Structure-activity relationship (SAR) studies on the mutagenic properties of 2,7-diaminofluorene and 2,7-diaminocarbazole derivatives

Article abstract of DOI:10.1016/j.bmcl.2020.127662

We discovered that 2,7-diaminofluorene or 2,7-diaminocarbazole moiety can be employed as a core structure of highly effective NS5A inhibitors that are connected through amide bonds to proline-valine-carbamate motifs. Amide bonds can be easily cleaved via various metabolic pathways upon administration into the body, and metabolites containing 2,7-diaminofluorene and 2,7-diaminocarbazole core structures have been known to be strong mutagens. To avoid the mutagenesis issue of these core structures, we examined various functional groups at the C9 or N9 position of 2,7-diaminofluorene or 2,7-diaminocarbazole, respectively, through the Ames test in TA98 and TA100 mutants of Salmonella typhimurium LT-2. We discovered that, through proper alkyl substitution at the C9 or N9 position, 2,7-diaminofluorene and 2,7-diaminocarbazole moieties can be successfully employed in drug discovery without necessarily causing mutagenicity problems.

Full text of DOI:10.1016/j.bmcl.2020.127662

Bioorg. Med. Chem. Lett. 31 (2021) 127662
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-activity relationship (SAR) studies on the mutagenic properties of
2,7-diaminofluorene and 2,7-diaminocarbazole derivatives
,
,
,
,*
Byeong Wook Kima 1, Hwa Leeb 1, Gyochang Keumb *, B. Moon Kima
a Department of Chemistry, College of Natural Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
b Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology (KIST), Hwarangno 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of
Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
We discovered that 2,7-diaminofluorene or 2,7-diaminocarbazole moiety can be employed as a core structure of
highly effective NS5A inhibitors that are connected through amide bonds to proline-valine-carbamate motifs.
Amide bonds can be easily cleaved via various metabolic pathways upon administration into the body, and
metabolites containing 2,7-diaminofluorene and 2,7-diaminocarbazole core structures have been known to be
strong mutagens. To avoid the mutagenesis issue of these core structures, we examined various functional groups
at the C9 or N9 position of 2,7-diaminofluorene or 2,7-diaminocarbazole, respectively, through the Ames test in
TA98 and TA100 mutants of Salmonella typhimurium LT-2. We discovered that, through proper alkyl substitution
at the C9 or N9 position, 2,7-diaminofluorene and 2,7-diaminocarbazole moieties can be successfully employed
in drug discovery without necessarily causing mutagenicity problems.
Ames test
Aromatic amines
HCV NS5A inhibitor
9H-Fluorene (or fluorene) is a polycyclic compound, which has a
violet fluorescence. As indicated in its name, fluorene has been used for
the preparation of various organic dyes.1 9H-Carbazole (or carbazole)
has similar structure as fluorene except for the nitrogen atom at the 9
position. Like fluorene, carbazole has also been utilized for many ap-
plications including dyes,2 drugs,3,4 and ligands.5 Among various de-
rivatives, 2,7-diaminofluorene and 2,7-diaminocarbazole have received
most attention because they can be utilized as an excellent scaffold for
symmetric or pseudo-symmetric molecules. Chemical applications of
2,7-diaminofluorene and 2,7-diaminocarbazole derivatives are various
including chemical adsorbent,6 nanoparticle for photodynamic ther-
apy,7 ATP sensing probe,8 and covalent organic frameworks (COFs) for
CO2 capture,9 etc. Among various usage of 2,7-diaminofluorene or 2,7-
diaminocarbazole derivatives, we focused on their potential as key
scaffolds for the construction of physiologically active molecules.
Daclatasvir (Fig. 1), which had been reported by Bristol-Myers
Squibb in 2010, was approved in 2015 by the US Food and Drug
Administration as an effective hepatitis C virus (HCV) non-structural
protein 5A (NS5A) inhibitor.10 Despite its extremely high efficacy,
daclatasvir loses its inhibitory activity over mutated NS5A proteins.11 To
circumvent this problem, many pharmaceutical companies and labora-
tories worldwide have searched for and reported a number of new NS5A
inhibitors based on the structure of daclatasvir.12–20
We have reported a new class of HCV NS5A inhibitors equipped with
benzidine21 and biaryl sulfate22 as core structures, maintaining the
proline-valine-carbamate motif of daclatasvir. In our continued
research, we were curious if 2,7-diaminofluorene and 2,7-diaminocarba-
zole could be utilized as an important bridging moiety of a drug can-
didates, particularly in relation with HCV NS5A inhibitors. Compounds
embedding these structures showed extremely potent inhibitory activ-
ities toward a variety of HCV genotype23 (Fig. 2).
