Optimization of a novel potent and selective bacterial DNA helicase inhibitor scaffold from a high throughput screening hit

Article abstract of DOI:10.1016/j.bmcl.2013.04.055

Benzobisthiazole derivatives were identified as novel helicase inhibitors through high throughput screening against purified Staphylococcus aureus (Sa) and Bacillus anthracis (Ba) replicative helicases. Chemical optimization has produced compound 59 with nanomolar potency against the DNA duplex strand unwinding activities of both B. anthracis and S. aureus helicases. Selectivity index (SI = CC₅₀/IC₅₀) values for 59 were greater than 500. Kinetic studies demonstrated that the benzobisthiazole-based bacterial helicase inhibitors act competitively with the DNA substrate. Therefore, benzobisthiazole helicase inhibitors represent a promising new scaffold for evaluation as antibacterial agents.

Full text of DOI:10.1016/j.bmcl.2013.04.055

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3481–3486
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of a novel potent and selective bacterial DNA helicase
inhibitor scaffold from a high throughput screening hit
a
a,
Bing Li a, , Ramdas Pai , Daniel Aiello , Ming Di a, Marjorie H. Barnes a, Norton P. Peet a,
⇑
Terry L. Bowlin a, Donald T. Moir a,
a Microbiotix Inc., One Innovation Drive, Worcester, MA 01605, USA
⇑
a r t i c l e i n f o
a b s t r a c t
Article history:
Benzobisthiazole derivatives were identified as novel helicase inhibitors through high throughput screen-
ing against purified Staphylococcus aureus (Sa) and Bacillus anthracis (Ba) replicative helicases. Chemical
optimization has produced compound 59 with nanomolar potency against the DNA duplex strand
unwinding activities of both B. anthracis and S. aureus helicases. Selectivity index (SI = CC50/IC50) values
for 59 were greater than 500. Kinetic studies demonstrated that the benzobisthiazole-based bacterial
helicase inhibitors act competitively with the DNA substrate. Therefore, benzobisthiazole helicase inhib-
itors represent a promising new scaffold for evaluation as antibacterial agents.
Received 26 February 2013
Revised 12 April 2013
Accepted 22 April 2013
Available online 30 April 2013
Keywords:
Optimization
DNA helicase
DNA replication
Inhibitor
Ó 2013 Elsevier Ltd. All rights reserved.
The increasing prevalence of antibiotic resistant strains of
bacterial pathogens presents a major unmet medical need. For
example, the incidence of skin and soft tissue infections with com-
munity acquired methicillin-resistant Staphylococcus aureus
(MRSA) has increased over fivefold since 2003.1 In addition, a re-
cent report revealed that 28% of enterococci cultured from 25
North American intensive care units (ICUs) were resistant to van-
comycin (VRE).2 The development of new antibiotics against
underexploited targets with novel mechanisms of action is a vital
part of the solution to these problems because such antibiotics will
not be affected by preexisting target based resistance mechanisms.
The replicative DNA helicase is an essential component of the DNA
replication pathway, acting early and catalyzing a rate limiting
step in replication, but it is currently untargeted by antibacterial
agents.
Replicative DNA helicase is a member of a drug-validated path-
way and, along with gyrase, topoisomerase IV, and DNA polymer-
ase III, is essential to bacteria.3–7 The primary structures of
bacterial replicative helicases differ significantly from those of
their eukaryotic and human counterparts,8,9 indicating that bacte-
ria-specific inhibitors of helicase may be developed. These features
make it particularly attractive as a target for the discovery of new
antibacterial therapeutics.
The replicative DNA helicases from Escherichia coli, S. aureus,
and Pseudomonas aeruginosa have been targeted previously in
anti-infective screens,10–17 but few hits have been described, and
none have progressed further in drug development due to poor po-
tency and inadequate selectivity. Two distinct X-ray crystal struc-
tures have been reported: one shows a hexameric DnaB helicase in
complex with a helicase binding fragment of primase,18 and an-
other shows that the DnaB hexamer adopts a closed spiral staircase
quaternary structure in complex with ATP mimic GDP-AlF4 and
ssDNA.19 The two structures suggest that helicase may exist in
both inactivated and activated forms during the bacterial DNA rep-
lication process. Structure-based approaches to target both the
inactivated and activated forms of DnaB helicase may aid in the
discovery of novel bacterial DNA helicase inhibitors.
