Optimization of potency and pharmacokinetics of tricyclic indole derived inhibitors of HCV NS5B polymerase. Identification of ester prodrugs with improved oral pharmacokinetics

Article abstract of DOI:10.1016/j.bmc.2013.11.007

HCV infections are the leading causes for hepatocellular carcinoma and liver transplantation in the United States. Recent advances in drug discovery have identified direct acting antivirals which have significantly improved cure rates in patients. Current

Full text of DOI:10.1016/j.bmc.2013.11.007

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Optimization of potency and pharmacokinetics of tricyclic indole
derived inhibitors of HCV NS5B polymerase. Identification of ester
prodrugs with improved oral pharmacokinetics
⇑
Srikanth Venkatraman , Francisco Velazquez, Stephen Gavalas, Wanli Wu, Kevin X. Chen,
Anilkumar G. Nair, Frank Bennett, Yuhua Huang, Patrick Pinto, Yueheng Jiang, Oleg Selyutin,
Bancha Vibulbhan, Qingbei Zeng, Charles Lesburg, Jose Duca, Larry Heimark, Hsueh-Cheng Huang,
Sony Agrawal, Chuan-kui Jiang, Eric Ferrari, Cheng Li, Joseph Kozlowski, Stuart Rosenblum,
Neng-Yang Shih, F. George Njoroge
Merck Research Laboratories, K15, 2015 Galloping Hill Road, Kenilworth, NJ 07033, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
HCV infections are the leading causes for hepatocellular carcinoma and liver transplantation in the United
States. Recent advances in drug discovery have identified direct acting antivirals which have significantly
improved cure rates in patients. Current efforts are directed towards identification of novel direct acting
antiviral targeting different mechanism of actions which could become part of all oral therapies. We
recently disclosed the identification of a novel tricyclic indole derived inhibitors of HCV NS5B polymerase
that bound to the enzyme close to the active site. In this manuscript we describe further optimization of
potency and pharmacokinetics (PK) of these inhibitors to identify compounds in low nM potency against
gt-1b. These analogs also demonstrate excellent PK in rats and monkeys when administered as a
dimethyl ethyl amino ester prodrug.
Received 12 September 2013
Revised 27 October 2013
Accepted 4 November 2013
Available online xxxx
Keywords:
HCV NS5B
Polymerase
Tricyclic indole
Prodrug
Ó 2013 Published by Elsevier Ltd.
Dimethylamino ethyl
1. Introduction
inhibitors such as filbuvir,5 ANA598,6 VCH-7597 VX222, BI207127
and ABT333 are currently undergoing clinical evaluation and have
Hepatitis C virus is the primary causative agent for non-A, non B
hepatitis.1 More than 80% of all infections turn chronic at the risk of
developing severe liver disease resulting in hepatocellular carci-
noma.2 Recent advances in development of direct acting antivirals
have significantly improved SVR (sustained virologic response) in
patients providing a new hope for cure in infected patients.3 Cur-
rently discovery of new treatments are directed towards interferon
sparing all oral therapies that are well tolerated with shorter dura-
tion of treatment. Direct acting antivirals derived from inhibition
of different mechanism are combined to achieve high rates of
cure.3
demonstrated robust viral load reductions in humans. Nucleoside
derived inhibitors such as Sofusbuvir8 have also demonstrated
excellent efficacy and being evaluated as a component of all oral
regimens.
We recently disclosed the discovery of a tricyclic indole derived
HCV NS5B polymerase inhibitor that bound to the enzyme close to
the active site9 Analogs derived from this scaffold demonstrated
excellent enzyme inhibition and modest cellular activity and PK
in animals. In this manuscript we disclose further optimization of
potency and PK leading to compounds with low single digit repli-
con activity and excellent oral exposures.
The HCV NS5B RNA dependent RNA polymerase has evolved as
a primary target for identification of novel inhibitors for drug inter-
vention. Proof of concept for this target was initially demonstrated
by Nesbuvir (HCV-796)4 which demonstrated significant viral load
reduction in humans. However, it was discontinued from further
development due to adverse side effects. Other non-nucleoside
2. Chemistry
Compounds described in this manuscript were synthesized fol-
lowing synthetic routes shown in following schemes. Scheme 1
outlines the synthesis of tricyclic indole and functionalization at
the N1 nitrogen.
Alkylation of 2-bromo-5-fluorophenol with diethyl acetal of
bromoacetaldehyde followed by cyclization of the resulted ether
⇑
Corresponding author. Tel.: +1 908 740 2420.
E-mail address: Srikanth.Venkatraman@merck.com (S. Venkatraman).
0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.11.007
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S. Venkatraman et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
OH
N
N
O
O
N
Br
a
b
Br
Br
b
CHO
a
N
H
Cl
Cl
Boc
F
F
F
14
5
3
4
N
O
O
O
OH
NH
O
O
COOC2H5
COOC2H5
c
d
O
O
N3
c
N3
COOH
F
F
F
O
N
7
F
N
6
8
F
N
N
N
Cl
O
Cl
N
f
OCH3
e
N
H
COOC2H5
15
H
16
(HO)2B
CH3O
COOC2H5
N
H
N
F
N
H
Scheme 2. Reagents and conditions: (a) (i) Boc2O, DMAP; (ii) NBS, Bz2O2, CCl4
reflux, 54%; (b) (i) 11, Cs2CO3, DMF; (ii) TFA, CH2Cl2, 76%; (c) aq LiOH, THF, 48%.
9
NH
N
O
O
is shown in Scheme 2. Commercially available 4-chloro-5-methyl-
benzene-1,2-diamine was refluxed with trimethyl orthoformate to
form 6-chloro-5-methyl-1H-benzo[d]imidazole.14 The imidazole
nitrogen was protected with Boc group and the resulting com-
pound was brominated using NBS and benzoyl peroxide to yield
bromide 14, which was converted to imidazole derivative 16 as
previously described.
Scheme 3 outlines the syntheses of tricyclic indole analogs
derived from N1 indazole and 3-aminoindazole. Acetylation of
5-fluoro-2-methylaniline with acetic anhydride formed acetanilide
17 which was selectively brominated at the 4-position using bro-
mine in acetic acid. Compound 17 was diazotized with isoamyl ni-
trite in the presence of potassium acetate which underwent
cyclization to form acetylated indazole 18. Refluxing 18 with a
solution of aq HCl resulted in deacetylation to form 1H-indazole
19.15 Halogen metal exchange of 19 followed by reaction with
DMF resulted in formation of aldehyde 20. The indazole 20 was
protected with Boc group and reduced to alcohol 21 by treatment
with sodium borohydride in excellent yield. This two-step se-
quence effectively homologated the aryl bromide 19 to benzylic
derivative 21. Treatment of alcohol 21 with methanesulfonyl chlo-
ride and triethylamine resulted in formation of benzylic chloride
22 which was converted to the indazole analog 24 using chemistry
previously described.
Amino indazole derived inhibitor 31 was synthesized starting
from 2,4-difluorotoluene. Treatment with NIS in TFA resulted in
regioselective iodination to yield iodide 26.16 Palladium catalyzed
cyanation of 26 with zinc cyanide and (PPh3)4Pd installed the ni-
trile group forming 2,4-difluoro-5-methylbenzonitrile (27). Reflux-
ing a solution of 27 with hydrazine formed 3-amino indazole
derivative 28 which was exhaustively Boc-protected using Boc2O
and DMAP. The resultant tri-Boc protected compound was bromi-
nated using NBS and benzoyl peroxide to form bromo compound
29 which was converted to inhibitor 31 following similar proce-
dures outlined in Scheme 1.
The methodology used for the syntheses of ester prodrugs is
shown in Scheme 4. Deprotonation of 2,2-dimethylamino ethanol
with butyl lithium followed by reaction with lactone 32 and puri-
fication using reverse phase HPLC resulted in ester derivative 33 as
the corresponding trifluoro acetate salt.
O
O
h
g
COOH
N
H
O
N
H
11
F
F
10
HN
N
N
O
O
O
O
O
OH
O
i
j
Br
N
F
F
O
NC
F
F
F
H2N
F
12
13
Scheme 1. Reagents and conditions: (a) (i) Cs2CO3, (C2H5O)2CH–CH2Br, DMF,
150 °C; (ii) PPA, toluene, reflux, 51%; (b) BuLi, ether, ꢀ78 °C, DMF, 94%; (c)
N3CH2COOC2H5, DBU, LiCl, THF, rt, 81%; (d) CH3SO2Cl, Et3N, ꢀ78 °C ? rt, 86%; (e)
Xylenes reflux, 70%; (f) (i) NIS, DMF, 93%; (ii) Pd(PPh3)2Cl2, K2CO3, H2O/DME, 80%;
(g) (i) HCl, dixoane reflux; (ii) aq LiOH, THF reflux, 85%; (h) EDCI, Et3N, DMF, 78%; (i)
Cs2CO3, DMF, 51%;(j) aq LiOH, THF reflux, 81%.
with polyphosphoric acid yielded furan 4.10 Halogen metal ex-
change with butyl lithium followed by reaction with DMF formed
aldehyde 5, which was condensed with ethyl azido acetate using
DBU and LiCl to form azido alcohols 6 as a mixture of diastereo-
mers. The mixtures of alcohols were eliminated using methanesul-
fonyl chloride and triethylamine to form azido ester11 7 which was
cyclized to indole 8 by refluxing in xylenes.12On cooling to room
temperature, indole 8 crystallized from the reaction mixture which
was filtered. Iodination of indole 8 with NIS resulted in selective
iodination at C(3) positions which was coupled with 2-methoxy
pyridin-3-yl boronic acid to yield 9.13 The methyl ether of 9 was
demethylated with 4 M HCl in dioxane to form the pyridone which
was further saponified using aqueous lithium hydroxide to form
carboxylic acid 10. Treatment of resultant acid with EDCI induced
an intramolecular cyclization to form tetracyclic lactone 11. N-
alkylation of 11 with 3-(bromomethyl)-4-fluorobenzonitrile in
DMF resulted in N-alkylated lactone intermediate which was fur-
ther hydrolyzed using aqueous lithium hydroxide to form desired
inhibitor 13. 7-Fluoro substituted indole was synthesized starting
from 2-bromo-4 fluorophenol and using similar reaction condi-
tions to assemble tricyclic indole of type 9. It was alkylated with
5-fluorobenzyl bromine and hydrolyzed with LiOH. It was further
demethylated using 4 M HCl in dioxane as previously described.