Although inhibitors can readily be constructed from 2,7-diamino-
fluorene or 2,7-diaminocarbazole through formation of a series of
amide bonds and have extremely potent inhibitory activities, it is sus-
ceptible to cleavage by proteolysis once administered into the body, and
the resulting metabolites may be potential mutagens.24–26 The muta-
genicity of 2,7-diaminofluorene and 2,7-diaminocarbazole core struc-
tures must be inspected for use in the discovery of new NS5A inhibitors.
Therefore, we decided to explore the structure–activity-relationships
(SAR) on the mutagenic properties of the 9-substituted 2,7-diamino-
fluorene and 2,7-diaminocarbazole. In this study, through the Ames
test,27 we revealed our systematic approach to circumvent the muta-
genicity problems encountered in the use of 2,7-diaminofluorene and
2,7-diaminocarbazole derivatives, which warranted them as suitable
* Corresponding authors.
E-mail addresses: gkeum@kist.re.kr (G. Keum), kimbm@snu.ac.kr (B.M. Kim).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2020.127662
Received 21 August 2020; Received in revised form 20 October 2020; Accepted 28 October 2020
Available online 20 November 2020
0960-894X/© 2020 Elsevier Ltd. All rights reserved.

B.W. Kim et al.
Bioorganic & Medicinal Chemistry Letters 31 (2021) 127662
dialkyl substituents is described in Scheme 3. We introduced two alkyl
groups through SN2 reaction of compound 2 to obtain compounds
11a–d. Reduction of the nitro groups of 11a–d gave compounds 12a–d.
Scheme 4 describes the preparation of 9-alkyl substituted carbazole
derivatives. In the synthesis of 2,7-diaminocarbazole derivatives, 2,7-
dibromocarbazole (compound 13) was used as starting material. Com-
pound 13 was converted to N-alkylated carbazole (14a–d) from SN2
reaction with proper alkyl bromides. Buchwald-Hartwig amination of
aromatic bromide (14a–d) with the use of diphenylmethanimine fol-
lowed by imine hydrolysis afforded 16a–d. Transformation from aryl
bromide to diamine via Buchwald-Hartwig amination and imine hy-
drolysis was repetitive in following Schemes (vide infra). Synthesis of 9-
(10-morpholinodecyl)-9H-carbazole-2,7-diamine (20) is described in
Scheme 5. Compound 17 was prepared via SN2 reaction of compound 13
with 1,10-dibromodecane. Additional SN2 reaction between compound
17 and morpholine gave compound 18 albeit in a moderate yield.
Following the synthetic procedure mentioned in Scheme 4, we con-
verted compound 18 to compound 20. Scheme 6 describes the synthetic
procedure for compounds 23a and 23b, which were prepared from para-
alkoxy-substituted benzyl chlorides. SN2 reaction between compound
13 and para-substituted benzyl chloride (21a and 21b) afforded 22a and
22b, respectively. These compounds were converted to diamine (23a
and 23b) through above-mentioned methods in Scheme 4. Scheme 7
describes the preparation of 2,7-diaminocarbazole derivatives (27a and
27b) containing meta-substituted benzyl group at the 9 position. N-
Alkylation of compound 13 with 1-(bromomethyl)-3-methoxybenzene
afforded compound 24 and demethylation of the resulting 24 furnished
compound 25. After proper O-alkylation (26a and 26b), compounds 27a
and 27b were synthesized via the same procedure described in Scheme
4. Compounds 30a–e have similar structure as dialkylated fluorene
derivatives (12a–d). Scheme 8 describes the preparation of compounds
30a–e. These compounds (30a–e) were also prepared via the same way
as described in Scheme 4: i) alkylation, ii) Buchwald-Hartwig amination,
and iii) imine hydrolysis.
Fig. 1. Chemical structure of daclatasvir.
Fig. 2. Chemical structure and EC50 values of compound 1.
new core structures of NS5A inhibitors.
All diamine compounds used in the Ames test27 were synthesized via
the following synthetic schemes (Schemes 1–8) (See details in the Sup-
plementary Material).