We have previously discovered a coumarin-based DNA helicase
inhibitor series through a high throughput screening campaign,
and chemical optimization yielded compounds with antibacterial
activities against several Gram-positive species including multiple
clinically relevant ciprofloxacin-resistant MRSA strains.20,21 Herein
we report chemical optimization and biological evaluation of a no-
vel series of DNA bacterial helicase inhibitors based on a benzobis-
thiazole scaffold.
Benzobisthiazole derivatives were identified as novel inhibitors
through high throughput screening against S. aureus (Sa) and Bacil-
lus anthracis (Ba) helicases. The screening hit compound 1 (Fig. 1)
⇑
Corresponding authors. Tel.: +1 508 757 2800; fax: +1 508 757 1999 (B.L.).
E-mail addresses: bli@microbiotix.com (B. Li), dmoir@microbiotix.com (D.T.
demonstrated antihelicase activities (IC50 (Ba) = 5
lM and IC50
(Sa) = 12 M) and minimal cytotoxicity (CC50 > 100
l
l
M). Initial
Moir).
investigation of structure-activity relationships (SARs) focused on
the benzamide portion of the benzobisthiazole scaffold.
Current address: University of Massachusetts Medical School, Worcester, MA
01605, USA.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.055

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B. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3481–3486
S
N
The effect of replacement of the phenyl ring with various groups
O
S
was also investigated in the B. anthracis DNA helicase assay, and
the results are shown in Table 2. Replacement of the phenyl ring
with alkyl, arylalkyl, naphthyl or heteroaryl groups (compounds
34–44) significantly decreased potency, except for compound 45
with a pyrazine replacement, which exhibited modest activity
N
S
N
O
NH
O
1
(IC50 = 28 lM).
IC50 (Ba) = 5 µM
IC50 (Sa) = 12 µM
CC50 > 100 µM
The most active B. anthracis helicase inhibitor, compound 33,
also exhibited potent inhibitory activity versus S. aureus DNA heli-
case (IC50 = 0.4
M), while compound 16, which bears a 3-OCH3 group on the phe-
nyl ring, inhibited S. aureus DNA helicase with an IC50 value of
6.6 M. To evaluate the SARs on the methylthio side of the benz-
lM) without detectable cytotoxicty (CC50 > 100
l
Figure 1. The structure of HTS hit 1.
l
obisthiazole core structure, we synthesized a series of analogs of
two precursors 33 and 16, by further transforming the methylthio
group to various amines, and the synthesis is shown in Scheme 1.
Commercially available aminobenzobisthiazole compound 46
was treated with corresponding benzoyl chlorides, to yield amides
16 and 33.22 Treatment of compounds 16 and 33 with KMnO4 un-
der acidic conditions produced sulfones 47 and 48, respectively,
which were treated with amines to produce benzobisthiazole ana-
logs 49–63.23,24 To evaluate whether the amide bond was required
for antihelicase activity, amino compound 46 was converted to
benzylamino analog 64 through reductive amination, and an imine
analog 65 was also synthesized through a condensation reaction
(Scheme 2).25 The biological activities of these analogs were eval-
uated vs both B. anthracis and S. aureus DNA replicative helicases,
and cytotoxicities were measured in a serum free MTT assay.21 Re-
sults are summarized in Table 3.
Thirty-two analogs (compounds 4–35) with various substitutions
on the phenyl ring of the benzamide portion and twelve analogs
(compounds 36–47) with phenyl ring replacements were pur-
chased from an outside vendor (Life Chemicals Inc.). Their biolog-
ical activities were evaluated in a fluorescence resonance transfer
(FRET)-based helicase strand unwinding assay20 to measure con-
centration-dependent inhibition of B. anthracis DNA replicative
helicase, and the results are summarized in Tables 1 and 2.
