Design and synthesis of pyridine-substituted itraconazole analogues with improved antifungal activities, water solubility and bioavailability

Article abstract of DOI:10.1016/j.bmcl.2011.06.062

To improve antifungal activities, water solubility and bioavailability, a series of novel analogues of itraconazole-containing pyridine rings were designed and synthesized. Their antifungal activities were evaluated in vitro against six clinically important fungi by measuring the minimal inhibitory concentrations (MICs). Most of the compounds showed more potent antifungal activities than that of itraconazole. In particular, the analogues 30d, 30c, 31c, and 36d exhibited much higher solubility and bioavailability than that of itraconazole. The bioavailability of 36d (42.2%) was five times higher than that of itraconazole (8%) and was negative for genetic toxicology in the Ames test.

Full text of DOI:10.1016/j.bmcl.2011.06.062

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4779–4783
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of pyridine-substituted itraconazole analogues
with improved antifungal activities, water solubility and bioavailability
⇑
Yu Liu , Zining Liu , Xufeng Cao, Xin Liu, Huili He, Yushe Yang
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
To improve antifungal activities, water solubility and bioavailability, a series of novel analogues of itrac-
onazole-containing pyridine rings were designed and synthesized. Their antifungal activities were eval-
uated in vitro against six clinically important fungi by measuring the minimal inhibitory concentrations
(MICs). Most of the compounds showed more potent antifungal activities than that of itraconazole. In
particular, the analogues 30d, 30c, 31c, and 36d exhibited much higher solubility and bioavailability than
that of itraconazole. The bioavailability of 36d (42.2%) was five times higher than that of itraconazole (8%)
and was negative for genetic toxicology in the Ames test.
Received 15 April 2011
Revised 13 June 2011
Accepted 14 June 2011
Available online 2 July 2011
Keywords:
Itraconazole analogues
Synthesis
Ó 2011 Published by Elsevier Ltd.
Antifungal
Bioavailability
Invasive fungal infections have increased during the past two
decades as a consequence of the rising number of immunocompro-
mised hosts, such as HIV-infected individuals, transplant recipients
and patients given immunosuppressive agents or broad-spectrum
antibiotic therapy.1 For the treatment of these infections, new anti-
fungal triazole drugs, such as itraconazole and posaconazole, have
been developed for clinical use. However, the main shortcomings
of these drugs are their poor water solubility and very low bio-
availability.2 For example, itraconazole injection is solubilized by
hydroxypropyl-b-cyclodextrin as a molecular inclusion complex,
which cannot be administrated to patients with severe renal dys-
function. Posaconazole is only be taken by oral suspension. There
is thus a great medical need for an injectable antifungal agent with
a broad spectrum for the treatment of severe mycoses infections.
To improve the low water solubility and bioavailability of tria-
zole antifungal agents, there have been many efforts to overcome
the problems by using a prodrug approach.3 We focused our efforts
on replacing one of the phenyl rings of itraconazole with a hydro-
philic group to obtain a series of novel analogues of itraconazole
with good water solubility. Because the pyridine ring is a simple
bioisostere for phenyl and has been widely used in drug design,
we reasoned that replacement of the phenyl ring on the itraconaz-
ole side chain with pyridine would produce a new class of anti-
fungal agents with improved antifungal activities, water solubility
(especially under acidic conditions) and bioavailability.
In this Letter, we report our efforts related to the synthesis of a
series of novel analogues of itraconazole containing a pyridine ring
as antifungal agents, as well as evaluation of their antifungal activ-
ity, solubility and pharmacokinetic profiles. This work resulted in
the discovery of 30d, 30c, 31c, and 36d, all of which exhibited
much higher solubility and bioavailability than itraconazole. The
synthetic strategy for these analogues is outlined in Figure 1.4
When R was alkyl, the molecule was disconnected at site a, but
when R was alkyl with a hydroxyl, the molecular was disconnected
at site b.
The preparation of the triazolone with various alkyl side chains
(8a–d) is outlined in Scheme 1.5 Methoxyphenyl piperazine 1 was
reacted with 2-chloro-5-nitropyridine in the presence of Et3N at
room temperature (rt) to give nitropyridine 2 in high yield. The
nitro group was then reduced to an amino group with 10% Pd/C
under H2 atmosphere. Next, aminopyridine 3 was converted to
phenylcarbamate 4 by reaction with phenyl chloroformate in
dichloromethane. Phenylcarbamate 4 was treated with hydrazine
⇑
Corresponding author. Tel./fax: +86 21 50806786.