We describe the synthesis of 9-aryl substituted fluorene compounds
in Scheme 1. Compound 2 was reduced to 2,7-diaminofluorene with the
aid of iron oxide nanoparticles and hydrazine as reported in our previous
report28 and Boc protection of the resulting diamine afforded compound
3. After oxidation at the 9 position, the resulting ketone 4 was converted
to hydrazone 5 from treatment with p-TsNHNH2. Metal-free reductive
coupling29 of 5 with the corresponding arylboronic acid was performed
followed by Boc deprotection to give compounds 6a–f. Synthesis of 2,7-
diaminofluorene derivatives containing 9-alkyl or alkylidene sub-
stituents is described in Scheme 2. Compounds 7a–c were synthesized
from compound 3 via Knoevenagel condensation with various alde-
hydes. Compounds 7a–c were converted to diamine through Boc
deprotection (8a–b) or dehydrogenation followed by Boc deprotection
(10b–c). Synthesis of 2,7-diaminofluorene derivatives containing 9,9-
The mutagenicity of several 2,7-diaminofluorene derivatives and
2,7-diaminocarbazole derivatives was examined using the Ames test
(see details in the Supplementary Material) in strains TA98 and TA100,
both with and without S9 mix treatment. All compounds were tested at
six different concentrations from 4 to 5000 μM. The results are presented
in Tables 1 and 2, respectively, and the detailed mutagenic property of
each examined compound is collected in the Supplementary Materials.
Scheme 1. Reagents and conditions: (a) Fe3O4, hydrazine monohydrate, EtOH, 75 ◦C, 2 h, 96%; (b) Boc2O, NaOH, 1,4-dioxane, H2O, 23 ◦C, 24 h, 84%; (c) Cs2CO3,
DMSO, 23 ◦C, 18 h, 63%; (d) p-TsNHNH2, MeOH, 60 ◦C, 4 h, 95%; (e) K2CO3, 1,4-dioxane, reflux, 2 h, 58–78%; (f) TFA, DCM, 23 ◦C, 2 h, 35–62%.
2

B.W. Kim et al.
Bioorganic & Medicinal Chemistry Letters 31 (2021) 127662
Scheme 2. Reagents and conditions: (a) t-BuOK, xylene, reflux, 1 h or KOH, DME, reflux, 4 h, 54–66%; (b) TFA, DCM, 23 ◦C, 2 h, 41–49%; (c) Pd/C, H2, MeOH,
23 ◦C, 12 h, 51–58%.
Scheme 3. Reagents and conditions: (a) Cs2CO3, DMF, 0 ◦C to 23 ◦C, 18 h, 40–64%; (b) Fe3O4, hydrazine monohydrate, EtOH, 75 ◦C, 2 h, 17–39%.
Scheme 4. Reagents and conditions: (a) NaH, DMF, 23 ◦C, 12 h, 69–98%; (b) Pd2(dba)3∙CHCl3, t-BuXPhos, t-BuONa, diphenylmethanimine, toluene, 100 ◦C, 16 h,
35–77%; (c) 4.0 M HCl/MeOH, 23 ◦C, 1 h, 86–93%.
Scheme 5. Reagents and conditions: (a) NaH, 1,10-dibromodecane, DMF, 0 ◦C to 23 ◦C, 16 h, 99%; (b) K2CO3, morpholine, MeCN, reflux, 10 h, 47%; (c)
Pd2(dba)3∙CHCl3, t-BuXPhos, t-BuONa, diphenylmethanimine, toluene, 100 ◦C, 16 h, 73%; (d) 4.0 M HCl/MeOH, 23 ◦C, 4 h, 69%.
3

B.W. Kim et al.
Bioorganic & Medicinal Chemistry Letters 31 (2021) 127662
Scheme 6. Reagents and conditions: (a) NaH, DMF, 0 ◦C to 23 ◦C, 12 h, 99%; (b) Pd2(dba)3∙CHCl3, t-BuXPhos, t-BuONa, diphenylmethanimine, toluene, 100 ◦C, 16
h, 69–79%; (c) 4.0 M HCl/MeOH, 23 ◦C, 1 h, 86–91%.
