [M+Na]+; Anal.
(C21H15F2NO4) C, H, N, F.
(1H, br s, NH), 7.40 (1H, dd, J = 4.2, 9.0 Hz), 7.27 (1H,
dd, J = 2.4, 9.0 Hz), 7.12 (1H, dt, J = 2.4, 9.0 Hz), 2.97
(2H, t, J = 6.0 Hz), 2.68 (2H, t, J = 6.0 Hz), 2.27 (2H, p,
J = 6.0 Hz); 13C NMR (CDCl3): d 191.7, 134.7, 132.8,
126.2, 116.4, 116.1, 113.9, 113.8, 106.0, 105.7, 38.6, 25.3,
21.7; MS (ESI): m/z 226.0 [M+Na]+; Anal. (C12H10FNO)
C, H, N.
3.12.1. 6-Chloro-2,3,4,9-tetrahydrocarbazol-1-one (15c).
1
Yield 69%; mp 223–225 °C; H NMR (CDCl3): d 9.56
(1H, br s, NH), 7.61 (1H, s), 7.40 (1H, d, J = 9.0 Hz),
7.29 (1H, t, J = 9.0 Hz), 2.97 (2H, t, J = 6.0 Hz), 2.68
(2H, t, J = 6.0 Hz), 2.27 (2H, p, J = 6.0 Hz); 13C NMR
(CDCl3): d 191.7, 136.3, 132.4, 128.9, 127.6, 127.0,
126.2, 120.8, 114.0, 38.6, 25.2, 21.6; MS (ESI): m/z
242.0 [M+Na]+; Anal. (C12H10ClNO + 0.2H2O) C, H,
N, Cl.
3.9.3. [6-Chloro-9-(4-fluorobenzyl)-4-oxo-1,2,4,9-tetrahy-
drocarbazol-3-ylidene]-hydroxyacetic acid (6c). Yield
64%; mp 210–212 °C; 1H NMR (acetone-d6): (enol
form:ketone form = 2:1) enol form d 8.18–8.03 (1H,
m), 7.60–7.49 (1H, m), 7.34–7.25 (3H, m), 7.15–7.08
(2H, m), 5.59 (2H, s), 3.27 (2H, t, J = 6.3 Hz), 3.15
(2H, t, J = 6.3 Hz); ketone form d 5.65 (2H, s); MS
(FAB): m/z 400.0 [M+H]+; Anal. (C21H15ClFNO4) C,
H, N, F.
3.12.2. 7-Chloro-2,3,4,9-tetrahydrocarbazol-1-one (15d).
1
Yield 48%; mp 218–221 °C; H NMR (CDCl3): d 9.46
(1H, br s, NH), 7.57 (1H, d, J = 9.0 Hz), 7.46 (1H, d,
J = 1.8 Hz), 7.11 (1H, dd, J = 1.8, 9.0 Hz), 2.99 (2H, t,
J = 6.0 Hz), 2.68 (2H, t, J = 6.0 Hz), 2.28 (2H, p,
J = 6.0 Hz); 13C NMR (CDCl3): d 191.6, 138.4, 133.0,
131.9, 129.7, 124.6, 122.5, 121.7, 112.7, 38.5, 25.2,
21.7; HRMS (ESI): Calcd for (C12H10ClNO)Na+:
242.0343. Found: 242.0332.
3.9.4. [7-Chloro-9-(4-fluorobenzyl)-4-oxo-1,2,4,9-tetrahy-
drocarbazol-3-ylidene]-hydroxyacetic acid (6d). Yield
57%; mp 191–193 °C; 1H NMR (CDCl3): (enol form:ke-
tone form = 6.4:1) enol form d 8.22 (1H, d, J = 9 Hz),
7.35–7.29 (3H, m), 7.05–7.02 (3H, m), 5.31 (2H, s),
3.26 (t, 2H, J = 6.9 Hz), 3.01 (t, 2H, J = 6.9 Hz); ketone
form d 8.12 (1H, m), 3.52 (2H, t), 2.94 (2H, t); MS
(FAB): m/z 399.9 [M+H]+; Anal. (C21H15ClFNO4) C,
H, N.