N1 substituents that were not readily available were synthe-
sized using methods shown below. The synthesis of imidazole 16
Synthesized analogs were initially evaluated in an enzyme as-
say to determine IC50. Compounds demonstrating excellent en-
zyme inhibition were evaluated in the replicon based cellular
assay to determine their ability to reduce HCV viral RNA levels
by 50% to generate EC50. Selected compounds displaying good en-
zyme potencies and cellular activities were evaluated for oral
exposure in rats using the rapid rat assay.17 Compounds
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S. Venkatraman et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
Br
F
Br
F
demonstrating acceptable oral exposures in rats were further eval-
uated in full PK in rats and monkeys. Synthesized prodrugs were
evaluated in rats and monkeys using similar paradigm monitoring
both the concentration of the ester prodrug and the acid metabo-
lite in plasma.
c
a
b
N
N
H2N
F
AcHN
Ac
17
18
CHO
F
OH
Br
F
N
e
d
N
N
N
3. Results and discussion
F
N
N
H
Boc
H
21
20
19
We recently disclosed the discovery of 4–5 furan fused tricyclic
indole derivatives of type 34 as potent inhibitors of HCV polymer-
ase that bound to the enzyme close to the active site.9,18 Even
though compounds in this class had excellent enzyme inhibition
activity, they demonstrated modest cellular potencies. Moreover,
they have an embedded furan ring that could potentially lead to
reactive metabolites resulting from CYP mediated oxidation of
the benzofuran. It is well established that furans readily undergo
N
O
O
Cl
O
g
N
F
f
N
h
N
F
Boc
22
F
N
N
H
P450 mediated oxidation forming 2-butene-1,4-dials which are fur-
23
NH
O
ther conjugated by proteins and glutathione. Eventhough benzofu-
rans are metabolized differently compared to furans, given the
plethora of toxicity literature of furans early investigation of oxida-
tive metabolites deemed prudent.19
O
COOH
N
F
Microsomal incubation of 34 resulted in formation of minor
metabolites with mass consistent to 38 and 40 (Fig. 1). It is
conceivable that 40 could be formed from the opening of epoxide
intermediate 37 by water. Structure 38 was envisioned to be
formed from compound 34 via oxidation of pyridone ring followed
by rearrangement leading to pyrrole derivative. To further confirm
the formation of reactive intermediate analog 34 was incubated
with human microsomes in the presence of methoxyl amine.
Metabolites resulting from addition of methoxyl amine and
glutathione 39 and 41 were observed in MS at low concentrations
indicating potential formation of epoxide intermediate 37. We
therefore needed methods to suppress the formation of this
intermediate. One of strategies widely adapted to suppress CYP
mediated oxidations is the introduction of strategically placed
fluorine atoms. The incorporation of fluorine atom not only mini-
mizes oxidation of C–H at the carbon bearing the fluorine atom
but also reduces metabolism at carbons in the vicinity. Based on
this premise we synthesized C(6) and C(7) fluorinated analogs 35
and 36.
F
N
N
H
24
I
NC
F
i
k
j
F
F
F
F
F
25
26
27
N
O
(Boc)2N
N
O
H2N
N
Br
g
l
O
N
F
N
N
F
F
H
(Boc)2N
Boc
28
29
F
N
N
Boc
30
NH
O
The C(7) fluoro analog 35 (IC50 = 17 nM, EC50 = 2600 nM) dem-
onstrated a comparable enzyme activity to the desfluoro analog
34 (IC50 = 31 nM, EC50 = 160 nM) however, the cellular activity of
the fluorinated analog was ꢁ15-fold less active than hydrogen
derivative. Whereas, the C(6) fluorinated derivative 36 (IC50 = 2 -
nM, EC90 = 90 nM) retained similar potency to the desfluoro analog
34. Furthermore, incubation of 36 with human microsomes sup-
pressed oxidation metabolites and did not form any glutathione
addition products. Thus, the introduction of fluorine at C(6) had
the beneficial effect of minimizing oxidation metabolite at the fur-
an ring, yet retaining cellular potency. We therefore decided to de-
velop 36 as our new lead and optimize potency by modification of
N1 moieties.
O
COOH
h
N
F
47%
H2N
F
N
N
H
31
Scheme 3. Reagents and conditions: (a) (i) Ac2O, toluene, (ii) Br2, acetic acid
82%;(b) isoamyl nitrite, CH3COOK, CHCl3 18-crown-6; (c) aq HCl reflux; (d) (i) NaH,
THF, 0 °C (ii) BuLi, ꢀ78 °C then DMF, 86%; (e) (i) Boc2O, DMAP (ii) NaBH4, MeOH,
68%; (f) CH3SO2Cl, Py, CH2Cl2, 59%; (g) (i) 11, Cs2CO3, DMF, 96%; (ii) HCl; dioxane (h)
aq LiOH, THF; (i) NIS, TFA, 77%; (j) Zn(CN)2, Pd(PPh3)4, DMF, 42%; (k) N2H4,
Isopropnol, reflux, 10%; (l) (i) Boc2O, DMAP; (ii) NBS, Bz2O2, 56%.
Previous SAR studies in the desfluoro series of inhibitors had
demonstrated that the introduction of 2-fluoro-5-carbamoyl ben-
zyl N1 moiety improved cellular activity of the resulting inhibi-
tors.9 We therefore incorporated this moiety in the C(6)
fluoroindole series resulting in analog 13 (IC50 = 4 nM, EC50 = 15 -
nM). This modification resulted in improvement in cellular po-
tency. Evaluation of 13 in full rat PK demonstrated a moderate
NH
N
O
O
N
O
O
HO
O
THF
N
BuLi,-78
0 °C
N
O
F
N
F
.
TFA
O
81%
exposure with an AUC = 1.7 lM h when dosed at 3 mg/kg and a
F
F
N
low bioavailability of 1.5%. Analysis of plasma samples for metab-
olites demonstrated no glutathione adducts further validating
microsomal incubation results. Having identified potent inhibitors
in the cellular assay we investigated modification of N1 substitu-
N
N
H
N
H
33
32
Scheme 4.
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S. Venkatraman et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
NH
NH
NH
O
O
O
O
O
O
COOH
COOH
COOH
N
N
N
F
F
F
F
F
F
F
F
34
35
36
IC50 = 17nM
EC50 = 2600nM
CC50 = 11 µM
IC50 = 2 nM
EC90 (Rep) = 90 nM
CC50 = 12 µM
IC50 = 31 nM
EC50 = 160 nM
CC50 = 10 µM
NH
O
O
HN
OH
O
O
COOH
+
microsomal incubation
N
34
N
H
O
F
F
37
38
H2O
HN
NH2OCH3
HN
Glutathione
HN
HO
HO
MeOHN
HO
GS
HO
O
O
O
O
O
O
OH
O
OH
O
OH
O
N
N
N
F
F
F
F
F
F
40
39
41
Figure 1. Microsomal incubation results of benzofuran derived inhibitors.
tion to improve oral PK in animals. We reasoned the poor oral
exposure of 13 resulted from reduced absorption. We hypothe-
sized the presence of primary amide potentially contributed to re-
duced oral absorption. We therefore replaced the carboxamide
functionality with bicyclic heterocyclic isosteres containing hydro-
gen bond donors. These results are summarized in Table 1.
Replacement of N-2-fluoro-5-carbamoyl benzyl group in analog
13 with benzimidazole moiety resulted in analog 42 (IC50 = 5 nM;
EC50 = 30 nM) with similar binding and cellular activity as amide
analog. Introduction of fluorine at 5 position of benzimidazole re-
sulted in analog 43 (IC50 = 3 nM; EC50 = 7 nM) with improved cellu-
lar potency compared to 42. Replacement of fluorine with chlorine
was also equally well tolerated with the resulting analog 16 having
similar activity to the fluoro derivative 43. We next evaluated the
incorporation of a series of regioisomeric indazoles at N1. Incorpo-
ration of 3-amino-6-fluoro-1H-indazolylmethyl group at N1 re-
sulted in very potent derivative 31 (IC50 = 4 nM; EC50 = 3 nM)
with excellent enzyme binding and cellular potencies. Evaluation
of this compound for oral PK in rats and monkeys showed modest
AUC = 9.3 lM h and monkeys with AUC = 1.0 lM h. The corre-
sponding chloro (48) and methyl analogs (49) were also well toler-
ated and demonstrated adequate exposure in rats.
Replacement of N1 group with 6-(7-fluoro-4-oxo-1,4-dihydro-
quinolinyl)methyl moiety and 6-((2-fluoroquinolin-3-yl)methyl
resulted in analogs 46 (IC50 = 3 nM; EC50 = 7 nM) and 47 (IC50 = 4 -
nM; EC50 = 6 nM) with excellent binding and cellular activities. The
amino quinoline derivative 47 showed good oral exposure in rats
(AUC0–6h = 24
(AUC0–6h = 0.4
l
l
M h) but was poorly absorbed in monkeys
M h).
It was clear from synthesized analogs that most analogs demon-
strated poor exposure in monkeys. Our efforts to boost PK in rats
by making carboxylate salt 50 resulted in modest improvement
in oral AUC (Table 2). We therefore explored the synthesis of pro-
drugs to enhance oral exposure. As an initial screen we synthesized
novel ester prodrugs of analog 16 and evaluated them in rats. Ana-
log 16 was chosen as a potential candidate for prodrug investiga-
tion based on its potency and ease of synthesis. Synthesized
compounds were dosed orally at 10 mg/kg in rats and the AUC of
both the parent ester and the corresponding acid were evaluated
integrating area under the curve over 0–6 h post dosing.
exposure in rats AUC = 2.1
monkeys AUC = 0 M h.
lM and no exposure in cynomologus
l
Introduction of regioisomeric amino indazole was also tolerated
resulting in compound 44 (IC50 = 5 nM; EC50 = 12 nM) with a mod-
est decrease in cellular activity. N-methylation of indazole nitrogen
in 31 also resulted in an analog with poor cellular activity
(IC50 = 6 nM; EC50 = 20 nM). However the incorporation of 6-flu-
oro-1H-indazole resulted in analogs 24 with good enzyme and cel-
lular potencies (IC50 = 3 nM; EC50 = 4 nM). It was also very
reassuring to find it was well absorbed in rats with an
As shown in the Table 2, esters derived from both primary and
secondary alcohols were synthesized. In addition, alcohols chosen
for ester formation contained both lipophilic and hydrophilic
groups to modulate aqueous and lipid solubilities. Thus, introduc-
tion of ethyl ester resulted in analog 51 (AUC0–6h = 0.0 lM h), which
displayed poor PK for both the parent ester and the acid metabolite.
The introduction of methoxyethyl ester resulted in analog 52 with
the acid metabolite showing an AUC0–6h = 1.1 lM h. This was
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S. Venkatraman et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
Table 1
Structure activity relationship of bicyclic heterocycles substitution at N1.
NH
O
O
COOH
N
F
R2
Entry
R2
Gt-1b IC50 (nM)
Gt-1b EC50 (nM)
15
CC50
2.5
(l
M)
AUC0–6h
1.7⁄
(lM h) rat
H2N
13
4
O
F
H
N
42
43
16
5
3
6
30
7
2.0
50
9.3
7.7
1.2
N
H
N
N
F
H
N
8
25
N
Cl
H2N
31
44
4
5
3
1
2
2.1
3.6
N
N
F
F
H
H
N
N
12
H2N
H2N
45
24
6
3
20
4
2
7.2
9.3
N
N
F
51
N
N
H
F
O
46
47
3
4
7
6
2
N
H
F
50
24
N
NH2
48
49
3
3
5
2
50
–
5.1
2.1
N
N
N
H
Cl
N
H
Compounds were orally dosed in Sprague Dawley rats at 10 mg/kg in MC and cynomolgus monkeys at 3 mg/kg in 0.4% MC.
partially better than dosing the ethyl ester 51, however it was no
better to the carboxylic acid 16. Even though the PK was not im-
proved compared to the corresponding acid analog it was clear
from this study that synthesized esters were substrates of esterases
and were being hydrolyzed to yield desirable acid. We next evalu-
ated secondary aliphatic esters derived from 2-methyl benzyl alco-
aliphatic esters 51, 53 and 54. We next decided to explore amine
containing esters and dose them as corresponding amine salts.