Scheme 7. Reagents and conditions: (a) NaH, 1-(bromomethyl)-3-methoxybenzene, DMF, 0 ◦C to 23 ◦C, 12 h, 99%; (b) NaI, TMSCl, MeCN, reflux, 3 h, 56%; (c)
K2CO3, DMF, 90 ◦C, 5 h, 37–92%; (d) Pd2(dba)3∙CHCl3, t-BuXPhos, t-BuONa, diphenylmethanimine, toluene, 100 ◦C, 16 h, 37–72%; (e) 4.0 M HCl/MeOH, 23 ◦C, 2
h, 71–82%.
Scheme 8. Reagents and conditions: (a) NaH, DMF, 0 ◦C to 23 ◦C, 12 h, 38–91%; (b) Pd2(dba)3∙CHCl3, t-BuXPhos, t-BuONa, diphenylmethanimine, toluene, 100 ◦C,
16 h, 36–95%; (c) 4.0 M HCl/MeOH, 23 ◦C, 1 h, 48–69%.
Compounds 6a–f, equipped with various aromatic rings at the C9
position of fluorene, were found to be non-mutagenic in TA98 and
TA100 strains in the absence of S9 mix. However, in the presence of S9
mix, these compounds turned to be mutagenic in TA98 strain. Com-
pounds containing a double bond at the C9 position (8a and 8b) were
found to be mutagenic regardless of treatment with S9 mix. Compounds
with a reduced double bond (10b and 10c) were mutagenic in TA 98
strain only in the presence of S9 mix. However, compounds possessing
dialkyl substituents at the C9 position of fluorene (12a–d) exhibited
varying degrees of mutagenicity depending on the length of the alkyl
groups. With dipropyl substituents, compound 12a showed mutagenic
activity in TA98 strain treated with S9 mix. In the cases of dibutyl, bis
(trifluoromethylpropyl), and bis(trifluoromethylbutyl) substituted
compounds 12b, 12c, and 12d, respectively, they were found to be
slightly mutagenic in TA98 strain treated with S9 mix only when the
concentration was higher than 5 mM. Therefore, it can be concluded that
the longer the length of alkyl chain is, the lower the probability of
mutation.
In the case of carbazole derivatives, compounds equipped with
monoalkyl substituents at the N9 position of carbazole (16a–d and 20)
showed a similar mutagenic pattern as in the fluorene derivatives; the
mutagenicity was dependent on the length and bulkiness of the alkyl
substituent. When the alkyl chains were longer than n-decyl, the cor-
responding carbazole derivatives were non-mutagenic. N-Benzyl de-
rivatives with an alkoxy substitution, such as compounds 23a, 23b, 27a,
and 27b, were all found to be mutagenic in TA98 strain treated with S9
mix, regardless of the position and type of the alkoxy groups attached to
the benzene ring. Bis(cyclopropyl)methyl-substituted derivative (30a)
4

B.W. Kim et al.
Bioorganic & Medicinal Chemistry Letters 31 (2021) 127662
substitution (30c–e), the alkyl chain length played a critical role in
determining their mutagenicity, regardless of the phenyl group.
According to the precedent research, 2-aminofluorene (2-AF) or N-
acetyl-2-aminofluorene (2-AAF) is transformed to N-hydroxy-2-AF or N-
hydroxy-2-AAF via CYP450 monooxygenase oxidation.25 N-Hydroxy-2-
AF or N-hydroxy-2-AAF can be metabolized to electrophilic species,
such as N-SO4-2-AF, N-acetoxyl-2-AF, and N-SO4-2-AAF.25 These elec-
trophiles can form DNA adducts through a reaction with the guanine
base, which we considered as a major mutation pathway in the amino-
fluorene case.
Table 1
Ames test results of 2,7-diaminofluorene derivatives.
Entry
R1
R2
Ames resulta,b
6a
6b
6c
6d
6e
6f
H
H
H
H
H
H
4-(i-Pr)-C6H4
4-(t-Bu)-C6H4
4-F-C6H4
ꢀ ,+,ꢀ ,ꢀ
ꢀ ,+,ꢀ ,ꢀ
ꢀ ,+,ꢀ ,ꢀ
ꢀ ,+,ꢀ ,-
4-Br-C6H4
4-CF3-C6H4
2-naphthyl
ꢀ ,+,ꢀ ,ꢀ
ꢀ ,+,ꢀ ,ꢀ
+,+,+,+
Compounds 6a–f, equipped with various aromatic ring substitutions
at the C9 position of 2,7-diaminofluorene, were found to be mutagenic
in TA98 strain treated with S9 mix. The fact that these compounds did
not act as mutagens in TA98 without S9 treatment indicated that the
formation of metabolites through DNA adduct can be a cause of muta-
tion. In addition, the negative result for mutation in TA100 strain treated
with S9 mixture indicated that the DNA adduct caused a frame-shift
mutation rather than a base-pair substitution.30 In the case of fluorene
derivatives substituted with dialkyl groups at the C9 position (com-
pounds 12a–d), shorter alkyl chain substituents tended to cause muta-
genicity. From the results of previous25 and current study, we
hypothesized that the mutation of 2,7-diaminofluorene can be pre-
vented by introducing sterically bulky dialkyl groups at the C9 position,
presumably because they prevent the guanine base from approaching
electrophilic metabolites.