3.12.3. Synthesis of 6(7)-halo-(1-oxo-1,3,4,9-tetra-
hydrocarbazol-2-ylidene)-hydroxyacetic acid ethyl esters
(29a–d). The title compounds were prepared from
2,3,4,9-tetrahydrocarbazol-1-one (15a–d) according to
Method B.

2952
X. Li, R. Vince / Bioorg. Med. Chem. 14 (2006) 2942–2955
3.13. (1-Oxo-1,3,4,9-tetrahydrocarbazol-2-ylidene)-
hydroxyacetic acid ethyl ester (29a)
20.4; MS (ESI): m/z 279.99 [M+Na]+; HRMS (ESI):
Calcd for (C14H11NO4)Na+: 280.0580. Found: 280.0586.
Yield 77%; mp 155–156 °C (156–157 °C37); 1H NMR
(CDCl3): d 8.9 (1H, br s, NH), 7.64 (1H, d), 7.40 (2H,
m), 7.20 (1H, m), 4.37 (2H, q, J = 6.9 Hz), 3.25 (2H,
m), 3.0 (2H, m), 1.42 (3H, t, J = 6.9 Hz); 13C NMR
(CDCl3): d 128.1, 121.8, 121.1, 112.7, 62.3, 24.5, 20.7,
14.5; HRMS (ESI): Calcd for (C16H15NO4)Na+:
308.0893. Found: 308.0896 [M+Na]+.
3.14.1. (6-Fluoro-1-oxo-1,3,4,9-tetrahydrocarbazol-2-yli-
dene)-hydroxyacetic acid (7b). Yield 69%; mp 190–
192 °C; 1H NMR (DMSO-d6): (enol form:ketone
form = 1.5:1) enol form d 11.89 (1H, br s), 7.50–7.37
(2H, m), 7.23–7.15 (1H, m), 2.93 (4H, m); ketone form
d 11.80 (1H, s), 4.47 (1H, dd, J = 4.5, 11 Hz), 2.35
(1H, m); 13C NMR (DMSO-d6): d 196.3, 187.7, 164.0,
162.9, 159.2, 135.8, 132.2, 125.7, 116.6, 115.0, 110.3,
106.3, 56.6, 27.7, 24.5, 20.5; MS (ESI): m/z 297.98
[M+Na]+; Anal. (C14H10FNO4 + 1H2O) C, H, N, F.
3.13.1. (6-Fluoro-1-oxo-1,3,4,9-tetrahydrocarbazol-2-yli-
dene)-hydroxyacetic acid ethyl ester (29b). Yield 66%;
1
mp 165–167 °C; H NMR (CDCl3): d 9.07 (1H, br s,
NH), 7.35 (1H, dd, J = 4.5, 9.0 Hz), 7.28 (1H, dd,
J = 2.4, 9.0 Hz), 7.15 (1H, dt, J = 2.4, 9.0 Hz), 4.10
(2H, q, J = 6.9 Hz), 3.24 (2H, t, J = 6.6 Hz), 2.99
(2H, t, J = 6.6 Hz), 1.42 (3H, t, J = 6.9 Hz); 13C
NMR (CDCl3): d 185.0, 163.0, 159.8, 158.0, 156.6,
135.7, 132.6, 129.7, 126.1, 117.4, 117.1, 113.8, 112.1,
106.2, 105.9, 62.4, 24.4, 20.6, 14.5; MS (ESI): m/z
358.1 [M+Na+MeOH]+, 326.0 [M+Na]+; Anal.
(C16H14FNO4) C, H, N.
3.14.2. (6-Chloro-1-oxo-1,3,4,9-tetrahydrocarbazol-2-yli-
dene)-hydroxyacetic acid (7c). Yield 69%; mp 188 °C
(dec.); 1H NMR (DMSO-d6): (enol form:ketone
form = 1.5:1) enol form d 11.98 (1H, br s), 7.77 (1H, m),
7.44–7.39 (1H, m), 7.33–7.29 (1H, m), 2.95 (4H, m); ke-
tone form d 11.91 (1H, s), 4.49 (1H, dd, J = 4.8, 11 Hz);
13C NMR (CD3OD): d 170.5, 128.7, 127.7, 126.0, 120.5,
114.0, 112.2, 24.5, 20.4; MS (ESI): m/z 290.1 [M-H]ꢀ;
Anal. (C14H10ClNO4 + 0.8H2O) C, H, N, Cl.