E-mail address: ysyang@mail.shcnc.ac.cn (Y. Yang).
Figure 1. Disconnections for the synthesis of itraconazole analogues.
Both authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.06.062

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Y. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4779–4783
Scheme 1. Reagents and conditions: (a) 2-chloro-5-nitropyridine, Et3N, CH3CN/MeOH (1:1, v/v), 95.8%; (b) H2, 10% Pd/C, THF, rt, 98%; (c) phenyl carbonochloridate, Py,
CH2Cl2, 5 °C, 80%; (d) NH2NH2 H2O, 1,4-dioxane, reflux, 95%; (e) formamidine acetate, DMF, 130 °C, 76%; (f) RBr, KOH, DMSO, rt, 40% ꢀ80%; (g) 48% HBr, reflux, 90–95%.
Scheme 2. Reagents and conditions: (a) NBS, NH4Ac, CH3CN, 0 °C to rt, 93.4%; (b) phenyl carbonochloridate, Py, CH2Cl2, 5 °C, 90%; (c) NH2NH2 H2O, 1,4-dioxane, reflux, 95%;
(d) formamidine acetate, DMF, 130 °C, 50%; (e) RBr, KOH, DMSO, rt; (f) 1-(4-methoxyphenyl)piperazine, Pd2(dba)3, t-BuONa, rac-BINAP, Tol, 50 °C, 50%-80%; (g) 48% HBr,
reflux, 90–95%.
Scheme 3. Reagents and conditions: (a) Et3N, CH3CN/MeOH (1/1, v/v), rt, 85%; (b) Boc2O, Na2CO3, 98%; (c) H2, 10% Pd/C, THF, rt, 95%; (d) phenyl carbonochloridate, Py, CH2Cl2,
5 °C, 93%; (e) NH2NH2.H2O, 1,4-dioxane, reflux, 96%; (f) formamidine acetate, DMF, 130 °C, 55%; (g) ROBs, Cs2CO3, DMF, reflux, 40–80%; (h) TFA, 95–98%.
hydrate to give semicarbazide 5, which was subsequently reacted
with formamidine acetate in DMF at 130 °C to give the triazolone
6. Finally, triazolone 6 was treated with bromoalkane in the
presence of KOH and DMF at rt to afford 7a–d. The methyl was
subsequently removed in refluxing 48% HBr (aq) to obtain 8a–d.
The synthesis of compounds 16a–d is shown in Scheme 2. Com-
pound 10, obtained by bromination of compound 9 in the presence
of NBS and NH4Ac, was converted to triazolone 13 via the similar
chemical procedure described in Scheme 1. Compounds 14a–d
were then coupled with p-methoxyphenyl piperazine by a Buch-
wald-Hartwig process to afford 15a–d. Next, the methyl ether
was removed in refluxing 48% HBr (aq) to get 16a–d.
Considering that the benzyl would be destroyed easily and the
hydroxyl could be brominated in the process of methyl ether re-
moval, the new synthesis route of compounds 25a–e, with a hydro-
xyl substituted alkyl as a side chain, is outlined in Scheme 3. The
commercially available 2-chloro-5-nitropyridine 17 was reacted
with 2 eq. of piperazine to afford the mono-N-arylated product

Y. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4779–4783
4781
Scheme 4. Reagents and conditions: (a) ROBs, Cs2CO3, DMF, reflux; (b) tert-butyl piperazine-1-carboxylate, Pd2(dba)3, t-BuONa, rac-BINAP, Tol, 50 °C; (c) TFA.
Scheme 5. Reagents and conditions: (a) KOH, DMF, 50 °C; (b) 4-bromophenol, KOH, DMF, 50 °C; (c) Pd2(dba)3, t-BuONa, rac-BINAP, Tol; (d) HCOOH, 10% Pd/C.
18, which was subsequently reacted with Boc2O to yield compound
19. Hydrogenation reduction of 19 provided amine 20, which was
subsequently converted to triazolone 23 by the similar three-step
sequence depicted in Scheme 1. Lastly, compound 23 was alkylated
with brosylate and then removed Boc protective group to afford
25a–e.