Dimethylamino ethyl ester prodrug had a profound effect on
exposure of these inhibitors in rats. Pharmacokinetic studies of
dimethylamino ethyl ester derivative 55 resulted in marked
improvement in AUC0–6h of the acid. The exposure for the metabolite
hol and 2-indanol resulting in esters 53 (AUC0–6h = 0.2
l
M h) and
acid increased from 1.1 to 32.5
ment in exposure of 16. Homologation of dimethylamino ethyl ester
l
M h. This was ꢁ30-fold improve-
54 (AUC0–6h = 0 M h). Both these esters displayed poor PK in rats.
l
A comparison of ester prodrugs 50–54 revealed the oxygen contain-
to dimethylamino propyl ester resulted in analog 56 which demon-
ing prodrug 52 had better AUC for the acid metabolite than
strated poor AUC0–6h = 0.7 lM h for the acid metabolite. However
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6
S. Venkatraman et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Table 2
improvement in PK rising the AUC0–6h from 1.2
h = 208 M h. A similar beneficial effect was observed in the inda-
zole analogs 31, 24 and 49. The 3-aminoindazole derivative 31
(AUC0–6h = 2.1 M h) had demonstrated an inadequate exposure
to be projected as a potential QD drug whereas the dimethylamino
ethyl ester product improved exposure (AUC0–24h = 118 M h)
making it suitable for QD dosing. Similarly analogs 33 and 61, pro-
drugs of 24 and 49 improved AUC from 9.3 and 5.1 M h to 222
and 79 M h respectively. Ester 62, prodrug of compound 47 re-
sulted in 30-fold improvement in exposure with an AUC0–24-
h = 651 M h.The prodrugs were also dosed in monkeys at 3 mg/
kg and assayed for concentration of ester and acid metabolite in
the plasma (Table 3). It is evident from the table that in most cases
the exposure was greatly improved compared to dosing the car-
boxylic acid as the sodium salt. In almost all cases the AUC0–24h
were between 10 and 20 lM h with exposures of acid metabolites
sustaining over 24 h supporting potential once a day dosing in
lM h to AUC0–24-
Effect of ester prodrugs on AUC of analog 16.
l
NH
NH
l
O
O
O
COR3
O
COOH
l
N
N
F
F
l
HN
HN
Cl
Cl
l
N
N
l
Entry R3
AUC0–6h-prodrug
M h)
AUC0–6h-acid
M h)
(
l
(l
ONa
O
50
51
52
–
2.1
CH3
O
0.1
0.2
0
CH3
O
1.1
humans.
O
53
54
0.3
0.0
0.2
0.0
CH3
4. Conclusions
O
O
We recently disclosed the identification of HCV NS5B polymer-
ase inhibitors that bound to the enzyme close to the active site, de-
rived from 6H-furo[2,3-e]indole-7-carboxylic acid core. Initial lead
characterized by compounds of type 34 (IC50 = 31 nM, EC50 = 160 -
nM) demonstrated excellent enzyme inhibition with modest repli-
con cellular potencies and pharmacokinetics. In addition, the
tricyclic indole core incorporated a fused furan moiety which
was a potential structural alert leading to reactive metabolites de-
rived from cytochrome P450 mediated oxidations.
Therefore, we first interrogated analog 34 for formation of reac-
tive species by incubating it with microsomes in the presence and
absence of glutathione and N-methoxylamine. Although the con-
versions were low, we could clearly detect glutathione and meth-
oxy amine trapped products in mass spectrum. This required
further modification of the lead to eliminate or minimize these
undesired metabolites. We therefore investigated introduction of
fluorine atoms in the ring to reduce oxidation.
CH3
N
N
55
56
57
58
0.5
9.1
0.8
0.2
32.5
0.7
CH3
CH3
N
O
O
CH3
CH3
32.6
5.5
CH3 CH3
O
N
O
Esters 55–58 were orally dosed in Sprague Dawley rats at 10 mg/kg in 0.4% MC as
the corresponding trifluoroacetate salts.
the exposure for the ester prodrug was good (AUC0–6h = 9.1 lM h).
This clearly indicated that the hydrolysis of the propyl ester was rate
limiting and was inefficiently converted to the acid. One could spec-
ulate the inefficient hydrolysis of 56–16 was probably because 56
was a poor substrate for the enzyme responsible for hydrolyzing
the ester. We further explored other prodrugs by incorporating 1-
dimethylamino 2-proponol and 2-morpholino ethanol yielding 57
Incorporation of fluorine at C(6) position was well tolerated
resulting in compound 36 (IC50 = 2 nM, IC50 = 90 nM). It was also
determined that introduction of C(6) fluorine atom suppressed oxi-
dation products thus generating further interest in optimizing of
the tricyclic indole lead.
(AUC0–6h = 32.6 lM h) and 58 (AUC0–6h = 5.5 lM h) indicating both
Previous studies had identified incorporation of 2-fluoro-5-car-
bamoyl benzyl group improved cellular potency. Incorporation of
this N1 group in 36 resulted in analog 13 (IC50 = 4 nM, IC50 = 15 -
were efficiently converted to the corresponding acid in vivo. It was
clear from this screening that polar esters were preferred and
dimethylamino ethyl and 1-dimethylamino 2-propyl esters were
most desirable. Since the introduction of 1-dimethylamino 2-propyl
ester resulted in the compound with a stereo center, we decided to
utilize dimethylamino ethyl ester for further investigation.
Having identified dimethyl amino ethyl ester as a desirable pro-
drug we introduced this in various analogs and evaluated these
compounds for full PK in rats and monkeys. Table 3 summarizes
the improvement in exposure of the parent by incorporation of
dimethyamino ethyl ester prodrugs. Various analogs with desir-
able cellular potencies were converted to the corresponding pro-
drug following procedure outline in Scheme 4 and dosed in rats
at 10 mg/kg in 0.4% methylcellulose. The concentration of the acid
metabolite was monitored in plasma over 24 h. It was very encour-
aging to observe a marked improvement in exposure of all deriva-
tives on administering the prodrug.
nM, AUC0–24h = 1.7 lM h). However inhibitor 13 was poorly ab-
sorbed when administered orally in rats at 3 mg/kg. In an
attempt to improve PK retaining the improved cellular activity
of 13 we explored introduction of novel bicyclic hetreocycles at
N1 that mimicked amide funtional groups. This had a profound
impact on the cellular activities of our compounds. The introduc-
tion of benzimidazole, and indazole moieties resulted in analogs
16, 31 and 24 that had improved potency to analog 13. These ana-
logs demonstrated modest PK in rats however had suboptimal PK
in monkeys. We therefore explored ester prodrugs and identified
dimethyl amino ethyl ester that greatly improved PK. These ester
derivatives were orally well absorbed and efficiently converted to
the corresponding acid metabolite in vivo, thus greatly improving
PK across species. The exact processing of the prodrug is still being
investigated. Due to the similarity to choline one could speculate
the role of choline esterases mediating the hydrolysis of these pro-
drugs. Thus we have optimized the potency and PK of our initial
tricyclic indole leads to identify analogs with excellent potency
and PK.
Synthesis of dimethylamino ethyl ester prodrug of 43 resulted
in compound 59 that improved the oral exposure in rats from
AUC0–6h = 7.7 to 159
lM h over 0–24 h. Similarly the formation of
dimethyl amino ethyl ester prodrug of 16 resulted in ꢁ175 fold
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S. Venkatraman et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
Table 3
Effect of dimethylaminoethyl ester prodrug on exposure of potent ticyclic inhibitors of HCV.
NH
NH
O
O
O
O
COOH
O
H
N
N
N
F
F
-
R2
R2
O
CF3COO
Entry
R2
Gt-1b EC50 (nM)
AUC0–24h
(l
M h) Acida (Rat)
AUC0–24h
14.6
(l
M h) Acida (Mky)
N
59
6
159
N
H
F
N
55
60
33
61
13
4
208
118
222
79
14
N
H
H2N
Cl
4.2
33
N
N
F
H
6
N
N
N
F
H
2
10.7
N
H
62
7
651
17
N
NH2
Dosed orally to cynomolgus monkeys (3 mg/kg) and Sprauge Dawley (10 mg/kg) in 0.4% MC. AUC represents area under the curve from 0 to 24 h.
a
(3 ꢂ 2 L). The combined organic layers were washed with aq NaOH
(2 M, 4 L). The organic layer was dried (MgSO4), filtered, concen-
trated in vacuo to yield 1-Bromo-2-(2,2-diethoxyethoxy)-4-fluoro-
benzene (362.00 g; Yield = 98%) that was used as it is in the next
step. 1H NMR (400 MHz, D6-DMSO) d 7.58 (d, 1H, J = 6.8 &
8.8 Hz), 7.11 (dd, 1H, J = 2.8 & 10.8 Hz), 6.76 (dt, 1H, J = 2.2 &
8.1 Hz), 4.81 (t, 1H, J = 5.1 Hz), 4.03 (d, 2H, J = 5.1 Hz), 3.73–3.56
(m, 4H), 1.13 (t, 6H, J = 6.6 Hz).
A solution of 1-Bromo-2-(2,2-diethoxyethoxy)-4-fluorobenzene
(352.00 g, 1.15 mol) in toluene (2500 mL, 2.3 mol) was treated
with polyphosphoric acid (370.00 g, 3.4 mol) and heated at reflux
for 5 h. The reaction mixture was concentrated in vacuo diluted
with water (3 L) and the extracted with EtOAc (4 L). The organic
layer was washed with aq NaOH (2 L), filtered, concentrated in va-
cuo and purified by distillation at reduced pressure to yield (4) 7-
bromo-4-fluorobenzofuran (125.00 g; Yield = 51%. bp 80 °C (1 mm/
Hg) as a colorless liquid which solidified at rt. 1H NMR (400 MHz,
CDCl3) d 7.67 (d, 1H, J = 2.2 Hz), 7.39 (dd, 1H J = 5.1 & 3.7 Hz),
6.94 (d, 1H, J = 2.2 Hz), 6.86 (t, 1H, J = 8.8 Hz).
5. Experimental
5.1. General
Dry solvents were purchased from Aldrich or Acros and used
without further purification. Other solvents or reagents were used
as obtained except when otherwise noted. Analytical thin layer
chromatographies (TLC) were performed on pre-coated silica gel
plates available from Analtech. Column chromatography were per-
formed using Merck silica gel 60 (particle size 0.040–0.055 lm,
230–400 mesh), or using Biotage or Isco chromatographic systems
with pre-packed silica columns. Purification of target compounds
were done on reverse phase HPLC C18 column. Elution was
achieved using water, and acetonitrile or water containing 0.1%
TFA and THF system. (Column:Waters:Delta Pk, P/No 11805, Wat
011805, 300 mm ꢂ 30 mm (L/ID) C18, 15
lM, 300 Å, 343K16006
(W): 30 mL/min flow; 30–70% ramp water/acetonitrile;
0 ? 40 min). Visualization was accomplished with UV light or by
staining with basic KMnO4 solution, methanolic H2SO4 or Vaughn’s
reagent. NMR spectra were recorded in CDCl3 or D6-DMSO unless
otherwise noted either in a 400 or 500 MHz (1H NMR), or 100 or
125 MHz (13C NMR). Mass spectra were obtained using electron
spray ionization methods. Where applicable 13C NMRs are reported
as fluorine coupled spectra unless otherwise indicated.