3.13.2. (6-Chloro-1-oxo-1,3,4,9-tetrahydrocarbazol-2-yli-
dene)-hydroxyacetic acid ethyl ester (29c). Yield 72%; mp
198–200 °C; H NMR (CDCl3): d 8.95 (1H, br s, NH),
7.63 (1H, s), 7.34 (2H, m), 4.38 (2H, q, J = 6.9 Hz),
3.24 (2H, t, J = 6.9 Hz), 2.99 (2H, t, J = 6.9 Hz), 1.42
(3H, t, J = 6.9 Hz); 13C NMR (CDCl3): d 185.0, 163.0,
158.0, 137.4, 132.2, 129.2, 128.4, 126.8, 121.0, 113.9,
112.0, 62.4, 24.4, 20.5, 14.5; MS (ESI): m/z 374.0
[M+Na+MeOH]+, 342.0 [M+Na]+; Anal. (C16H14-
ClNO4+ 0.4H2O) C, H, N.
3.14.3. (7-Chloro-1-oxo-1,3,4,9-tetrahydrocarbazol-2-yli-
dene)-hydroxyacetic acid (7d). Yield 64%; mp 208–
210 °C; 1H NMR (DMSO-d6): (enol form:ketone
form = 1.5:1) enol form d 11.94 (1H, br s), 7.73–7.68
(1H, m), 7.40 (1H, m), 7.10 (1H, m), 2.95 (4H, m); ke-
tone form d 11.86 (1H, s), 4.48 (1H, dd), 2.34 (2H, m);
1H NMR (CD3OD): d 11.48 (1H, br s), 7.62 (1H, d,
J = 9 Hz), 7.43 (1H, dd, J = 2.1, 0.6 Hz), 7.08 (1H, dd,
J = 9, 1.8 Hz), 3.18 (2H, t, J = 6 Hz), 3.00 (2H, t,
J = 6 Hz); 13C NMR (DMSO-d6): d 196.3, 187.6,
162.9, 139.8, 139.3, 131.9, 131.6, 129.7, 124.4, 123.8,
121.3, 112.9, 110.2, 73.0, 61.0, 56.5, 27.6, 24.5, 20.5;
MS (ESI): m/z 314.0 [M+Na]+; Anal. (C14H10ClNO4+
0.8 H2O) C, H, N.
1
3.13.3. (7-Chloro-1-oxo-1,3,4,9-tetrahydrocarbazol-2-yli-
dene)-hydroxyacetic acid ethyl ester (29d). Yield 67%;
1
mp 171–173 °C; H NMR (CDCl3): d 8.86 (1H, br s,
NH), 7.57 (1H, d, J = 9 Hz), 7.41 (1H, d, J = 1.2 Hz),
7.14 (1H, dd, J = 1.2, 9 Hz), 4.39 (2H, q, J = 6.9 Hz),
3.24 (2H, t, J = 6.9 Hz), 3.01 (2H, t, J = 6.9 Hz), 1.42
(3H, t, J = 6.9 Hz); 13C NMR (CDCl3): d 132.0, 124.5,
122.7, 122.3, 112.5, 62.4, 24.4, 20.6, 14.5; HRMS
(ESI): Calcd for (C16H14ClNO4)Na+: 342.0504. Found:
342.0515 [M+Na]+.
3.14.4. Synthesis of 9-(4-fluorobenzyl) 2,3,4,9-tetrahydro-
carbazol-1-ones (30a–d). The title compounds were pre-
pared from 2,3,4,9-tetrahydrocarbazol-1-one (15a–d)
according to Method D. The crude product was purified
by flash chromatography (EtOAc/hexanes 1/30–1/20).
3.15. 9-(4-Fluorobenzyl)-2,3,4,9-tetrahydrocarbazol-1-
one (30a)
3.13.4. Synthesis of 6(7)-halo-(1-oxo-1,3,4,9-tetrahy-
drocarbazol-2-ylidene)-hydroxyacetic acids (7a–d). The
title compounds were prepared from 6(7)-halo-(1-oxo-
1,3,4,9-tetrahydrocarbazol-2-ylidene)-hydroxyacetic acid
ethyl ester (29a–d, 1 mmol) according to method C. The
crude product was recrystallized with EtOAc/MeOH to
give the title compound.