8a
8b
+,+,ꢀ ,ꢀ
+,+,ꢀ ,ꢀ
ꢀ ,+,ꢀ ,+
10b
10c
H
H
12a
12b
12c
12d
(CH2)2CH3
(CH2)3CH3
(CH2)3CF3
(CH2)4CF3
(CH2)2CH3
(CH2)3CH3
(CH2)3CF3
(CH2)4CF3
ꢀ ,+,ꢀ ,ꢀ
ꢀ ,+,ꢀ ,ꢀ
ꢀ ,+,ꢀ ,ꢀ
ꢀ ,ꢀ ,ꢀ ,ꢀ
a
Ames results means the test result of TA98 ¡S9, TA98 + S9, TA100 ¡S9, and
TA100 + S9, respectively.
In the case of 2,7-diaminofluorene, introduction of dibutyl-, bis(tri-
fluoromethylpropyl)-, or bis(trifluoromethylbutyl)- chains, as in com-
pounds 12b, 12c, and 12d, respectively, diminished mutation
propensity. However, the carbazole moiety needed to be substituted
with a longer N-alkyl chain than an n-decyl group (as in 16c, 16d and
20) to avoid mutagenicity issues. These results agree well with our hy-
pothesis because carbazole needed to be substituted with a longer chain
monoalkyl group than fluorene with longer-than-propyl dialkyl groups
at the C9 position. Therefore, to block the formation of DNA adduct,
carbazole has to be substituted with a longer alkyl chain than that of
fluorene. In the case of carbazoles substituted with a symmetric sec-
ondary alkyl group (30a and 30b), which mimics the dialkyl group of
fluorene derivatives, compounds substituted with a long branched alkyl
chain, such as (1-butyl)pentyl substitution (mimicking 9,9-dipentyl
groups at the fluorene) (30b), may avoid mutagenic issues, but not
those with a short chain (30a). The importance of the alkyl chain length
in determining the mutagenicity of compounds could be explained by
the (1-phenyl)alkyl substitution cases (30c–e). Regardless of the phenyl
group, mutagenicity pattern was consistent with the length of the alkyl
chain. With methyl (30c) or propyl (30d) substitution, the compounds
were found to be mutagenic, but compounds with longer substituent (e.
g. nonyl group, 30e) were non-mutagenic. Our hypothesis was also
confirmed by the results of N-benzyl derivatives with an alkoxy substi-
tution (23a, 23b, 27a and 27b). In these cases, any changes in the
alkoxy group did not affect the Ames results of the four compounds
because those variations did not affect the bulkiness of the compounds.
In this study, using the Ames test, we investigated the important
factors affecting the mutagenicity of the aniline derivatives 2,7-diamino-
fluorene and 2,7-diaminocarbazole, which are often employed in drug
discovery. Ames test results showed that mutagenicity problems with
derivatives of 2,7-diaminofluorene and 2,7-diaminocarbazole can be
solved by equipping these derivatives with a long alkyl chain at a proper
location. The results imply that 2,7-diaminofluorene and 2,7-diamino-
carbazole can successfully be employed in drug discovery as long as
they are equipped with proper substituents. In addition, several of the
examined compounds showed extremely high antiviral activities,23
proving that fluorene and carbazole can be used as effective core
structures of HCV NS5A inhibitors.
b
+: positive, ꢀ : negative.
Table 2
Ames test results of 2,7-diaminocarbazole derivatives.