5.1.2. 4-Fluorobenzofuran-7-carbaldehyde (5)
A
solution of 7-bromo-4-fluorobenzofuran (4) (124.12 g,
577.25 mmol) in ether (2.0 L) was cooled to ꢀ78 °C and treated
drop wise with a solution of 2.5 M of n-Butyllithium in Hexane
(235.5 mL) and stirred at ꢀ78 °C for 15 min. To this reaction mix-
ture was added N,N-Dimethylformamide (89.393 mL, 1.15 mol)
and stirred at ꢀ78 °C for 30 min. The reaction mixture was
quenched with methanol (23.383 mL, 577.25 mmol) and warmed
to rt. It was diluted with ether (300 ml) and the organic layer
was washed with water (300 mL). The seperated organic layer
was dried (MgSO4) filtered, concentrated in vacuo to yield 4-Fluor-
5.1.1. 7-Bromo-4-fluorobenzofuran (4)
A solution of 2-Bromo-5-fluorophenol (228.00 g, 1.19 mol),
Potassium carbonate (247.47 g, 1.79 mol) in N,N-Dimethylformam-
ide (3.00 L) was treated with 2-Bromo-1,1-diethoxyethane
(197.54 mL, 1.31 mol) and heated at 135 °C for 7 h. The reaction
mixture was concentrated in vacuo and extracted with EtOAc
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8
S. Venkatraman et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
obenzofuran-7-carbaldehyde (5) (89.00 g; Yield = 93.9%). Analysis
of the product by 1H NMR indicated that the reaction mixture
was pure. It was therefore taken to the next step as it is. 1H NMR
D6-DMSO) d 12.53 (s, 1H), 8.07 (d, 1H, J = 1.8 Hz), 7.14 (d, 1H,
J = 10.4 Hz), 7.11 (d, 1H, J = 2.4 Hz), 4.35 (q, 2H, J = 7.3 Hz), 1.37
(t, 3H, J = 7.3 Hz).
10.35 (s, 1H), 7.81 (dd, 1H, J = 5.2
J = 2.0 Hz), 7.07 (t, 1H, J = 8.8 Hz), 6.95 (d, 1H, J = 2.2 Hz).
&
8.0 Hz), 7.76 (d, 1H,
A solution of ethyl-4-fluoro-8-iodo-6H-furo[2,3-e]indole-7-car-
boxylate (22.000 g, 58.962 mmol), 2-methoxypyridin-3-ylboronic
acid (13.527 g, 88.444 mmol), (PPh3)2PdCl2 (4.13 g, 5.88 mmol) in
1,2-dimethoxyethane (250.0 mL) was degassed for 2 min and stir-
red at rt for 15 min. The yellow reaction mixture was treated with
a solution of potassium carbonate (30.53 g, 220.9 mmol) in water
(250.0 mL) and stirred at 90 °C for 3 h. The yellow reaction turned
orange dark with the disappearance of starting material (TLC). The
reaction mixture was diluted with EtOAc (1000 mL) and washed
with aq. NaOH (500 mL, 1 M), dried (MgSO4), filtered, concentrated
in vacuo, and purified by chromatography SiO2 (THF/Hexanes 0–
60%) to yield (9) ethyl-4-fluoro-8-(2-methoxypyridin-3-yl)-6H-
furo[2,3-e]indole-7-carboxylate (16.65 g; Yield = 80%) as pale
brown solid. 1H NMR (400 MHz, D6-DMSO), d 12.53 (s, 1H), 8.20
(dd, 1H, J = 1.8 & 5.2 Hz), 7.85 (d, 1H, J = 2.2 Hz), 7.79 (dd, 1H,
J = 1.6 & 7.2 Hz), 7.14 (d, 1H, J = 9.8 Hz), 7.08–7.04 (m, 1H), 7.04
(d, 1H, J = 2.1 Hz), 4.15 (q, 2H, J = 6.8 Hz), 3.72 (s, 3H), 1.08 (t, 3H,
J = 7.3 Hz)
5.1.3. Ethyl 2-azido-3-(4-fluorobenzofuran-7-yl)-3-
hydroxypropanoate (6)
To a solution of 4-fluorobenzofuran-7-carbaldehyde (5, 12.71 g,
77.45 mmol), lithium chloride (6.567 g, 154.9 mmol) and diazabi-
cyclo[5.4.0]undec-7-ene (23.16 mL, 154.9 mmol) was added
a
solution of ethyl azidoacetate (20.00 g, 154.9 mmol; added as a
30% solution in CH2Cl2) at 0 °C and stirred for 2–3 h. The comple-
tion of the reaction was followed by TLC (EtOAc/Hexanes 1:4).
On complete consumption of starting material it was diluted with
ethyl acetate (1 L) and washed with water and aqueous HCl (1 M,
400 mL). The combined organic layers were dried (MgSO4), filtered,
concentrated in vacuo and dried (MgSO4). The organic layer was
concentrated in vacuo and purified by chromatography SiO2
(EtOAc/Hexanes) to yield (6) ethyl 2-azido-3-(4-fluorobenzofu-
ran-7-yl)-3-hydroxypropanoate (18.3 g; Yield = 81%) as a colorless
oil and mixture of diastereomers.
5.1.7. 4-Fluoro-8-(2-oxo-1,2-dihydropyridin-3-yl)-6H-furo[2,3-
e]indole-7-carboxylic acid (10)
5.1.4. (Z)-Ethyl 2-azido-3-(4-fluorobenzofuran-7-yl)acrylate (7)
A solution of (6) ethyl-2-azido-3-(4-fluorobenzofuran-7-yl)-3-
hydroxypropanoate (15.7 g, 53.5 mmol) and methanesulfonyl
chloride (8.29 mL, 107 mmol) in methylene chloride (87.7 mL,
1.37 mmol) at ꢀ30 °C was treated drop wise with a solution of tri-
ethylamine (52.2 mL, 375.0 mmol) in methylene chloride (100 mL).
The reaction mixture was stirred at ꢀ30 °C for 3 h, diluted with aq.
saturated sodium bicarbonate and methylene chloride (400 mL).
The organic layer was separated and washed with water, aq HCl
(1 M) and brine. The organic layer was dried (MgSO4), filtered, con-
centrated in vacuo, and purified by chromatography (SiO2, 10%
EtOAc in (1:1) Hexanes/CH2Cl2) to yield (7) (Z)-ethyl 2-azido-3-
(4-fluorobenzofuran-7-yl)acrylate (12.6 g; Yield = 86%). 1H NMR
(400 MHz, D6-DMSO) d 8.14 (dd, 1H, J = 5.6 & 8.8 Hz), 8.10 (d, 1H,
J = 2.2 Hz), 7.19 (s, 1H), 7.13 (t, 1H, J = 8.8 Hz), 7.10 (d, 1H,
J = 2.2 Hz), 4.32 (q, 2H, J = 6.6 Hz), 1.32 (t, 3H, J = 6.6 Hz).
A solution of (9) ethyl 4-fluoro-8-(2-methoxypyridin-3-yl)-6H-
furo[2,3-e]indole-7-carboxylate (4.50 g, 12.7 mmol) in methanol
(10.00 mL, 246.9 mmol) was treated with a solution of 4 M Hydro-
gen chloride in dioxane (100 mL) and heated at 90 °C for 3 h in a
pressure tube. The reaction mixture was concentrated in vacuo
and the residue was purified by chromatography SiO2 (THF/Hex-
anes 0–100%) to obtain ethyl 4-fluoro-8-(2-oxo-1,2-dihydropyri-
din-3-yl)-6H-furo[2,3-e]indole-7-carboxylate as a colorless solid.
The reaction was repeated such that a total of 17 g of ethyl 4-flu-
oro-8-(2-methoxypyridin-3-yl)-6H-furo[2,3-e]indole-7-carboxyl-
ate was completely used up.
A solution of ethyl 4-fluoro-8-(2-oxo-1,2-dihydropyridin-3-yl)-
6H-furo[2,3-e]indole-7-carboxylate (810.00 mg, 2.38 mmol) in
water (25.00 mL, 1388 mmol), THF (25.00 mL, 346.7 mmol) and
methanol (25.00 mL, 780.2 mmol) was treated with lithium
hydroxide monohydrate (499.41 mg, 11.901 mmol) and heated at
80 °C for 1 h. As the reaction proceeds a colorless solid precipitates
out. Analysis of the reaction mixture with TLC (acetone/hexanes
70%) showed disappearance of starting material. The reaction mix-
ture was acidified, filtered and dried in vacuo to yield (10) 4-flu-
oro-8-(2-oxo-1,2-dihydropyridin-3-yl)-6H-furo[2,3-e]indole-7-
carboxylic acid (627.00 mg; Yield = 85%) as colorless solid used in
the next reaction.
5.1.5. Ethyl 4-fluoro-6H-furo[2,3-e]indole-7-carboxylate (8)
150 mL of Xylenes was heated at 165 °C. To this boiling solution
was added drop wise a solution of (Z)-ethyl 2-azido-3-(4-fluor-
obenzofuran-7-yl)acrylate (7, 11.2 g, 40.7 mmol) in Xylenes
(70.00 mL, 189.4 mmol). The reaction mixture was stirred for addi-
tional 20.0 min and allowed to cool to rt. when colorless solid (8)
Ethyl 4-fluoro-6H-furo[2,3-e]indole-7-carboxylate precipitated
(7.00 g; Yield = 69.6%). The solid was filtered and washed with hex-
anes and dried. 1H NMR (400 MHz, D6-DMSO) d 12.31 (s, 1H), 7.99
(d, 1H, J = 1.7 Hz), 7.30 (d, 1H, J = 2.5 Hz), 7.12–7.08 (m, 2H), 4.34
(q, 2H, J = 6.8 Hz), 1.33 (t, 3H, J = 6.8 Hz).