1
Yield 66% (semi-solid); H NMR (CDCl3): d 7.60 (1H,
d), 7.45 (2H, m), 7.00 (3H, m), 6.8 (2H, m), 5.6 (2H,
d), 2.9 (2H, m), 2.5 (2H, m), 2.1 (2H, m); 13C NMR
(CDCl3): d 192.1, 164, 160, 139, 135, 130.3, 128.7,
127.2, 125.3, 121.7, 121, 115 (m), 113, 111, 47.5, 40.4,
25.1, 22.3; HRMS (ESI): Calcd for (C19H16FNO)Na+:
316.1108. Found: 316.1123.
3.14. (1-Oxo-1,3,4,9-tetrahydrocarbazol-2-ylidene)-
hydroxyacetic acid (7a)
3.15.1. 6-Fluoro-9-(4-fluorobenzyl) 2,3,4,9-tetrahydrocar-
bazol-1-one (30b). Yield 80%; mp 122–123 °C; 1H NMR
(CDCl3): d 7.25 (2H, m), 7.08 (3H, m), 6.93 (2H, t,
J = 9.0 Hz), 7.75 (2H, s), 2.99 (2H, t, J = 6.0 Hz), 2.66
(2H, t, J = 6.0 Hz), 2.34 (2H, p, J = 6.0 Hz); 13C NMR
(CDCl3): d 192.2, 164, 156, 136.0, 134.0, 131.1, 128.6,
128.5, 116.4, 116.1, 115.8, 115.5, 112.1, 111.9, 106.1,
1
Yield 55%; mp 177–179 °C; H NMR (CDCl3): (enol
form:ketone form = 6.2:1) enol form d 8.92 (1H, br s),
7.68 (1H, d, J = 8.4 Hz), 7.44 (2H, t, J = 6 Hz), 7.20
(1H, m), 3.28 (2H, t, J = 6 Hz), 3.15 (2H, t, J = 6 Hz);
ketone form d 8.10 (1H, s), 3.49 (2H, t), 3.08 (2H, m);
13C NMR (CDCl3): d 129.6, 122.4, 121.6, 112.9, 26.5,

X. Li, R. Vince / Bioorg. Med. Chem. 14 (2006) 2942–2955
2953
105.8, 47.8, 40.3, 24.9, 22.2; MS (ESI): m/z 334.1
J = 6.9 Hz); 13C NMR (CDCl3): d185.9, 163.1, 158.9,
138.9, 129.8, 128.6, 128.3, 126.8, 125.8, 121.1, 116.0,
115.7, 112.3, 62.4, 48.0, 24.4, 20.8, 14.5; MS (ESI): m/z
482.0 [M+Na+MeOH]+, 450.0 [M+Na]+; Anal.
(C23H19ClFNO4) C, H, N, Cl.
[M+Na]+; Anal. (C19H15F2NO) C, H, N, F.
3.15.2. 6-Chloro-9-(4-fluorobenzyl)-2,3,4,9-tetrahydrocar-
1
bazol-1-one (30c). Yield 84%; mp 134–135 °C; H NMR
(CDCl3): d 7.64 (1H, s), 7.32–7.26 (2H, m), 7.08 (2H, m),
6.92 (2H, m), 5.75 (2H, s), 2.99 (2H, t, J = 6.0 Hz), 2.66
(2H, t, J = 6.0 Hz), 2.23 (2H, p, J = 6.0 Hz); 13C NMR
(CDCl3): d 192.2, 163.8, 160.5, 137.7, 133.9, 130.9, 129.3,
128.6, 127.5, 126.3, 120.9, 115.8, 115.5, 112.2, 47.7, 40.3,
24.9, 22.1; MS (ESI): m/z 350.0 [M+Na]+; Anal.
(C19H15ClFNO) C, H, N.