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Y. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4779–4783
Table 1
In vitro antifungal activities of triazole derivativesa
Compound
MIC (lg/mL)
C. albicans (2)
C. parapsilosis (2)
C. krusei (2)
C. neoformans (2)
A. fumigatus (2)
A. flavus (2)
30a
30b
30c
30d
35a
35b
35c
35d
35e
31a
31b
31c
31d
36a
36b
36c
36d
<0.0038–0.015
<0.0038–0.003
0.0075–0.015
0.03
0.0625–0.25
0.0625–0.25
<0.0625
<0.0625
<0.0625
0.03
0.015–0.03
0.0075–0.015
0.0625–0.25
0.0625–1
<0.0156–0.03
<0.0156–0.0156
0.0156
0.25–1
0.125–1
0.5–1
0.0625–0.25
0.0625–0.5
0.25–1
0.125–2
2–>32
2–4
0.0625–1
0.0625–1
0.125–0.5
0.0625–0.25
0.125
0.0625–0.25
0.03–0.25
<0.0156
0.5
0.0625–0.125
0.125–0.25
0.25
0.25
0.125–0.25
>32
1–8
4
1–2
0.5
0.125–0.25
0.125–0.25
0.5
1–2
0.5
0.25–1
0.25–1
0.25–0.5
0.125–0.25
0.125–0.5
0.5–1
0.25–0.5
0.25–0.5
0.5–1
0.125–1
0.0625–0.125
0.125–0.25
0.5–>32
0.25–2
4–16
0.25–1
0.5
0.0625–0.25
0.125–0.5
0.0625
0.125–0.25
0.03–0.25
0.125–0.5
<0.0156
0.125–1
2–8
0.0625–0.25
<0.0625–0.125
0.0625–0.125
<0.0625–0.0625
1
<0.0625–0.125
<0.0625–0.25
0.0625–1
0.5–1
0.25–2
0.25–1
0.5–2
0.5–1
0.0156–0.25
<0.0156–0.0625
<0.0156–0.03
0.25–0.5
0.25
0.125–32
0.0625–0.25
<0.0156–0.03
0.03–0.0625
<0.0156
0.5–1
0.03
<0.0156–0.0156
ITZb
0.125–0.5
0.25–0.5
0.0625–0.25
1
0.25
0.125
a
The tested fungal species each included 2 strains. Candida albicans: JLC30364, JLC31374; Candida parapsilosis: JLC30365, JLC30275; Candida krusei: JLC30366, JLC30386;
Cryptococcus neoformans: JLC50122, JLC50123; Aspergillus fumigatus: JLC30506, JLC30883; Aspergillus flavus: JLC30784, JLC40437.
b
ITZ: Itraconazole.
The corresponding compounds 28a–d were also obtained from
intermediate 13 (Scheme 4). Briefly, compound 13 was alkylated
with brosylates in the presence of Cs2CO3 to achieve compounds
26a–d, which were coupled with mono-N-Boc protected pipera-
zine by the Buchwald-Hartwig reaction. Finally, removing the
Boc protective group provided the mono-N-arylated piperazines
28a–d.
showed less activity. The hydroxyl compounds 35b and 35c also
showed less activity against A. flavus.
Next, we investigated the N atom position effect of the pyridine
ring on the antifungal activity. Comparing antifungal activities of
compounds with the same R group, we found the activities were
influenced by the N atom position on the pyridine ring, but the
relationship between N atom position and antifungal activity was
not clear. For example, for all 12 tested fungal species, the activity
of 30a was more potent than that of 31a, but 30c was less potent
than 31c and 30b and 31b were similar.
Compounds 30d, 30c, 31c, and 36d, which have more potent
antifungal activities, were chosen to test water solubility at differ-
ent pH values. As shown in Table 2, the water solubility of tested
compounds was sufficiently improved over itraconazole, especially
in acidic conditions. These results indicated that these compounds
might have good solubility in the human stomach and so their
absorption in the human might be improved. The introduction of
the pyridine ring may be the reason for this improved water
solubility.
The absorption of selected compounds after a single dose was
studied in rats and the results are given in Table 3. As can be
seen, all compounds showed much higher plasma levels (Cmax
and AUC0–t) than that of itraconazole. The fact that the pyridine
ring and the hydroxyl group incorporated in the analogues both
have higher hydrophilic properties may be the reason for these
results. Furthermore, the bioavailability was greatly improved
than that of itraconazole in all of the studied compounds. These
combined results confirmed our expectations that the improve-
The target compounds 30a–d6 and 31a–d7 were synthesized
via a nucleophilic substitution of 29 and 8a–d or 16a–d (Scheme
5). Compounds 33a–e and 34a–d, with hydroxyl substituted alkyl
side chains, were synthesized via Pd-catalyzed C–N coupling reac-
tions of 32 and mono-N-arylated piperazines 25a–e and 28a–d.