5.1.8. 4-Fluorofuro[2,3-e]pyrido[30,20:5,6]pyrano[3,4-b]indol-
7(6h)-one (11)
A solution of (10) 4-fluoro-8-(2-oxo-1,2-dihydropyridin-3-yl)-
6H-furo[2,3-e]indole-7-carboxylic acid (8.00 g, 25.6 mmol) and N-
5.1.6. Ethyl-4-fluoro-8-(2-methoxypyridin-3-yl)-6H-furo[2,3-
e]indole-7-carboxylate (9)
(3-Dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride
(9.82 g, 51.2 mmol) in N,N-Dimethylformamide (153.85 mL) was
treated with triethylamine (35.71 mL, 256.2 mmol) and stirred
overnight at rt. The mixture was concentrated in vacuo to remove
the DMF and triethyl amine and the resulting residue was diluted
with methanol (100.00 mL) when a colorless solid seperated out. It
was filtered and dried to yield (11) 4-Fluorofuro[2,3e]pyr-
ido[30,20:5,6]pyrano[3,4-b]indol-7(6h)-one (5.90 g; Yield = 78%) 1H
NMR (400 MHz, D6-DMSO) d 13.23 (s, 1H), 9.10 (dd, 1H, J = 1.6 &
7.2 Hz), 8.43 (dd, 1H, J = 2.0 & 4.4 Hz), 8.19 (d, 1H, J = 2.4 Hz),
7.59 (dd, 1H, J = 4.8 & 7.6 Hz) 7.32 (m, 2H). LR–MS (ESI): calcd for
A solution of Ethyl-4-fluoro-6H-furo[2,3-e]indole-7-carboxyl-
ate (8, 15.88 g, 64.23 mmol) in DMF (100.00 mL) was treated
with N-Iodosuccinimide (15.90 g, 70.66 mmol) and stirred at rt
for 2 h. The reaction mixture was diluted with water (1000 mL)
and extracted in EtOAc (1000 mL). The organic layer was washed
with water (1000 mL), aq sodium thiosulfate (5% aq soln 1 L) and
dried (MgSO4). The organic layer was dried (MgSO4), filtered,
concentrated in vacuo to yield ethyl-4-fluoro-8-iodo-6H-
furo[2,3-e]indole-7-carboxylate (22.30 g; Yield = 93%) as a buff
colored solid used in the next reaction. 1H NMR (400 MHz,
C
16H8FN2O3 [M+H]+ 295.1; found 295.
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S. Venkatraman et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
9
5.1.9. 4-Fluoro-6-[(2-fluoro-5-cyanophenyl)methyl]furo[2,3-
e]pyrido[3,4-B]indol-7(6H)-one (12)
purified by reverse phase HPLC (water containing 0.01% TFA and
CH3CN) to yield 16 (100 mg, 48%) as a colorless solid 1H NMR
(400 MHz, D6-DMSO) d 9.32 (s, 1H), 8.07 (s, 1H), 7.98 (d, 1H,
J = 2.2 Hz), 7.70 (dd, 1H, J = 2.2 & 7.2 Hz), 7.45 (dd, 1H, J = 1.4 &
6.6 Hz), 7.42 (d, 1H, J = 11 Hz), 7.11 (d, 1H, J = 2.2 Hz), 6.56 (s,
1H), 6.37 (t, 1H, J = 6.6 Hz), 6.06 (s, 2H). 13C NMR (125 MHz, D6-
DMSO) d 162.3, 161.3, 158.2, 157.9, 155.1, 153.1, 149.0, 148.9,
144.2, 142.6, 140.5, 135.7, 135.6, 134.4, 134.0, 133.1, 131.7,
126.72, 126.65, 125.7, 116.0, 115.6, 111.6, 110.9, 110.7, 109.8,
104.8, 103.3, 92.7, 92.5, 46.5; MS (LRMS, ESI) Calcd for C24H15-
ClFN4O4 [M + H]+ 477.1; Found 477.3.
A suspension of lactone 11 (230 mg, 0.79 mmol), 3-(bromo-
methyl)-4-fluorobenzonitrile (334 mg, 1.56 mmol) in DMF
(5.0 mL, 64.4 mmol) was treated with Cs2CO3 (510 mg, 1.56 mmol)
and stirred at rt for 2 h. The reaction mixture was diluted with
water (50 mL) and extracted with EtOAc (300 mL). The organic
layer was separated and concentrated in vacuo. The residue was di-
luted with methanol (20 mL) when a colorless solid separated out.
The colorless solid was filtered and concentrated in vacuo to yield
12 (170 mg, 51%). 1H NMR (400 MHz, D6-DMSO) 9.30 (d, 1H,
J = 7.3 Hz), 8.48 (br s, 1H), 8.29 (bs, 1H), 7.85 (bs, 1H), 7.70–7.63
(m, 2H), 7.52 (t, 1H, J = 9.8 Hz), 7.30 (bs, 1H), 7.26 (bd, 1H,
J = 5.0 Hz), 6.07 (s, 2H). LR–MS (ESI): calcd for C24H12F2N3O3
[M+H]+ 428.4; found 428.1 [M+1]+
5.1.14. N-(4-Bromo-5-fluoro-2-methylphenyl)acetamide (17)
A solution of 5-fluoro-2-methylaniline (25 g, 200 mmol) in tol-
uene (250 mL) was treated with Ac2O (25 mL, 226 mmol) and
heated at reflux for 1 h. The reaction mixture was concentrated
in vacuo when a colorless solid precipitated out which was filtered
and washed with ether and hexanes. The colorless solid was taken
in acetic acide (150 mL) and treated with a solution of Br2 (9.6 mL,
186 mmol) in acetic acid (20 mL) and stirred at rt for 12 h. The
reaction mixture was diluted with water when a colorless solid
17 (40 g, 81.6%) precipitated out. It was filtered and extensively
washed.
5.1.10. 6-(5-Carbamoyl-2-fluorobenzyl)-4-fluoro-8-(2-oxo-1,2-
dihydropyridin-3-yl)-6H-furo[2,3-e]indole-7-carboxylic acid
(13)
A solution of 12 (90 mg, 0.21 mmol) in THF (20 mL) was treated
with LiOHꢃH2O (36 mg, 0.84 mmol) and heated at reflux for 14 h.
The reaction mixture was acidified with aqueous HCl (1 M soln)
and stirred fot 10 min A colorless solid separated out which was fil-
tered and used as it is. (yield 80 mg, 81%) 1H NMR (400 MHz, D6-
DMSO), d 12.93 (s, 1H), 11.74 (s, 1H), 7.92 (d, 1H, J = 2.4 Hz), 7.87
(s, 1H), 7.82–7.78 (m, 1H), 7.64 (dd, 1H, J = 2.1 & 6.8 Hz), 7.47 (d,
1H, J = 10.8 Hz), 7.40 (d, 1H, J = 7.6 Hz), 7.31–7.27 (m, 3H), 7.07
(d, 1H, J = 2.4 Hz), 6.32 (t, 1H, J = 6.8 Hz), 5.92 (s, 2H); 13C NMR
(125 MHz, D6-DMSO) d 166.5, 162.4, 162.3, 161.4, 160.3, 154.9,
153.0, 149.0, 148.9, 144.1, 140.5, 135.9, 135.8, 134.3, 130.7,
130.6, 128.3, 128.2, 127.98, 127.94, 126.69, 126.66, 125.7, 125.3,
125.2, 115.8, 115.1, 114.9, 110.8, 110.6, 109.7, 104.9, 103.2, 92.7,
92.5, 42.44, 42.41; LR–MS (ESI): calcd for C24H16F2N3O5 [M+H]+
464.1; found 464.0.
5.1.15. 1-(5-bromo-6-fluoro-1H-indazol-1-yl)ethanone (18)
A solution of 17 (10.00 g, 40.6 mmol) in CHCl3 (100 mL) was
treated with Ac2O (11.5 mL, 122 mmol), KOAc (8.00 g, 81.5 mmol),
18-crown-6 (540 mg, 2.04 mmol) amyl nitrite (12.3 mL, 871 mmol)
and heated at 65 °C for 12 h. The reaction mixture was cooled to rt
and treated with EtOAc (500 mL) washed with water and dried
(MgSO4), filtered concentrated in vacuo and purified by chromatog-
raphy (SiO2, EtOAc/Hexanes) to yield 18 as a colorless solid. 1H
NMR (400 MHz, D6-DMSO) d 8.41 (s, 1H), 8.29 (d, 1H, J = 6.6 Hz),
8.08 (d, 1H, J = 9.5 Hz), 2.69 (s, 3H).
5.1.11. tert-Butyl-5-(bromomethyl)-6-chloro-1H
5.1.16. 5-Bromo-6-fluoro-1H-indazole (19)
benzo[d]imidazole-1-carboxylate (14)
A solution of 18 (5.0 g, 19.5 mmol) was taken in aqueous HCl
(3 M, 100 mL) and methanol (20 mL) and heated at 90 °C for 3 h.
The reaction mixture was cooled to rt and basified with aqueous
NaOH (1 M). A colorless solid precipitated out which was filtered
and dried to yield 5-bromo-6-fluoro-1H-indazole as a colorless
solid.
A solution of tert-butyl-6-chloro-5-methyl-1H-benzo[d]imidaz-
ole-1-carboxylate14 (2.00 g, 7.5 mmol) in CCl4, was treated with
NBS (1.74 g, 9.75 mmol) and benzoyl peroxide (273 mg,
1.13 mmol) and heated at reflux for 14 h. The reaction mixture
was concentrated in vacuo and purified by chromatography
(SiO2, EtOAc/Hexanes) to yield 14 (1.4 g, 54%) as a viscous oil.
5.1.17. 6-Fluoro-1H-indazole-5-carbaldehyde (20)
5.1.12. 6-((6-Chloro-1H-benzo[d]imidazol-5-yl)methyl)-4-
fluorofuro[2,3-e]pyrido[30,20,5,6]pyrano[3,4-b]indol-7(6H)-one
(15)
A solution of 19 (3.5 g, 16.28 mmol) in THF (200 mL) was trea-
ted with NaH (60% mineral oil, 1.17 g) at 0 °C and stirred at rt for
20 min. The reaction mixture was cooled to ꢀ78 °C and treated
with BuLi (2.5 M in Hexanes, 8.14 mL) dropwise. The mixture
was stirred at temperature for 30 min treated with DMF
(64 mmols) and slowly warmed to rt when the viscous solution
turn homogenous and stirring was efficient. Analysis of TLC (40%
EtOAc/Hexanes) indicated complete conversion of starting mate-
rial to product. The reaction mixture was acidified with aqueous
1 M HCl and taken up in EtOAc (500 mL) washed with aqueous
HCl (1 M, 100 mL), brine (100 mL) dried (MgSO4), filtered concen-
trated in vacuo to yield 20 (2.3 g, 86%) 1H NMR (400 MHz, D6-
DMSO) d 13.52 (s, 1H), 10.16 (s, 1H), 8.37 (d, 1H, J = 6.7), 8.32 (s,
1H), 7.46 (d, 1H, J = 11.6 Hz).
A solution of 11 (843 mg, 2.86 mmol), 14 (1.10 g, 3.2 mmol),
and Cs2CO3 (1.17 g, 3.58 mmol) in DMF (20.0 mL) was stirred at
rt for 12 h and concentrated in vacuo The residue was dissolved
in EtOAc (300 mL) and washed with brine, dried (MgSO4), filtered
concentrated in vacuo and purified by chromatography (SiO2,
EtOAc/Hexanes) to yield N-alkylated compound. It was further dis-
solved in CH2Cl2 (20 mL) and treated with TFA (20 mL) and stirred
at rt for 0.5 h. The reaction mixture was concentrated in vacuo to
yield 15 a colorless solid with poor solubility in most solvents that
was used in the next reaction without further purification (1.00 g,
76%).