3.16.3. [7-Chloro-9-(4-fluorobenzyl)-1-oxo-1,3,4,9-tetra-
hydrocarbazol-2-ylidene]-hydroxyacetic acid ethyl ester
(31d). Yield 71%; mp 146–148 °C; H NMR (CDCl3): d
7.82 (1H, d, J = 9 Hz), 7.30 (1H, d, J = 1.2 Hz), 7.15–
7.07 (3H, m), 6.96 (2H, t, J = 9 Hz), 5.73 (2H, s),
4.37(2H, q, J = 6 Hz), 3.15 (2H, t, J = 7.5 Hz), 3.00
(2H, t, J = 7.5 Hz), 1.40 (3H, t, J = 6 Hz); 13C NMR
(CDCl3): d185.7, 163.1, 158.6, 134.1, 130.7, 128.6,
128.5, 123.5, 122.9, 122.2, 116.0, 115.7, 112.1, 110.6,
62.3, 47.9, 24.5, 20.8, 14.5; HRMS (ESI): Calcd for
(C23H19ClFNO4)Na+: 450.0879. Found: 450.0888.
1
3.15.3. 7-Chloro-9-(4-fluorobenzyl)-2,3,4,9-tetrahydrocar-
1
bazol-1-one (30d). Yield 80%; mp 95–97 °C; H NMR
(CDCl3): d 7.55 (1H, d, J = 9 Hz), 7.29 (1H, d,
J = 1.5 Hz), 7.08 (3H, m), 6.91 (2H, t, J = 9 Hz), 5.69
(2H, s), 2.99 (2H, t, J = 6.0 Hz), 2.63 (2H, t, J = 6.0 Hz),
2.21 (2H, p, J = 6.0 Hz); 13C NMR (CDCl3): d 191.9,
163.8, 160.6, 139.7, 133.8, 133.1, 130.5, 130.2, 128.6,
123.9, 122.7, 121.7, 115.8, 115.6, 110.8, 47.7, 40.2, 24.9,
22.1; HRMS (ESI): Calcd for (C19H15ClFNO)Na+
350.0718: Found: 350.0715.
3.16.4. Synthesis of [9-(4-fluorobenzyl)-1-oxo-1,3,4,9-tet-
rahydrocarbazol-2-ylidene]-hydroxyacetic acids (8a–d).
The title compounds were prepared from 6(7)-halo-[9-
(4-fluorobenzyl)-1-oxo-1,3,4,9-tetrahydrocarbazol-2-yli-
dene]-hydroxyacetic acid ethyl ester (31a–d) according
to Method C. The reaction was complete by refluxing
for 10 h and the crude product was crystallized with
diethyl ether to give the title compound.
3.15.4. Synthesis of 6(7)-halo-[9-(4-fluorobenzyl)-1-oxo-
1,3,4,9-tetrahydrocarbazol-2-ylidene]-hydroxyacetic acid
ethyl esters (31a–d). The title compounds were prepared
from 9-(4-fluorobenzyl)-2,3,4,9-tetrahydrocarbazol-1-
one (30a–d) according to Method B. The crude product
was recrystallized with diethyl ether to afford the title
compound.
3.17. [9-(4-Fluorobenzyl)-1-oxo-1,3,4,9-tetra-
hydrocarbazol-2-ylidene]-hydroxyacetic acid (8a)
Yield 67%; mp 146–149 °C; 1H NMR (CDCl3): (enol
form:ketone form = 2.8:1) enol form 7.76 (1H, t,
J = 7.5 Hz), 7.52 (1H, q, J = 7.2 Hz), 7.43 (1H, q,
J = 8 Hz), 7.27 (1H, m), 7.14 (2H, t, J = 8.4 Hz), 7.00 (2H,
t, J = 9 Hz), 5.84 (2H, s), 3.30 (2H, m), 3.22 (2H, m); ketone
form d 3.52 (2H, t, J = 6 Hz), 3.14 (2H, t, J = 6 Hz); 13C
(CDCl3): d 181.7, 164.1, 153.1, 142.2, 135.4, 129.8, 128.5,
124.7, 122.5, 122.2, 121.7, 121.4, 117.9, 116.0, 115.7, 111.4,
111.2, 48.2, 47.9, 26.6, 24.0, 20.8; MS (ESI): m/z 388.1
[M+Na]+; Anal. (C21H16FNO4) C, H, N.