Substituents on the phenyl ring of the benzamide portion
dramatically affected the antihelicase activity of the benzobisthiaz-
oles (Table 1). In general, compounds with bulky substituents
(compounds 2–6) were inactive versus B. anthracis helicase, while
smaller substituents, such as F, Cl, Br, CN, CH3, CO2CH3, OCH3, and
OCH2CH3 were tolerated at the 3- or 4-positions (compounds
7–20). Substituents at the 2-position of the phenyl ring were not
tolerated except for the 2-CH3 group (compound 25). Disubstitu-
tion at the 3,4- or 3,5-positions with CH3 or OCH3 groups on the
phenyl ring was tolerated. For example, compounds 29–32 with
substituents 3,4-(CH3)2, 3,4-(OCH2CH2O), 3,4-(OCH3)2, and 3,5-
Compared to the methylthio compound 16, methylamino ana-
log 49 exhibited improved biological activity versus both Ba and
Sa DNA helicases, and its cytotoxicity (CC50) was greater than
100 lM. Increasing the size of the alkylamino group revealed that
(OCH3)2 displayed 1.7–3.2 lM IC50 values versus B. anthracis heli-
case, while compounds with disubstitution at the 2,4- or 2,6-posi-
tions
(26–28) exhibited weak or no inhibitory activity. Compound 33,
with 3,4,5-(OCH3)3 substitution on the phenyl ring, showed the
best potency with an IC50 value of 0.7 lM in this initial investiga-
propylamino was tolerated; however, isopropylamino, cyclohexyl-
amino and benzylamino compounds (51, 53, 54) showed signifi-
cantly reduced activity, while the isobutylamino compound 52
displayed modest inhibition versus both of the helicases. Interest-
ingly, compounds 55–58, which bear additional polar groups, such
as N(CH3)2, morpholino, or OH groups through a linker attaching to
the aminobenzobisthiazole core, exhibited improved potency with
tion of probing the substitution effect on the antihelicase activity.
Table 1
Bacillus anthracis helicase inhibition by benzobisthiazole compounds 1–33
S
S
N
S
2
3
1
NH
O
4
R
N
Compd
R
IC50 Ba helicase (
lM)
Compd
R
IC50 Ba helicase (lM)
1
2
3
4
5
6
7
8
3-N[-(C@O)CH2CH2(C@O)-]
4-N(CH2CH3)2
4-tBu
4-OPh
4-OiPr
4-SO2N(CH2CH3)2
4-CH3
4-CN
4-OCH2CH3
4-Cl
4-COOCH3
4-N(CH3)2
4-OCH3
5.0
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
3-F
3-NO2
3-Br
2-OPh
2-Cl
2-Br
2-F
2-CH3
2,4-(OCH3)2
2,6-(OCH3)2
2,6-F2
3,4-(CH3)2
3,4-(OCH2CH2O)
3,4-(OCH3)2
3,5-(OCH3)2
3,4,5-(OCH3)3
3.3
2.8
2.3
>100
>100
>100
>100
>100
7.6
11.0
6.9
5.0
3.7
2.4
2.2
1.3
>100
>100
>100
ꢀ50
6.6
>100
>100
64.5
3.2
3.0
2.1
1.7
0.7
9
10
11
12
13
14
15
16
17
4-N[-(C@O)CH2CH2(C@O)-]
3-N(CH3)2
3-OCH3
36.0
12.5
5.3
3-Cl

B. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3481–3486
3483
Table 2
Bacillus anthracis helicase inhibition by benzobisthiazole compounds 34–45
S
N
S
S
N
NH
O
R1
Compd
R1
IC50 Ba helicase (
lM)
Compd
R1
IC50 Ba helicase (
lM)
N
N
34
Cyclopropyl
>100
40
>100
CH3
OCH3
35
36
CH2Ph
CH2CH2Ph
O
47
>100
41
42
2-Furan
2-Thiophene
CH3
102
90
N
37
>100
>100
43
>50
N
O
O
Cl
H3C
O
38
44
>50
Cl
N
N
CH3
H3CO
39
>50
45
2-Pyrazine
28
O
O
S
S
S
N
S
O
S
S
O
a
b
N
N
S
N
S
N
S
N
N
H
NH2
H
R1
R1
N
16 R1 = 3-OCH3
47 R1 = 3-OCH3
48 R1 = 3,4,5-(OCH3)3
46
33 R1 = 3,4,5-(OCH3)3
R2
N
R3
c
S
O
N
S
N
N
H
R1
−
49 63
Scheme 1. Reagents and conditions: (a) benzoyl chlorides, CH2Cl2, yields 31–95%; (b) aq KMnO4, glacial HOAc, dioxane, 73% for 47, and 44% for 48; (c) R2R3NH, DMF, 80–
100 °C, yields 20–83%.
IC50 values ranging between 1.0–2.3
l
M and 1.0–4.2
l
M versus Ba
To determine the mode of inhibition for the benzobisthiazole
scaffold, a dilution series of compound 49 was added to the heli-
case reaction in the presence of varying concentrations of the
two substrates, ATP and oligonucleotide. The IC50 value of com-
and Sa helicases, respectively. This finding suggests that the polar
groups make additional, favorable interactions with the helicase
enzymes.