5.1.13. 6-((6-Chloro-1H-benzo[d]imidazol-5-yl)methyl)-4-
fluoro-8-(2-oxo-1,2-dihydropyridin-3-yl)-6H-furo[2,3-e]indole-
7-carboxylic acid (16)
A suspension of 15 (200 mg, 0.43 mmol) in THF (10 mL) and
water (10 mL) was treated with LiOHꢃH2O (100 mg, 2.38 mmol)
and stirred at rt for 2 h. The reaction mixture was quenched with
aq HCl (1 M, 5 mL) and concentrated in vacuo. The residue was
5.1.18. tert-Butyl-5-(chloromethyl)-6-fluoro-1H-indazole-1-
carboxylate (22)
A solution of the crude product 20 (2.3 g, 14.0 mmol) was take
in THF (100 mL) was treated with Boc2O (3.55 g, 16.28 mmol) and
DMAP (300 mg) and stirred at rt for 3 h. The reaction mixture was
concentrated in vacuo and the residue purified by chromatography
(SiO2, EtOAc/Hexanes) to yield tert-butyl-6-fluoro-1H-inadzole-1-
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10
S. Venkatraman et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
carboxylate (3.55 g, 81%). The boc-protected compound was taken
in methanol (50 mL) and cooled to 0 °C and treated with NaBH4
(1.02 g, 26.9 mmol) and stirred at that temperature for 1 h. The
reaction mixture was diluted with aq HCl (200 mL) and extracted
into EtOAc (200 mL). The combined organic layers were dried
(MgSO4), filtered concentrated in vacuo and purified by chroma-
tography (SiO2, EtOAc/Hexanes) to yield alcohol (3.00 g, 84%) as a
colorless solid. The alcohol (3.00 g, 11.27 mmol) was taken in CH2-
Cl2 (50 mL) at 0 °C and treated with pyridine (4.56 mL,
56.33 mmol) and methanesulfonyl chloride (1.31 mL, 16.90 mmol)
and stirred at rt for 16 h. The reaction mixture was concentrated in
vacuo and the residue was dissolved in EtOAc (300 mL). The organ-
ic layer was washed with aq HCl (1 M, 100 mL), brine (100 mL)
dried (MgSO4) filtered concentrated in vacuo and purified by chro-
matography (SiO2, EtOAc/Hexanes) to yield tert-butyl 5-(chloro-
methyl)-6-fluoro-1H-indazole-1-carboxylate (1.9 g, 59%). 1H NMR
(400 MHz, CDCl3) d 8.14 (s, 1H), 7.91 (d, 1H, J = 10.3 Hz), 7.78 (d,
1H, J = 7.2 Hz), 4.73 (s, 2H), 1.72 (s, 9H).
colorless oil. 1H NMR (400 MHz, CDCl3) d 7.55 (t, 1H, J = 8.0 Hz),
6.79 (dd, 1H, J = 8.7, 8.4 Hz), 2.2 (s, 3H).
5.1.22. 2,4-Difluoro-5-methylbenzonitrile (27)
A solution of 1,5-difluoro-2-iodo-4-methylbenzene (26, 7.11 g,
28.0 mmol), Zn(CN)2 (1.97 g, 16.8 mmol) and Pd(PPh3)4 (3.23 g,
2.8 mmol) in DMF (40 mL) was heated at 90 °C for 1.5 h. The reac-
tion mixture was concentrated in vacuo and the residue was taken
in water (400 mL) and extracted with ether (400 mL). The com-
bined organic layer was washed with excess aq ammonium
hydroxide solution. The separated organic layers were dried and
purified b chromatography to yield purified product which con-
tained triphenylphospine oxide. The crude mixture was further
purified by sublimation (45 °C, 1 mm/Hg) to yield 27 (1.8 g, 42%)
as a colorless solid. 1H NMR (400 MHz, CDCl3) d 7.47 (t, 1H,
J = 8.0 Hz), 6.92 (t, 1H, J = 9.2 Hz), 2.27 (s, 3H).
5.1.23. 6-Fluoro-5-methyl-1H-indazol-3-amine (28)
A
solution of 27 (1.40 g, 9.1 mmol), hydrazine (0.7 mL,
5.1.19. 6-((1-(tert-butoxycarbonyl)-6-fluoro-1H-indazol-5-
yl)methyl)-4-fluorofuro[2,3-e]pyrido[30,20,5,6]pyrano[3,4-
b]indol-7(6H)-one (23)
22.30 mmol) in isopropanol (50 mL, 653 mmol) was heated at reflux
for 24 h. The reaction mixture was concentrated in vacuo and the
residue was treated with aq. Na2S2O3 (200 mL) and extracted with
EtOAc (200 mL). The organic layer was dried (MgSO4), filtered con-
centrated in vacuo and the residue was purified by chromatography
(SiO2, Acetone/Hexanes) to yield 6-fluoro-5-methyl-1H-indazol-3-
amine (360 mg, 25%). 1H NMR 11.26 (s, 1H), 7.60 (d, 1H, J = 7.6 Hz),
6.92 (d, 1H, 10.8 Hz), 5.27 (s, 2H), 2.22 (s, 3H).
A suspension of 11 (900 mg, 3.05 mmol) in DMF (40 mL) was
treated with tert-butyl 5-(chloromethyl)-6-fluoro-1H-indazole-1-
carboxylate (22, 1.088 g, 3.82 mmol) and Cs2CO3 (1.50 g,
4.58 mmol) and stirred overnight at rt. The reaction mixture was
concentrated in vacuo extracted with CH2Cl2 (600 mL). The com-
bined organic layer was washed with water, dried (MgSO4), filtered
concentrated in vacuo and purified by chromatography (THF/Hex-
anes) to yield (1.60 g, 96.4%). The product was dissolved in CH2Cl2
(40 mL) and TFA (40 mL) and stirred at rt for 1 h. The reaction mix-
ture was concentrated in vacuo and used in the next reaction as it
is.
5.1.24. tert-Butyl-5-(bromomethyl)-3-(bis-(tert-
butoxycarbonyl)amino)-6-fluoro-1H-indazole-1-carboxylate
(29)
A solution of 28 (330 mg, 1.99 mmol) and Boc2O (2.61 g,
12 mmol) and DMAP (48.8 mg, 0.39 mmol) in acetonitrile (15 mL)
was stirred at reflux for 2 h and concentrated in vacuo. The residue
was purified by chromatography (SiO2, EtOAc/Hexanes) to yield
tert-butyl 5-(bromomethyl)-3-(bis-(tert-butoxycarbonyl)amino)-
6-fluoro-1H-indazole-1-carboxylate (640 mg, 68%) as a viscous oil
that was used in the next step without further purification.
A solution of N-Boc-protected compound (630 mg, 1.35 mmol),
NBS (337 mg, 1.89 mmol) and benzoylperoxide (66 mg, 0.3 mmol)
in CCl4 (20 mL, 207 mmol) was heated at reflux for 3 h. The reac-
tion mixture was concentrated in vacuo and the residue was dis-
solved in EtOAc (300 mL). The organic layer was washed with
aqueous NaS2O3, dried (MgSO4), filtered concentrated in vacuo
and purified by chromatography (SiO2, EtOAc/Hexanes) to yield
29 (610 mg, 83%) as a colorless oil which also contained unreacted
starting material.
5.1.20. 4-Fluoro-6-((6-fluoro-1H-indazol-5-yl)methyl)-8-(2-oxo-
1,2-dihydropyridin-3-yl)-6H-furo[2,3-e]indole-7-carboxylic
acid (24)
A suspension of 23 (900 mg, 2.04 mmol) in THF (40 mL) and
water (40 mL) was treated with LiOHꢃH2O (426 mg, 10.2 mmol)
and stirred at rt for 3.5 h. The reaction mixture was acidified with
aqueous HCl and concentrated in vacuo. The residue was directly
purified on reverse phase HPLC (CH3CN, water containing 0.01%
TFA) to yield 24 as a colorless solid. 1H NMR (500 MHz, D6-DMSO)
d 13.06 (bs, 2H), 11.78 (s, 1H), 7.96 (s, 1H), 7.94 (d, 1H, J = 2.5 Hz),
7.69 (dd, 1H, J = 2.0 & 7.0 Hz), 7.49 (d, 1H, J = 11.0 Hz), 7.43 (dd, 1H,
J = 1.6 & 6.5 Hz), 7.38 (d, 1H, J = 10.5 Hz), 7.08 (d, 1H, J = 2.0 Hz),
7.03 (d, 1H, J = 7.0 Hz), 6.35 (t, 1H, J = 7.0 Hz), 5.99 (s, 2H). 13C
NMR (125 MHz, D6-DMSO) d 162.5, 161.4, 160.0, 158.0, 154.8,
152.9, 149.0, 148.9, 144.0, 140.5, 135.7, 135.6, 134.3, 133.5,
126.91, 126.88, 125.8, 119.6, 119.4, 119.3, 119.03, 118.98, 115.5,
110.7, 110.5, 109.7, 104.9, 103.2, 95.9, 95.7, 92.9, 92.7, 42.91,
42.87; MS (LRMS, ESI) Calcd for C24H15F2N4O4 [M+1]+ 461.1; Found
461.3.
5.1.25. 5-((3-(Bis(tert-butoxycarbonyl)amino)-1-(tert-
butoxycarbonyl)-6-fluoro-1H-indazole-5-yl)methyl)-4-
fluorofuro[2,3-e]pyrido[30,20,5,6]pyrano[3,4-b]indol-7(6H)-one
(30)
A
suspension of 11 (294 mg, 1.00 mmol), 29 (592 mg,
1.09 mmol) and Cs2CO3 (488.7 mg, 1.5) in DMF (20 mL) was stirred
a rt for 4 h and concentrated in vacuo. The residue was dissolved in
EtOAc (200 mL) and washed with aq HCl (100 mL), brine (100 mL),
dried (MgSO4), filtered, concentrated in vacuo and purified by
chromatography (SiO2, Acetone/Hexanes) to yield 30 (490 mg,
65%) as a colorless solid. 1H NMR (400 MHz, CD3OD), d 9.17 (dd,
1H, J = 1.5 & 7.3 Hz), 8.34 (dd, 1H, J = 5.2 & 2.2 Hz), 7.95–7.92 (m,
2H), 7.45 (dd, 1H, J = 5.1 & 8.1 Hz), 7.27 (d, 1H, J = 10.3 Hz), 7.08
(d, 1H, J = 2.2 Hz), 6.71 (d, 1H, J = 6.6 Hz), 6.08 (s, 2H), 1.68 (s,
9H), 1.05 (s, 18H).
5.1.21. 1,5-Difluoro-2-iodo-4-methylbenzene (26)
A solution of 2,4-difluorotoluene (4.72 g, 36.8 mmol) in TFA
(12.3 mL, 159.5 mmol) was treated with NIS (9.59 g, 42.6 mmol)
at 0 °C and stirred at room temperature overnight. The reaction
mixture was concentrated in vacuo and the residue was dissolved
in hexanes. The organic layer was washed with aqueous sodium
thiosulfate (3 ꢂ 30 mL) and brine (3 ꢂ 30 mL). The organic layer
was separated dried (MgSO4), filtered concentrated in vacuo and
purified by bulb to bulb distillation to yield 26 (7.2 g, 77%) as a
Please cite this article in press as: Venkatraman, S.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.007

S. Venkatraman et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
11
5.1.26. 6-((3-Amino-6-fluoro-1H-indazol-5-yl)methyl)-4-fluoro-
8-(2-oxo-1,2-dihydropyridin-3-yl)-6H-furo[2,3-e]indole-7-
carboxylic acid (31)
7.17–7.12 (m, 1H), 7.08 (d, 1H, J = 2.0 Hz), 6.37–6.33 (m, 1H),
6.31 (t, 1H, J = 6.8 Hz), 5.90 (s, 2H).