Entry
R1
R2
Ames resulta,b
16a
16b
16c
16d
20
H
H
H
H
H
(CH2)2CH3
(CH2)6CH3
(CH2)8CH3
(CH2)10CH3
+,+,+,+
ꢀ ,+,ꢀ ,ꢀ
ꢀ ,+,ꢀ ,ꢀ
ꢀ ,ꢀ ,ꢀ ,ꢀ
ꢀ ,ꢀ ,ꢀ ,ꢀ
23a
23b
27a
27b
30a
H
H
H
H
ꢀ ,+,ꢀ ,ꢀ
ꢀ ,+,ꢀ ,ꢀ
ꢀ ,+,ꢀ ,ꢀ
ꢀ ,+,ꢀ ,ꢀ
ꢀ ,+,ꢀ ,+
30b
30c
30d
30e
(CH2)3CH3
C6H5
(CH2)3CH3
CH3
ꢀ ,ꢀ ,ꢀ ,ꢀ
ꢀ ,+,ꢀ ,+
ꢀ ,+,ꢀ ,ꢀ
ꢀ ,ꢀ ,ꢀ ,ꢀ
C6H5
(CH2)2CH3
(CH2)8CH3
C6H5
a
Ames results means the test result of TA98 –S9, TA98 + S9, TA100 –S9, and
TA100 + S9, respectively.
b
+: positive, ꢀ negative.
also showed mutagenic activity in both TA98 and TA100 strains treated
with S9 mix. A compound with a longer substitution such as a 5-nonyl
group (30b) showed no mutagenicity in TA98 and TA100 strains
treated with and without S9 mix. In compounds with (1-phenyl)alkyl
5

B.W. Kim et al.
Bioorganic & Medicinal Chemistry Letters 31 (2021) 127662
BMS-790052 in humans: in vitro and in vivo correlations. Hepatology. 2011;54(6):
1924–1935.
Declaration of Competing Interest
12 Coburn CA, Meinke PT, Chang W, et al. Discovery of MK-8742: an HCV NS5A
inhibitor with broad genotype activity. ChemMedChem. 2013;8(12):1930–1940.
13 DeGoey DA, Randolph JT, Liu D, et al. Discovery of ABT-267, a pan-genotypic
inhibitor of HCV NS5A. J Med Chem. 2014;57(5):2047–2057.
14 Link JO, Taylor JG, Xu L, et al. Discovery of ledipasvir (GS-5885): a potent, once-
daily oral NS5A inhibitor for the treatment of hepatitis C virus infection. J Med Chem.
2014;57(5):2033–2046.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
15 Link JO, Taylor JG, Trejo-Martin A, et al. Discovery of velpatasvir (GS-5816): a
potent pan-genotypic HCV NS5A inhibitor in the single-tablet regimens Vosevi((R))
and Epclusa((R)). Bioorg Med Chem Lett. 2019;29(16):2415–2427.
16 Meanwell NA, Philip S. Portoghese medicinal chemistry lectureship. curing hepatitis
c virus infection with direct-acting antiviral agents: the arc of a medicinal chemistry
triumph. J Med Chem. 2016;59(16):7311–7351.
This research was supported by the National Research Foundation
(NRF) of Korea (NRF-2017M3A9F6029755). This research was also
supproted by Bio & Medical Technology Development Program of the
NRF of Korea (NRF-2012M3A9A9054975)
17 Tong L, Yu W, Chen L, et al. Discovery of ruzasvir (MK-8408): a potent, pan-genotype
HCV NS5A inhibitor with optimized activity against common resistance-associated
polymorphisms. J Med Chem. 2017;60(1):290–306.
Appendix A. Supplementary data
18 Wagner R, Randolph JT, Patel SV, et al. Highlights of the structure-activity
relationships of benzimidazole linked pyrrolidines leading to the discovery of the
hepatitis C virus NS5A inhibitor pibrentasvir (ABT-530). J Med Chem. 2018;61(9):
4052–4066.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2020.127662.
19 Walker J, Crosby R, Wang A, et al. Preclinical characterization of GSK2336805, a
novel inhibitor of hepatitis C virus replication that selects for resistance in NS5A.
Antimicrob Agents Chemother. 2014;58(1):38–47.
References
20 Zhong M, Peng E, Huang N, et al. Discovery of ravidasvir (PPI-668) as a potent pan-
genotypic HCV NS5A inhibitor. Bioorg Med Chem Lett. 2016;26(18):4508–4512.
21 Bae IH, Choi JK, Chough C, et al. Potent hepatitis C virus NS5A inhibitors containing
a benzidine core. ACS Med Chem Lett. 2014;5(3):255–258.