Similarly, compared to the methylthio compound 33, methyl-
amino analog 59 also displayed improved helicase inhibitory activ-
pound 49 varied less than twofold (2.0–3.8 lM) in response to
changes in the ATP concentration (Fig. 2A), but varied considerably
(>6.5-fold) when oligonucleotide concentrations were altered
(Fig. 2B). These results suggest that compound 49 is noncompeti-
tive with ATP but competitive with the DNA substrate.26 Indeed,
a Dixon plot (Fig. 2C) of the DNA variation results is consistent with
ity (Ba helicase IC50 = 0.2
detectable cytotoxicity (CC50 > 100
(CC50/IC50) were more than 500. Analogs 60–63 all inhibited Ba
and Sa helicases with IC50 values between 0.2 and 0.3 M. Further-
more, compound 63 did not show cytotoxicity (CC50 > 100 M).
lM; Sa helicase IC50 = 0.2
lM) without
lM), and selectivity indices
l
l
competitive inhibition with a Ki value of 2 lM, which is in good
Compounds 64 and 65, in which the amide linker was replaced
with ANHCH2A and AN@CHA linkers, lost activities versus both
Ba and Sa helicases, suggesting that the amide bond is required
for inhibitor binding, probably because of favorable hydrogen
bonding interactions.
agreement with the IC50 value at the lowest oligonucleotide con-
centration tested (3 nM). Similar results were also obtained for
compounds 59 and 63 in kinetic studies (data not shown). These
results indicate that helicase inhibitors with the benzobisthiazole
scaffold act with a different mechanism of action than the
previously reported coumarin-based helicase inhibitors, which

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B. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3481–3486
S
S
N
S
N
OCH3
OCH3
a
S
N
H
N
S
S
N
H3CO
NH2
b
64
S
N
S
46
S
N
N
OCH3
OCH3
H3CO
65
Scheme 2. Reagents and conditions: (a) 3,4,5-trimethoxybenzaldehyde, Na(OAc)3BH, glacial HOAc, DMSO, rt, 48%; (b) 3,4,5-trimethoxybenzaldehyde, toluene, reflux, 67%.
Table 3
Bacillus anthracis and Staphyloccocus aureus helicase inhibition by benzobisthiazole compounds 16, 33 and 49–65
R1
S
O
N
S
N
N
H
R2
M)
Compd
R1
R2
IC50 B. anthracis helicase (
l
IC50 S. aureus helicase (
l
M)
HeLa CC50 in SFM (lM)
16
49
50
51
52
53
54
55
S-CH3
NH-CH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
12.5
0.5
1.1
65.0
13.0
90
6.6
2.2
1.3
80
12.4
>100
<0.08; 15.6
nd
44
nd
nd
NH-propyl
NH-isopropyl
NH-isobutyl
NH-cyclohexyl
NH-Bn
9.0
>100
>100
1.0
75
1.0
NHCH2CH2N(CH3)2
N
O
56
3-OCH3
0.5
2.2
9.6
NH
N
57
3-OCH3
2.3
3.0
>100
O
58
33
59
60
NHCH2CH2OH
S-CH3
NH-CH3
3-OCH3
2.2
0.7
0.2
0.2
4.2
0.4
0.2
0.2
>100
>100
>100
<0.78
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
NHCH2CH2N(CH3)2
N
61
O
3,4,5-(OCH3)3
0.2
0.2
5.7
NH
N
N
62
63
3,4,5-(OCH3)3
0.2
0.2
0.2
0.3
2.8
NHCH2CH2OH
3,4,5-(OCH3)3
>100
O
S
S
H
N
S
O
64
65
>100
>100
>100
>100
nd
nd
N
N
O
O
S
S
S
O
O
N
N
N
are noncompetitive with both ATP and DNA.21 However, neither
the coumarin nor the benzobisthiazole inhibitors have any signifi-
cant effect on the single-strand stimulated ATPase activity of S.