3.16. [9-(4-Fluorobenzyl)-1-oxo-1,3,4,9-tetrahydrocarbazol-
2-ylidene]-hydroxyacetic acid ethyl ester (31a)
1
Yield 71%; mp 132–134 °C; H NMR (CDCl3): d 7.66
(1H, d, J = 9.0 Hz), 7.36 (2H, m), 7.12 (3H, m), 6.94
(2H, m), 5.78 (2H, s), 4.37 (2H, q, J = 7.2 Hz), 3.16
(2H, dt, J = 1.8, 6.0 Hz), 3.04 (2H, dt, J = 1.2, 6.0 Hz),
1.41 (3H, t, J = 7.5 Hz); 13C NMR (CDCl3): d 185.9,
158.3, 140.7, 131.0, 130.2, 128.6, 128.0, 125.0, 121.9,
121.1, 115.9, 115.6, 112.5, 111.1, 62.3, 47.8, 24.5, 20.9,
14.5; HRMS (ESI): Calcd for (C23H20FNO4)H+:
394.1449. Found: 394.1456.
3.17.1. [6-Fluoro-9-(4-fluorobenzyl)-1-oxo-1,3,4,9-tetra-
hydrocarbazol-2-ylidene]-hydroxyacetic acid (8b). Yield
62%; mp 164–165 °C; 1H NMR (CDCl3): (enol form:ke-
tone form = 2.7:1) enol form d 7.53–7.10 (7H, m), 5.95
(2H, s), 3.42 (2H, t, J = 6 Hz), 3.28 (2H, t, J = 6 Hz); ke-
tone form d 3.64 (2H, t, J = 6.6 Hz), 3.21 (2H, t,
J = 6.6 Hz); 13C NMR (CDCl3): d 182.0, 163.9, 162.7,
160.7, 159.9, 156.8, 153.4, 138.7, 134.4, 133.3, 131.7,
128.5, 124.7, 119.2, 118.8, 118.1, 117.8, 116.0, 115.8,
112.7, 106.6, 48.4, 26.5, 23.9, 20.6; MS (ESI): m/z
382.2 [MꢀH]ꢀ; Anal. (C21H15F2NO4) C, H, N.
3.16.1. [6-Fluoro-9-(4-fluorobenzyl)-1-oxo-1,3,4,9-tetra-
hydrocarbazol-2-ylidene]-hydroxyacetic acid ethyl ester
1
(31b). Yield 70%; mp 151–152 °C; H NMR (CDCl3): d
7.30 (2H, m), 7.10 (3H, m), 6.95 (2H, m), 5.77 (2H, s),
4.38 (2H, q, J = 7.5 Hz), 3.15 (2H, t, J = 6.9 Hz), 2.98
(2H, t, J = 6.9 Hz), 1.41 (3H, t, J = 7.5 Hz); 13C NMR
(CDCl3): d 186.0, 163.1, 158.8, 137.3, 128.5, 117.3,
116.9, 115.9, 115.6 112.3, 112.2, 106.3, 106.0, 62.4,
48.0, 24.4, 20.8, 14.5; MS (ESI): m/z 466.1 [M+Na+Me-
OH]+; 434.1 [M+Na]+; Anal. (C23H19F2NO4) C, H,
N, F.
3.17.2. [6-Chloro-9-(4-fluorobenzyl)-1-oxo-1,3,4,9-tetra-
hydrocarbazol-2-ylidene]-hydroxyacetic acid (8c). Yield
76%; mp 147–149 °C; 1H NMR (CDCl3): (enol form:ke-
tone form = 2.4:1) enol form d 7.90 (1H, m), 7.64–7.47
(2H, m), 7.33–7.16 (4H, m), 6.0 (2H, s), 3.47 (2H, m),
3.34 (2H, m); ketone form d 3.67 (2H, t, J = 6.6 Hz),
3.26 (2H, t, J = 6.6 Hz); 13C NMR (CDCl3): d 182.1,
163.8, 153.5, 133.9, 130.0, 129.0, 128.5, 128.3, 127.4,
125.4, 121.6, 117.7, 116.1, 115.8, 112.6, 48.4, 26.5,
23.9, 20.6; MS (ESI): m/z 398 [MꢀH]ꢀ; HRMS (ESI):
3.16.2. [6-Chloro-9-(4-fluorobenzyl)-1-oxo-1,3,4,9-tetra-
hydrocarbazol-2-ylidene]-hydroxyacetic acid ethyl ester
1
(31c). Yield 50%; mp 167–170 °C; H NMR (CDCl3): d
7.64 (1H, s), 7.29 (2H, m), 7.07 (2H, m), 6.94 (2H, m),
5.76 (2H, s), 4.38 (2H, q, J = 6.9 Hz), 3.15 (2H, t,
J = 6.9 Hz), 2.98 (2H, t, J = 6.6 Hz), 1.41 (3H, t,

2954
X. Li, R. Vince / Bioorg. Med. Chem. 14 (2006) 2942–2955
Calcd for (C21H15ClFNO4)Na+: 422.0566. Found:
422.0540.