A solution of 30 (490 mg, 0.65 mmol) was treated with 4 M soln
of HCl in dioxane (25 mL) and stirred at rt for 4 h. The reaction mix-
ture was concentrated in vacuo and used as it is in the next step.
The crude solid (100 mg, 0.22 mmol) in THF (4.00 mL) and
water (4.00 mL) was treated with LiOHꢃH2O (64 mg, 1.53 mmol)
and stirred at rt for 12 h. The reaction mixture was concentrated
in vacuo and purified by reverse phase HPLC (water containing
0.01% TFA and CH3CN) to yield 31 as a colorless solid (48 mg,
47%) as a colorless solid. 1H NMR (400 MHz, D6-DMSO) d 1H NMR
13.04 (b, 1H), 11.78 (bs, 1H), 7.92 (d, 1H, J = 2.4 Hz), 7.70 (dd, 1H,
J = 6.8 & 2.5 Hz), 7.44 (d, 1H, J = 14.8 Hz), 7.43–7.41 (m, 1H), 7.13
(d, 1H, J = 7.6 Hz), 7.08 (d, 1H, J = 2.4 Hz), 7.03 (d, 1H, J = 11.2 Hz),
6.35 (t, 1H, J = 6.8 Hz), 5.91 (s, 2H). 13C NMR (125 MHz, D6-DMSO).
d 162.5, 161.5, 160.7, 158.8, 154.8, 152.8, 149.0, 148.9, 148.7,
143.9, 140.6, 140.6, 140.5, 135.9, 135.8, 134.2, 127.0, 126.9,
125.9, 119.55, 119.49, 116.37, 116.2, 111.0, 110.6, 110.4, 109.80,
109.79, 104.9, 103.2, 92.7, 43.10, 43.05. MS (LRMS, ESI) Calcd for
5.1.30. 6-((1H-Benzo[d]imidazol-5-yl)methyl)-4-fluoro-8-(2-
oxo-1,2-dihydropyridin-3-yl)-6H-furo[2,3-e]indole-7-carboxylic
acid (42)
1H NMR (500 MHz, D6-DMSO) d, 13.08 (s, 1H), 11.82 (s, 1H),
9.46 (s, 1H), 7.99 (d, 1H, J = 2.0 Hz), 7.80 (d, 1H, J = 8.5 Hz), 7.66
(dd, 1H, J = 6.5 & 2.0 Hz), 7.52 (d, 1H, J = 10.5 Hz), 7.51 (s, 1H),
7.44 (dd, 1H, J = 6.5 & 2.0 Hz), 7.39 (d, 1H, J = 9.5 Hz), 7.07 (d, 1H,
J = 2.0 Hz), 6.35 (t, 1H, J = 7.0 Hz), 6.07 (s, 2H). 13C NMR
(125 MHz, D6-DMSO) d 162.6, 161.4, 154.9, 152.9, 149.0, 148.9,
144.1, 140.7, 140.5, 136.3, 135.5, 135.4, 134.4, 131.0, 130.0,
126.69, 126.67, 125.7, 124.5, 115.7, 114.6, 111.9, 110.7, 110.5,
109.8, 104.9, 103.2, 92.9, 92.7, 47.6; LR–MS (ESI): calcd for C24H16-
FN4O4 [M+H]+ 443.12; found 443.22.
5.1.31. 4-Fluoro-6-((6-fluoro-1H-benzo[d]imidazol-5-
yl)methyl)-8-(2-oxo-1,2-dihydropyridin-3-yl)-6H-furo[2,3-
e]indole-7-carboxylic acidꢃHCl (43)
C
24H16F2N5O4 [M+1]+ 475.4; Found 475.3.
1H NMR (500 MHz, D6-DMSO) d 13.07 (s, 1H), 11.82 (s, 1H), 9.32
(s, 1H), 7.96 (s, 1H), 7.78 (d, 1H, J = 9.0 Hz), 7.67 (d, 1H, J = 6.5 Hz),
7.52 (s, 1H, J = 10.5 Hz), 7.44 (d, 1H, J = 6.0 Hz), 7.09 (s, 1H), 6.96 (d,
1H, J = 6.0 Hz), 6.35 (t, 1H, J = 6.5 Hz), 6.07 (s, 2H). 13C NMR
(125 MHz, D6-DMSO) d 162.4, 161.3, 158.4, 156.4, 155.0, 153.0,
149.0, 148.9, 144.2, 141.8, 140.5, 135.7, 135.6, 134.4, 131.0,
130.9, 128.1, 126.7, 126.6, 125.7, 124.6, 124.4, 116.0, 112.60,
112.56, 110.8, 110.6, 109.8, 104.9, 103.2, 101.3, 101.1, 92.8, 92.6,
42.87, 42.82; MS (LRMS, ESI) Calcd for C24H15F2N4O4 [M+1]+
461.1; Found 461.2.
5.1.27. 2-(Dimethylamino)ethyl 4-fluoro-6-((6-fluoro-1H-
indazol-5-yl)methyl)-8-(2-oxo-1,2-dihydropyridin-3-yl)-6H-
furo[2,3-e]indole-7-carboxylate (33)
A solution of N,N dimethylamino ethanol (603 lL, 6.00 mmol) in
THF (1.6 mL) at ꢀ20 °C was treated with a solution of BuLi (2.5 M
in Hexanes, 2 mL) and stirred for 15 min. To this solution was
added 32 (700 mg, 1.54 mmol) and stirred at rt for 45 min. The
reaction mixture was quenched with TFA and concentrated in va-
cuo. The residue was purified by reverse phase HPLC to isolate
the TFA salt 33 as a colorless solid. The TFA salt was further con-
verted to HCl salt by adding aq HCl to the salt and concentrating
in vacuo (730 mg). 1H NMR (500 MHz, D6-DMSO), d, 12.20 (s,
1H), 10.45 (s, 1H), 7.99 (s, 1H), 7.99 (d, 1H, J = 2.5 Hz), 7.92 (dd,
1H, J = 7.0 & 2.0 Hz), 7.59 (d, 1H, J = 10.5 Hz), 7.53 (d, 1H, J = 6.5
& 2.0 Hz), 7.39 (d, 1H, J = 11.0 Hz), 7.17 (d, 1H, J = 7.0 Hz), 7.13 (d,
1H, J = 2.0 Hz), 6.48 (t, 1H, J = 6.5 Hz), 5.95 (s, 2H), 4.49 (bt, 2H,
J = 5.0 Hz), 3.28 (dd, 2H, J = 9.5 & 4.5 Hz), 2.72 (s, 3H), 2.71 (s,
3H). 13C NMR (125 MHz, D6-DMSO), d, 161.3, 160.8, 159.9, 158.0,
155.1, 153.2, 148.8, 148.7, 144.2, 140.9, 139.2, 139.0, 135.9,
135.8, 134.6, 133.5, 125.62, 125.59, 124.6, 119.43, 119.38, 119.28,
119.1, 118.9, 115.3, 111.1, 110.9, 109.2, 105.6, 103.3, 96.0, 95.8,
93.1, 92.9, 58.6, 54.8, 43.29, 43.25, 42.2; MS (LRMS, ESI) Calcd for
5.1.32. 6-((3-Amino-5-fluoro-1H-indazol-6-yl)methyl)-4-fluoro-
8-(2-oxo-1,2-dihydropyridin-3-yl)-6H-furo[2,3-e]indole-7-
carboxylic acid (44)
1H NMR (400 MHz, D6-DMSO), d, 11.76 (b, 1H), 11.23 (bs, 1H),
7.94 (d, 1H, J = 2.0 Hz), 7.67 (dd, 1H, J = 2.0 & 6.8 Hz), 7.51 (dd,
1H, J = 10.4 Hz), 7.46 (d, 1H, J = 10.8 Hz), 7.44 (s, 1H, J = 2.8 &
7.2 Hz), 7.09 (d, 1H, J = 2.4 Hz), 6.36 (d, 1H, J = 6.8 Hz), 6.34 (dd,
1H, J = 6.8 & 2.4 Hz), 5.98 (s, 2H). 13C NMR (125 MHz, D6-DMSO),
d, 162.4, 161.3, 154.9, 153.9, 152.9, 152.1, 149.0, 149.0, 148.9,
144.1, 140.5, 138.1, 135.7, 135.6, 134.3, 126.84, 126.81, 125.72,
125.71, 125.35, 125.19, 115.6, 112.14, 112.06, 110.7, 110.5,
109.68, 109.66, 107.11, 107.07, 104.8, 104.5, 104.4, 103.2, 92.8,
92.6, 43.14, 43.10. MS (LRMS, ESI) Calcd for C24H16F2N5O4 [M+1]+
475.4; Found 475.3.
C
28H24F2N5O4 [M+H]+ 532.2; Found 532.4.
5.1.33. 6-((3-Amino-6-fluoro-1-methyl-1H-indazol-5-
yl)methyl)-4-fluoro-8-(2-oxo-1,2-dihydropyridin-3-yl)-6H-
furo[2,3-e]indole-7-carboxylic acid (45)
5.1.28. 6-(2,5-Difluorobenzyl)-5-fluoro-8-(2-oxo-1,2-
dihydropyridin-3-yl)-6H-furo[2,3-e]indole-7-carboxylic acid
(35)
1H NMR (500 MHz, D6-DMSO), d, 12.90 (b, 1H), 11.76 (bs, 1H),
7.92 (dd, 1H, J = 2.0 & 0.5 Hz), 7.70 (d, 1H, J = 6 & 1 Hz), 7.43 (d,
1H, J = 11.0 Hz), 7.43–7.42 (m, 1H), 7.23 (d, 1H, J = 11.5 Hz), 7.10
(d, 1H, J = 7.5 Hz), 7.07 (dd, 1H, J = 0.5 & 1 Hz), 6.35 (t, 1H,
J = 6.5 Hz), 5.91 (s, 2H), 5.4 (b, 2H), 3.65 (s, 3H). 13C NMR
(125 MHz, D6-DMSO), d 162.45, 161.43, 160.72, 158.79, 154.74,
152.80, 149.00, 148.91, 148.09, 143.87, 140.54, 140.43, 140.32,
135.84, 135.74, 134.19, 126.92, 126.89, 125.88, 119.72, 119.66,
116.04, 115.90, 115.51, 110.82, 110.54, 110.34, 109.78, 104.87,
103.19, 94.56, 94.34, 92.82, 92.63, 43.01, 42.96, 34.53. MS (LRMS,
ESI) Calcd for C25H18F2N5O4 [M+1]+ 490.1; Found 490.4.