aureus helicase at concentrations over 100-fold higher than their
IC50 values (Fig. 2D).21 X-ray crystallography studies have revealed
two distinct hexameric DnaB helicase structures: one showed a flat
structure without DNA and NTP association, and the other formed
a closed spiral staircase quaternary structure in complex with DNA
and ATP mimic GDP-AIF4, suggesting that the former structure
represents an inactivated form of DnaB helicase, and the latter
one represents an activated form of DnaB helicase. The coumarin
type helicase inhibitors are kinetically noncompetitive with both
DNA and ATP substrates, suggesting that the coumarin-based
inhibitors are likely bound to the inactivated form of the helicase,
while the benzobisthiazole derivatives exhibit competitive kinetics
with the DNA substrate, suggesting that the benzobisthiazole-
based compounds may inhibit the activated form of the bacterial
helicase. Structural information for the inhibitor-DnaB helicase

B. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3481–3486
3485
Figure 2. Mode of inhibition of benzobisthiazole 49. A dilution series of compound 49 was examined in the FRET-based S. aureus helicase assay20 with varying concentrations
of ATP (A) or DNA (B) to determine IC50 values. Data were plotted in GraphPad Prism 5.0 using 4-parameter curve fitting. (C) The results from varying DNA are also shown in a
Dixon plot (1/V vs [I]). (D) Compounds 49 and 59 failed to exhibit significant inhibition of the single-stranded DNA stimulated ATPase activity of S. aureus helicase as detected
by malachite green.21
complex is needed to further elucidate the mechanism of action of
these small molecule helicase inhibitors and provide guidance for
the discovery of novel bacterial helicase inhibitors.
Dentistry of New Jersey (UMDNJ)) and Dr. Esther E. Biswas-Fliss
(UMDNJ and Department of Bioscience Technologies, Jefferson
School of Health Professions, Thomas Jefferson University) for pro-
viding B. anthracis helicase, for radiometric assays of the B. anthra-
cis helicase inhibition potency, and for helpful discussions. We
thank Mr. Steven C. Cardinale at Microbiotix for assistance with
the malachite green-based ATPase assay. This work was supported
by the National Institutes of Health/National Institute of Allergy
and Infectious Diseases (AI064974). The content of this publication
does not necessarily reflect the views or policies of the Department
of Health and Human Services.
In addition to potently inhibiting replicative helicase, many
benzobisthiazole analogs also inhibit the growth of bacterial cells
(data not shown). However, the growth inhibition manifests pri-
marily as a reproducible lengthened lag phase, delaying exponen-
tial growth, and is not sufficient to produce MIC values, possibly
due to poor bacterial cellular permeability and/or short half-life
of the parent compounds in cells. Nonetheless, incorporation of
radiolabeled precursors into DNA in permeabilized preparations
of B. anthracis cells27 was reduced significantly by compounds 62
and 63 with IC50 values of 10 and 17 lM, respectively (Supplemen-
Supplementary data
tary Fig. S1). These results indicate that the benzobisthiazoles inhi-
bit DNA synthesis in bacterial cells, and such inhibition could be
responsible for the modest growth inhibition observed in live cells.
In conclusion, we have identified a novel class of potent and
selective benzobisthiazole-based bacterial DnaB helicase inhibitors
through high throughput screening. Subsequent structure-activity
relationship analysis and chemical optimization led to substantial
improvement of biological activity and identification of nanomolar
compounds with selectivity indices of more than 500. To the best
of our knowledge, these compounds are the most potent bacterial
replicative helicase inhibitors reported to date. Further optimiza-
tion of the benzobisthiazole-based helicase inhibitors may provide
a novel small molecule drug for antibacterial therapy.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
04.055.
References and notes
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23. Synthetic procedures for preparation of compounds 47 and 48. Compound 16
(3.0 g, 10.0 mmol) was suspended in a mixture of glacial acetic acid (100 mL)
and dioxane (40 mL). A solution of KMnO4 (6.0 g, 38.0 mmol) in water (15 mL)
was added dropwise while stirring. After stirring at 55 °C for 24 h the mixture
was poured into a solution of NaHSO3 (15 g in 250 mL) and stirred for 15 min.
The resulting precipitate was collected by filtration, washed with water and
dried at 55 °C under vacuum overnight to give the desired product. The crude
product was refluxed with MeOH (250 mL) for 1 h and filtered while hot. The
precipitate was washed with MeOH and dried at 55 °C overnight under
vacuum to give compound 47 (3.15 g, yield 73%), mp = 284–285 °C, Rf = 0.35
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J = 8.7 Hz, 1H), 8.06 (d, J = 9.0 Hz, 1H), 7.73–7.72 (m, 2H), 7.48 (t, J = 8.4 Hz, 1H),
7.23 (dd, J = 2.1, 8.1 Hz, 1H), 3.87 (s, 3H), 3.65 (s, 3H). LC/MS m/z 420 (M+1)+.
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3.91 (s, 6H), 3.77 (s, 3H), 3.63 (s, 3H). LC/MS m/z 479.8 (M+1)+.