according to Method D. The crude product was recrys-
tallized with diethyl ether to give the final product as
white fine needles. Yield 92%; mp 158–160 °C; 1H
NMR (CDCl3): d 7.91 (1H, dd, J = 2.7, 9 Hz), 7.20
(1H, dd, J = 4.5, 9 Hz), 6.99 (1H, dt, J = 2.7, 9 Hz),
3.7 (3H, s), 2.93 (2H, t, J = 6 Hz), 2.56 (2H, t,
J = 6 Hz), 2.25 (2H, p, J = 6 Hz); 13C NMR (CDCl3):
d 167.7, 111.3, 111.0, 110.0, 109.9, 107.6, 107.3, 38.1,
30.5, 23.6, 22.7; MS (ESI): m/z 218 [M+H]+, 240
[M+Na]+.
3.17.3. [7-Chloro-9-(4-fluorobenzyl)-1-oxo-1,3,4,9-tetra-
hydrocarbazol-2-ylidene]-hydroxyacetic acid (8d). Yield
52%; mp 208–210 °C; 1H NMR (CDCl3): (enol form:ke-
tone form = 2.8:1) enol form d 7.63 (1H, t, J = 9 Hz),
7.36 (1H, d, J = 1 Hz), 7.19–7.17 (1H, m), 7.10–7.05
(2H, m), 7.01–6.98 (2H, m), 5.73 (2H, s), 3.24 (2H, m),
3.14 (2H, m); ketone form d 7.32 (1H, d, J = 1 Hz), 3.46
(2H, t, J = 6.6 Hz), 3.06 (2H, t J = 6.6 Hz); 13C NMR
(CDCl3): d 163.9, 128.5, 128.3, 123.5, 122.9, 116.1,
115.8, 111.2, 48.3, 26.4, 20.6; MS (ESI): m/z 422.0
[M+Na]+; Anal. (C21H15ClFNO4) C, H, N.
3.20. (6-Fluoro-9-methyl-4-oxo-1,2,4,9-tetrahydrocarbazol-
3-ylidene)-hydroxyacetic acid ethyl ester (21)
The title compound was prepared from 6-fluoro-9-meth-
yl-1,2,3,9-tetrahydrocarbazol-4-one (20) according to
Method E. Yield 70%; mp 161–162 °C; 1H NMR
(CDCl3): d 7.78 (1H, dd, J = 3, 9 Hz), 7.18 (1H, m),
6.97 (1H, dt, J = 3, 9 Hz), 4.36 (2H, q, J = 6 Hz), 3.66
(3H, s), 3.18 (2H, t, J = 6 Hz), 2.85 (2H, t, J = 6 Hz),
1.40 (3H, t, J = 6 Hz); 13C NMR (CDCl3): d 189, 162,
160, 158, 157, 153, 135, 126, 113, 112 (2), 111 (m),
105, 60, 31, 22, 20, 15; MS (ESI): m/z 340.1 [M+Na]+;
Anal. (C17H16FNO4) C, H, N.
3.17.4. 6-Fluoro-9-tosyl -1,2,3,9-tetrahydrocarbazol-4-one
(18). To a solution of 6-fluoro-1,2,3,9-tetrahydrocar-
bazol-4-one (12b, 203 mg, 1 mmol) in 2.5 mL of anhy-
drous DMF were added tosyl chloride (229 mg, 1.2
mmol), triethylamine (250 lL, 1.8 mmol), and DMAP
(9 mg, 0.07 mmol). The mixture was stirred at room tem-
perature under argon for 8 h. After the reaction was com-
plete, the mixture was poured into 40 mL saturated
ammonium chloride solution and extracted with methy-
lene chloride. The organic portion was dried over MgSO4.