1 Kurdyukova IV, Ishchenko AA. Organic dyes based on fluorene and its derivatives.
Russ Chem Rev. 2012;81(3):258–290.
22 You Y, Kim HS, Park JW, Keum G, Jang SK, Kim BM. Sulfur(vi) fluoride exchange as a
key reaction for synthesizing biaryl sulfate core derivatives as potent hepatitis C virus
NS5A inhibitors and their structure-activity relationship studies. RSC Adv. 2018;8
(55):31803–31821.
2 Chamberlain T. Dioxazine violet pigments. High Perform Pigments. 2001:185–194.
3 Eaton MJ, Lookingland KJ, Moore KE. The
σ ligand rimcazole activates
noradrenergic neurons projecting to the paraventricular nucleus and increases
corticosterone secretion in rats. Brain Res. 1996;733(2):162–166.
23 Kim HS, You Y, Mun J, et al. Structure-activity relationships of fluorene compounds
inhibiting HCV variants. Antiviral Res. 2020;174, 104678.
4 Bostrom IM, Nyman GC, Lord PE, Haggstrom J, Jones BE, Bohlin HP. Effects of
carprofen on renal function and results of serum biochemical and hematologic
analyses in anesthetized dogs that had low blood pressure during anesthesia. Am J
Vet Res. 2002;63(5):712–721.
24 Bomhard EM, Herbold BA. Genotoxic activities of aniline and its metabolites and
their relationship to the carcinogenicity of aniline in the spleen of rats. Crit Rev
Toxicol. 2005;35(10):783–835.
5 Rowland CA, Yap GPA, Bloch ED. Novel syntheses of carbazole-3,6-dicarboxylate
ligands and their utilization for porous coordination cages. Dalton Trans. 2020.
6 Mokhtari N, Afshari M, Dinari M. Synthesis and characterization of a novel fluorene-
based covalent triazine framework as a chemical adsorbent for highly efficient dye
removal. Polymer. 2020;195.
25 Heflich RH, Neft RE. Genetic toxicity of 2-acetylaminofluorene, 2-aminofluorene and
some of their metabolites and model metabolites. Mutat Res Rev Genet Toxicol. 1994;
318(2):73–174.
26 Purohit V, Basu AK. Mutagenicity of nitroaromatic compounds. Chem Res Toxicol.
2000;13(8):673–692.
7 Yin H-Q, Cao P-P, Wang X-Y, Li Y-H, Yin X-B. Computed tomography imaging-guided
tandem catalysis-enhanced photodynamic therapy with gold nanoparticle functional
covalent organic polymers. ACS Appl Bio Mater. 2020;3(4):2534–2542.
8 Ye R, Cui Q, Yao C, Liu R, Li L. Tunable fluorescence behaviors of a supramolecular
system based on a fluorene derivative and cucurbit[8]uril and its application for ATP
sensing. Phys Chem Chem Phys. 2017;19(46):31306–31315.
27 Ames BN, McCann J, Yamasaki E. Methods for detecting carcinogens and mutagens
with the Salmonella/mammalian-microsome mutagenicity test. Mutat Res. 1975;31
(6):347–364.
28 Kim S, Kim E, Kim BM. Fe3O4 nanoparticles: a conveniently reusable catalyst for the
reduction of nitroarenes using hydrazine hydrate. Chem Asian J. 2011;6(8):
1921–1925.
9 An S, Xu T, Peng C, Hu J, Liu H. Rational design of functionalized covalent organic
frameworks and their performance towards CO2 capture. RSC Adv. 2019;9(37):
21438–21443.
29 Barluenga J, Tomas-Gamasa M, Aznar F, Valdes C. Metal-free carbon-carbon bond-
forming reductive coupling between boronic acids and tosylhydrazones. Nat Chem.
2009;1(6):494–499.
10 Gao M, Nettles RE, Belema M, et al. Chemical genetics strategy identifies an HCV
NS5A inhibitor with a potent clinical effect. Nature. 2010;465(7294):96–100.
11 Fridell RA, Wang C, Sun JH, et al. Genotypic and phenotypic analysis of variants
resistant to hepatitis C virus nonstructural protein 5A replication complex inhibitor
30 Flückiger-Isler S, Kamber M. The ames II and ames MPF penta I assay: a liquid
microplate format modification of the classic ames test. Genotox DNA Repair. 2014:
23–41.
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