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22. Synthetic procedures for preparation of compounds 16 and 33. Compound 46
(10 g, 39.5 mmol) was suspended in pyridine (200 mL) and cooled to 0 °C in an
ice bath. Diisopropylethylamine (11.18 g, 86 mmol) was added, followed by
dropwise addition of 3-methoxybenzoyl chloride (6.8 g, 39.9 mmol). After
stirring at room temperature for 3 days the solvent was evaporated. The crude
product was sonicated with ethyl acetate (200 mL) and water (100 mL) for
10 min and filtered. The precipitate was stirred in chloroform (400 mL) at room
temperature and filtered. The filtrate was washed thrice with water, brine,
dried over sodium sulphate and evaporated to give a white solid 16 (9.4 g, yield
62%), mp = 237–238 °C, Rf = 0.71 (2:98::MeOH:CH2Cl2); 1H NMR (300 MHz,
DMSO-d6) d 13.08 (s, 1H), 7.93 (d, J = 8.7 Hz, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.73
(d, J = 2.7 Hz, 2H), 7.48 (t, J = 8.1 Hz, 1H), 7.23 (d, J = 8.1 Hz, 1H), 3.87 (s, 3H),
2.84 (s, 3H). LC/MS m/z 388.3 (M+1)+. Compound 33 was synthesized using the
same procedure. Yield 96%, mp = 231–232 °C, Rf = 0.76 (5:95::MeOH:CH2Cl2);
1H NMR (300 MHz, DMSO-d6) d 13.05 (s, 1H), 7.94 (d, J = 8.7 Hz, 1H), 7.85 (d,
J = 8.4 Hz, 1H), 7.53 (s, 2H), 3.90 (s, 6H), 3.77 (s, 3H), 2.84 (s, 3H).
24. General synthetic procedures for preparation of compounds 49–63. Compounds
47 and 48 (6.3 mmol) was treated with 20 equiv of corresponding amine in a
sealed tube at 70 °C for 24 h. Then the reaction mixture was evaporated to
dryness and dried at 50 °C under vacuum overnight. The resulting crude
mixture was purified by high-pressure liquid chromatography to obtain
compounds 49–63. Characterization data for compound 49: yield 62%,
mp = 301–302 °C, Rf = 0.1 (1:1::EtOAc:Hexane); 1H NMR (300 MHz, DMSO-d6)
d 13.17 (s, 1H), 8.73 (s, 1H), 7.74 (m, 3H), 7.56 (d, J = 9.0 Hz, 1H), 7.48 (t,
J = 9.0 Hz, 1H), 7.23 (dd, J = 8.1 Hz, 1H), 3.88 (s, 3H), 2.52 (s, 3H). LC/MS m/z
371.2 (M+1)+. Characterization data for compound 59: yield 38%, mp = 238–
239 °C, Rf = 0.87 (80:18:2::CHCl3:CH3OH:CH3NH2); 1H NMR (300 MHz, DMSO-
d6) d 8.24 (br, 1H), 7.67 (d, J = 9.0 Hz, 1H), 7.54 (s, 1H), 7.51 (d, J = 0.9 Hz, 2H),
3.88 (s, 6H), 3.74 (s, 3H), 2.97 (s, 3H). LC/MS m/z 431.1 (M+1)+.
25. Characterization data for compounds 64 and 65. For compound 64: yield 48%,
mp = 168–169 °C, Rf = 0.38 (50:50::EtOAc:n-Hexanes), 1H NMR (300 MHz,
DMSO-d6) d 8.67 (s, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.51 (d, J = 8.7 Hz, 1H), 6.73
(s, 2H), 4.54 (d, J = 4.8 Hz, 2H), 3.75 (s, 6H), 3.62 (s, 3H), 2.78 (s, 3H). LC/MS m/z
434.1 (M+1)+. For compound 65: yield 67%, mp = 207–208 °C, Rf = 0.68
(5:95::MeOH:CH2Cl2); 1H NMR (300 MHz, DMSO-d6) d 9.13 (s, 1H), 7.99 (s,
2H), 7.46 (s, 2H), 3.89 (s, 6H), 3.79 (s, 3H), 2.85 (s, 3H), LC/MS m/z 431.6 (M+1)+.
26. Wei, M.; Wynn, R.; Hollis, G.; Liao, B.; Margulis, A.; Reid, B. G.; Klabe, R.; Liu, P.
C.; Becker-Pasha, M.; Rupar, M.; Burn, T. C.; McCall, D. E.; Li, Y. J. Biomol. Screen.
2007, 12, 220.
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