The synthesis of 7-fluoro indole derivative was initiated from 2-
bromo-4-fluorophenol and converted to substituted tricyclic in-
dole of type 9 using similar reactions shown in Scheme 1. Thus ob-
tained regioisomeric indole of type 9 was alkylated with 2,5
difluorobenzyl bromide and demethylated using 4 M HCl in diox-
ane.9 1H NMR (400 MHz, D6-DMSO), d above 12 (b, 1H) 11.75 (s,
1H), 7.89 (s, 1H), 7.69 (d, 1H, J = 5.9 Hz), 7.42–7.37 (m, 2H), 7.33–
7.27 (m, 1H), 7.16–7.12 (m, 1H), 6.95 (s, 1H), 6.38–6.31 (m, 2H),
5.97 (d, 2H).
5.1.29. 6-(2,5-Difluorobenzyl)-4-fluoro-8-(2-oxo-1,2-
dihydropyridin-3-yl)-6H-furo[2,3-e]indole-7-carboxylic acid
(36)
5.1.34. 4-Fluoro-6-((6-fluoro-1H-indazol-5-yl)methyl)-8-(2-oxo-
1,2-dihydropyridin-3-yl)-6H-furo[2,3-e]indole-7-carboxylic
acid (46)
1H NMR (400 MHz, D6-DMSO), d above 12 (1H), 11.75 (s, 1H),
7.93 (d, 1H, J = 2.0 Hz), 7.66 (dd, 1H, J = 2.4 & 7.5 Hz), 7.48 (d, 1H,
J = 10.8 Hz), 7.41–7.39 (m, 1H), 7.30 (dt, 1H, J = 4.8 & 8.2 Hz),
1H NMR (500 MHz, D6-DMSO), d 12.52–11.32 (bm, 3H), 7.98 (dd,
2H, J = 7.5 & 5.0 Hz), 7.67 (dd, 1H, J = 2.0 & 7.2 Hz), 7.51–7.50 (bm,
Please cite this article in press as: Venkatraman, S.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.007

12
S. Venkatraman et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
2H), 7.43 (dd, 1H, J = 6.5 & 1.5 Hz), 7.40 (d, 1H, J = 11.0 Hz), 7.09 (d,
1H, J = 2.5 Hz), 6.34 (t, 1H, J = 6.5 Hz), 6.08 (d, 1H, J = 7.5 Hz), 5.99
(s, 2H). 13C NMR (125 MHz, D6-DMSO), d, 175.0, 162.5, 162.4,
161.3, 160.5, 154.9, 153.0, 149.0, 148.9, 144.1, 140.6, 140.4, 140.3,
140.2, 135.8, 135.7, 134.3, 126.7, 126.6, 125.7, 124.82, 124.77,
122.6, 122.5, 121.8, 115.8, 110.8, 110.6, 109.7, 108.5, 104.9, 104.1,
103., 103.7, 103.2, 92.8, 92.6, 42.48, 42.45, MS (LRMS, ESI) Calcd
for C26H16F2N3O5 [M+1]+ 488.1; Found 488.2.
5.1.39. 2-(Dimethylamino)ethyl 6-((3-amino-6-fluoro-1H-
indazol-5-yl)methyl)-4-fluoro-8-(2-oxo-1,2-dihydropyridin-3-
yl)-6H-furo[2,3-e]indole-7-carboxylate (60)
1H NMR (500 MHz, D6-DMSO) d 12.16 (s, 1H), 10.51 (s, 1H), 7.98
(d, 1H, J = 2.5 Hz), 7.90 (dd, 1H, J = 7.0 & 2.0 Hz), 7.54 (d, 1H,
J = 10.5 Hz), 7.51 (dd, 1H, J = 6.5 & 2.0 Hz), 7.46 (d, 1H, J = 7.0 Hz),
7.34 (d, 1H, J = 10.5 Hz), 7.12 (d, 1H, J = 2.0 Hz), 6.46 (t, 1H,
J = 7.0 Hz), 5.90 (s, 2H), 4.48 (bt, 2H, J = 4.5 Hz), 3.27 (bt, 2H),
2.70 (s, 6H). 13C NMR (125 MHz, D6-DMSO), d, 161.3, 160.7,
155.2, 153., 148.9, 148.8, 144.2, 141.5, 141.4, 141.0, 136.2, 136.1,
134.6, 125.26, 125.23, 124.8, 120.75, 120.71, 120.69, 119.9,
119.78, 119.75, 115.8, 111.1, 110.9, 109.43, 109.33, 109.31,
109.28, 105.5, 103.3, 96.8, 96.6, 93.0, 92.8, 58.5, 54.8, 43.22,
5.1.35. 6-((2-Aminoquinolin-4-yl)methyl)-4-fluoro-8-(2-oxo-
1,2-dihydropyridin-3-yl)-6H-furo[2,3-e]indole-7-carboxylic
acid (47)
1H NMR (500 MHz, D6-DMSO), d, 14.22 (s, 1H), 11.83 (s, 1H),
8.82 (bs, 1H), 8.28 (d, 1H, J = 8.5 Hz), 8.05 (bs, 1H), 7.98 (s, 1H),
7.87 (t, 1H, J = 7.5 Hz), 7.78 (d, 1H, J = 8.0 Hz), 7.71 (d, 1H,
J = 5.5 Hz), 7.63 (t, 1H, J = 7.5 Hz), 7.53 (d, 1H, J = 11.0 Hz), 7.46
(d, 1H, J = 5.5 Hz), 7.12 (s, 1H), 6.37 (t, 1H, J = 6.5 Hz), 6.32 (s,
2H), 5.94 (s, 1H). 13C NMR (125 MHz, D6-DMSO), d, 162.3, 161.3,
155.2, 153.7, 153.3, 152.4, 149.1, 149.0, 144.3, 140.7, 136.04,
135.94, 135.5, 134.5, 132.6, 126.3, 126.2, 125.7, 124.9, 124.7,
118.8, 117.6, 116.7, 111.0, 110.9, 110.0, 108.1, 104.8, 103.3, 92.8,
92.6, 46.3, MS (LRMS, ESI) Calcd for C26H18FN4O4 [M+1]+ 469.1;
Found 469.2.
43.17, 42.2. MS (LRMS, ESI) Calcd for
547.2; Found 547.4
C
28H25F2N6O4 [M+H]+
References and notes
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5.1.36. 4-Fluoro-6-((6-methyl-1H-indazol-5-yl)methyl)-8-(2-
oxo-1,2-dihydropyridin-3-yl)-6H-furo[2,3-e]indole-7-carboxylic
acid (49)
1H NMR (500 MHz, D6-DMSO), d, 12.87 (b, 2H), 11.78 (b, 1H),
7.95 (d, 1H, J = 2.5 Hz), 7.77 (s, 1H), 7.74 (dd, 1H, J = 7.0 & 2.0 Hz),
7.44 (dd, 1H, J = 6.6
& 2.0 Hz), 7.40 (s, 1H), 7.34 (d, 1H,
J = 10.5 Hz), 7.08 (d, 1H, J = 2.0 Hz), 6.39 (s, 1H), 6.37 (t, 1H,
J = 6.5 Hz), 5.91 (s, 2H), 2.54 (s, 3H). 13C NMR (125 MHz, D6-DMSO),
d, 162.4, 161.4, 154.8, 152.9, 149.0, 148.9, 144.0, 140.5, 139.2,
135.8, 135.7, 134.3, 133.6, 132.8, 129.6, 127.08, 127.06, 125.9,
121.1, 115.4, 114.9, 110.7, 110.5, 110.3, 109.7, 104.9, 103.3, 92.9,
92.7, 46.3, 19.5, MS (LRMS, ESI) Calcd for C25H18FN4O4 [M+1]+
457.1; Found 457.2.
5.1.37. 2-(Dimethylamino)ethyl 6-((6-chloro-1H-
benzo[d]imidazol-5-yl)methyl)-4-fluoro-8-(2-oxo-1,2-
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18. Anilkumar, G.; Lesburg, C. A.; Selyutin, O.; Rosenblum, S. B.; Zeng, Q.; Jiang, Y.;
Chan, T.-Y.; Pu, H.; Vaccaro, H.; Wang, L.; Bennett, F.; Chen, K.-X.; Duca, H.;
Gavalas, S.; Huang, Y.; Pinto, P.; Sannigrahi, M.; Velazquez, F.; Venkatraman, S.;
Vibulbhan, B.; Agrawal, S.; Butkiewicz, N.; Feld, B.; Ferrari, E.; He, Z.; Jiang, C.-
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dihydropyridin-3-yl)-6H-furo[2,3-e]indole-7-carboxylate (55)
1H NMR (500 MHz, D6-DMSO), 12.08 (s, 1H), 9.58 (s, 1H), 8.63 (s,
1H), 8.09 (d, 1H, J = 2.5 Hz), 7.93 (dd, 1H, J = 7.0 and 2 Hz), 7.90 (s,
1H), 7.56 (dd, 1H, J = 2.0 & 6.5 Hz), 7.47 (d, 1H, J = 10.5 Hz), 7.14 (d,
1H, J = 2.0 Hz), 6.54 (s, 1H), 6.50 (t, 1H, J = 7.0 Hz), 5.99 (s, 2H), 4.02
(bt, 2H), 3.27 (bt, 2H, J = 4.0 Hz), 2.73 (s, 6H). 13C NMR (125 MHz,
D6-DMSO), 161.4, 160.60 158.1,157.8, 155.4, 153.4, 148.9, 148.8,
144.4, 143.0, 141.1, 136.1, 136.0, 134.8, 130.2, 127.9, 127.3,
125.9, 125.3, 124.6, 115.8, 112.0, 111.3, 111.1, 109.3, 105.6,
103.4, 92.9, 92.7, 58.6, 55.1, 46.8, 42.5. MS (LRMS, ESI) Calcd for
C
28H24ClFN5O4 [M+H]+ 548.2; Found 548.4.
5.1.38. 2-(Dimethylamino)ethyl-4-fluoro-6-((6-fluoro-1H-
benzo[d]imidazol-5-yl)methyl)-8-(2-oxo-1,2-dihydropyridin-3-
yl)-6H-furo[2,3-e]indole-7-carboxylate (59)
1H NMR (400 MHz, D6-DMSO) d 10.49 (s, 1H), 9.43 (s, 1H), 7.99
(d, 1H, J = 2.2 Hz), 7.86 (dd, 1H, J = 2.0 & 7.20 Hz), 7.80 (d, 1H,
J = 9.5 Hz), 7.60 (d, 1H, J = 10.3 Hz), 7.51 (dd, 1H, J = 2.0 & 6.4 Hz),
7.11 (d, 1H, J = 2.0 Hz), 7.10 (d, 1H, J = 6.6 Hz), 6.45 (t, 1H,
J = 6.6 Hz), 6.01 (s, 2H), 4.45 (t, 2H, J = 5.1Hz), 3.25 (bq, 2H,
J = 4.4 Hz), 2.69 (s, 3H), 2.68 (s, 3H).
19. Hamberger, C.; Kellert, M.; Schauer, U. M.; Dekant, W.; Mally, A. Drug Metab.
Dispos. 2010, 38, 1698–1706.
Please cite this article in press as: Venkatraman, S.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.007