After concentrated, the crude product was purified by
flash chromatography (EtOAc/hexanes, 1/4) to give the
3.21. (6-Fluoro-9-methyl-4-oxo-1,2,4,9-tetrahydrocarbazol-
3-ylidene)-hydroxyacetic acid (22)
1
title compound. Yield 59%; mp 159–160 °C; H NMR
(CDCl3): d 8.10 (1H, dd, J = 4.5, 9.0 Hz), 7.91 (1H, dd,
J = 2.4, 9.0 Hz), 7.74 (2H, dt, J = 1.5, 8.4 Hz), 7.29–7.25
(2H, m), 7.07 (1H, td, J = 2.7, 9.0, 9.0 Hz), 3.31 (2H, t,
J = 6.0 Hz), 2.55 (2H, t, J = 6.3 Hz), 2.20 (2H, p,
J = 6.9 Hz); 13C NMR (CDCl3): d 194.8, 162, 152.3,
146.2, 135.4, 130.5, 126.8, 115.2 (2), 113.5 (2), 107.8,
38.1, 24.9, 23.5, 22.1; HRMS (EI): Calcd for (C19H16
FO3S)+: 357.0835. Found: 357.0845.
The title compound was prepared from (6-fluoro-9-meth-
yl-4-oxo-1,2,4,9-tetrahydrocarbazol-3-ylidene)-hydroxy-
acetic acid ethyl ester (21) according to Method C. After
cooling down, the mixture was diluted with water and
extracted with ethyl acetate. The organic portion was con-
centrated and purified by flash chromatography to give a
130 mg yellow solid as the title compound. Yield 68%; mp
1
205–207 °C; H NMR (DMSO-d6): (enol form:ketone
form = 3.5:1) enol form d 8.05–7.96 (2H, m), 7.52 (1H,
dt, J = 2.4, 9 Hz), 4.13 (3H, s), 3.42 (4H, m); ketone form
d 4.82 (1H, dd, J = 5, 12 Hz), 2.80–2.68 (2H, m); 13C
NMR (DMSO-d6): d 188.5, 165.0, 163.2, 158.9, 155.6,
135.3, 113.0, 111.4, 111.1, 108.7, 55.4, 45.9, 31.1, 23.2,
21.5; MS (ESI): m/z 301.2 [M+Na]+; Anal. (C15H12FNO4)
C, H, N.
3.18. N-Boc protected 6-chloro-1,2,3,9-tetra-
hydrocarbazol-4-one (19)
To a solution of 6-chloro-1,2,3,9-tetrahydrocarbazol-4-
one (12c, 219.5 mg, 1 mmol) in 2.5 mL anhydrous DMF
were added (Boc)2O (262 mg, 1.2 mmol), triethyl amine
(250 lL, 1.8 mmol), and DMAP (15 mg, 0.125 mmol).
The mixture was stirred at room temperature overnight.
After the reaction was complete, the mixture was diluted
with 20 mL ethyl acetate and poured into 20 mL saturated
sodium chloride solution. The organic portion was
washed with saturated NH4Cl and NaCl, and dried over
MgSO4. After removing the solvent, the crude product
was recrystallized with EtOAc/hexanes to give 300 mg
white fine needle crystals as the title compound. Yield
Acknowledgment
We thank Dr Colleen Jonsson and Sinu John of Southern
Research Institute for conducting the HIV integrase
assays.
1
References and notes
94%; mp 138–140 °C; H NMR (CDCl3): d 8.20 (1H, d,
J = 1.5 Hz), 7.93 (1H, d, J = 9.0 Hz), 7.20 (2H, m), 3.23
(2H, t, J = 6 Hz), 2.51 (2H, t, J = 6 Hz), 2.16 (2H, p,
J = 6 Hz), 1.60 (9H, s); 13C NMR (CDCl3): d 130.4,
125.2, 121.4, 116.4, 110.0, 86.1, 38.1, 28.5, 26.3, 23.6;
MS (ESI): m/z 342 [M+Na]+.
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