The benzyl ethers of 33a–e and 34a–d were removed with formic
acid and 10% Pd/C to yield the desired target compounds 35a–e
and 36a–d (Scheme 5).8
Minimum inhibitory concentrations (MICs) of the target com-
pounds were determined against four yeast (Candida albicans, Can-
dida parapsilosis, Candida krusei, and Cryptococcus neoformans) and
two mold species (Aspergillus fumigatus, Aspergillus flavus) by the
broth microdilution method in accordance with the guidelines in
the National Committee for Clinical Laboratory Standards (NCCLS)
documents.9 Each fungal species included two strains, and itraco-
nazole was used as the positive control. The minimum inhibitory
concentration values are summarized in Table 1.
From the biological data, it was clear that nearly all the com-
pounds showed excellent antifungal activities against the six fun-
gal species tested. For C. albicans, most of the tested compounds
showed a high level of activity with MIC values 2- to 45-fold lower
than that of itraconazole, except for compound 36a, which showed
slight weaker activity against one strain. The activity of com-
pounds 30b, 30c and 30d was higher or comparable to that of itr-
aconazole against C. parapsilosis with MIC values between
Table 2
Water solubility at different pH values
0.125 lg/mL and 0.5 lg/mL. The compounds 35a–b, bearing a hy-
Compound
Solubility in water (mg/mL)
pH 7.4
droxyl group on the side chain, have lower activity against C. par-
apsilosis. In addition, the activity of 30c, 30d and 36a was
comparable to itraconazole against C. Krusei. Furthermore, most
of the compounds showed higher activity than itraconazole against
C. neoformans. For mold species, the activity of compounds 30a–d
and 31b–c was higher or comparable to that of itraconazole against
A. fumigatus, whereas the hydroxyl compounds 35b–e and 36a
pH 1.2
30d
30c
31c
36d
ITZ
2.33
1.68
0.32
0.19
0.016
BDL
BDL
0.00025
BDL
0.0018
BDL: below the detection limit.

Y. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4779–4783
4783
Table 3
Pharmacokinetic parameters of compound absorption after a single dose
Compound
Dose (mg/kg)
Parameter
AUC0–t (ng h/mL)
Cmax (ng/mL)
Tmax (h)
t
(h)
CL (L/h/kg)
F (%)
½
ITZ
ITZ
po 10
iv 10
po 10
iv 5
po 10
iv 10
po 10
po 10
iv 2.5
6.28 4.90
3.00 1.41
27.5 21.9
343 22
2.57 0.86
22.0 15.7
2.18 0.56
1.43 0.31
1.26 0.44
4.18 1.34
3.45 1.18
3.41 1.34
2.30 0.30
8.0
21.7
35
19.3 9.5
1.41 0.45
1.27 0.11
30d
30d
30c
30c
31c
36d
36d
392 151
712 108
2.0
1606 389
3707 912
2706 477
7733 536
1221 303
1897 422
1123 279
1.88 1.55
159 17
297 70
3.0 1.4
3.0 0.8
42.2
2.20 0.63
Cmax = maximal concentration of drug; Tmax = time after dosing for maximal concentration to be reached; AUC = area under the concentration curve; t = half-life;
½
CL = clearance; and F = bioavailability.
J = 15.5 Hz), 121.1, 118.5, 115.2, 111.2 (d, J = 20.5 Hz), 107.0, 106.7, 105.1 (t,
ment of water solubility through the incorporation of a pyridine
ring could elevate oral bioavailability.
J = 25.5 Hz), 74.9, 67.5 (2C), 54.4, 50.4 (2C), 47.1, 45.3 (2C), 21.1 (2C). MS (ESI):
m/z 660.4 (M+1), 682.4 (M+Na). HRMS (EI): m/z C33H35F2O4N9 [M]+ 659.2780.
Compound 30d: 1H NMR (CDCl3): d 8.24 (d, J = 3.0 Hz, 1H), 8.21 (s, 1H), 7.87 (s,
1H), 7.71 (dd, J1 = 9.0 Hz, J2 = 2.7 Hz, 1H), 7.58 (s, 1H), 7.52–7.44 (m, 1H), 6.94–
6.74 (m, 7H), 4.70–4.69 (m, 2H), 4.42–4.38 (m, 1H), 3.99–3.94 (m, 1H), 3.83–
3.76 (m, 2H), 3.73 (t, J = 5.1 Hz, 4H), 3.65 (d, J = 7.5 Hz, 2H), 3.50–3.45 (m, 1H),
3.17 (t, J = 5.1 Hz, 4H), 2.19 (m, J = 6.6 Hz, 1H), 0.97 (d, J = 6.6 Hz, 6H). 13C NMR
Compound 36d has the highest bioavailability, it was selected
for further evaluation. Compound 36d was negative in the Ames
microbial mutagenesis assay (P >0.05).10 The acute toxicity of
36d was also assessed using a LD50 test in ICR mice weighing
18–22 g. Doses were given orally and mice were kept under obser-
vation for 10 days. The LD50 of 36d was 277.59 mg/kg, which was
considered low toxicity.11
In summary, a novel series of antifungal agents containing a
pyridine ring have been designed and synthesized. The antifungal
activity, water solubility and pharmacokinetic profiles of these
new compounds were greatly improved than those of itraconazole.
In particular, 36d showed more potent antifungal activity, better
solubility in water, higher bioavailability, lower LD50 and negative
genetic toxicity, suggesting a high potential for further develop-
ment as a novel triazole antifungal agent. Further evaluation is
necessary to determine the antifungal activity of 36d in vivo.
(CDCl3):
d 163.7 (dd, J1 = 250 Hz, J2 = 11.4 Hz), 160.4 (dd, J1 = 250 Hz,
J2 = 11.9 Hz), 158.3, 152.6, 152.4, 151.3, 146.0, 144.8, 142.3, 133.6, 132.9,
129.2, 121.7 (d, J = 11.8 Hz), 121.2, 118.5 (2C), 115.2 (2C), 111.2 (d, J = 21.0 Hz),
107.0, 106.7, 105.2 (t, J = 25.5 Hz), 74.9, 67.5 (2C), 54.4, 52.9, 50.4 (2C), 45.3
(2C), 28.2, 19.8 (2C). MS (ESI): m/z 674.3 (M+1), 696.3 (M+Na). HRMS (EI): m/z
C
34H37F2O4N9 [M]+ 673.2934.
7. Compound 31c: 1H NMR (CDCl3): d 8.31 (s, 1H), 8.20 (s, 1H), 8.17 (s, 1H), 8.09 (d,
J = 2.7 Hz, 1H), 7.89 (s, 1H), 7.53–7.45 (m, 1H), 7.41–7.37 (dd, J1 = 9.0 Hz,
J2 = 3.0 Hz, 1H), 6.98–6.79 (m, 6H), 4.75–4.70 (m, 2H), 4.55 (m, J = 6.8 Hz, 1H),
4.41 (m, J = 5.3 Hz, 1H), 4.00–3.95 (m, 1H), 3.83–3.75 (m, 2H), 3.50–3.45 (m,
1H), 3.38–3.37 (m, 4H), 3.27–3.25 (m, 4H), 1.40 (d, J = 6.9 Hz, 6H). 13C NMR
(CDCl3):
d 163.6 (dd, J1 = 250 Hz, J2=11.4 Hz), 160.3 (dd, J1 = 251 Hz,
J2 = 12.3 Hz), 152.6, 151.2, 150.5, 145.8, 139.9, 136.0, 132.3, 129.1, 125.4,
121.6 (d, J = 13.2 Hz), 118.4 (2C), 115.1 (2C), 113.6, 111.2 (d, J = 20.1 Hz), 106.7,
105.1(t, J = 25 Hz), 74.8, 67.5 (2C), 54.3, 50.4 (2C), 48.8 (2C), 46.7, 21.0 (2C). MS
(ESI): m/z 660.4 (M+1), 682.4 (M+Na).
8. Compound 36d: The mixture of compound 32 (0.25 g, 0.55 mmol), 28d (0.23 g,
0.55 mmol), Pd2(dba)3 (40 mg, 0.055 mmol), t-BuONa (80 mg, 0.083 mmol) and
rac-BINAP (0.1 g, 0.17 mmol) in 30 mL dry toluene was stirred overnight in
80 °C under Ar atmosphere. After cooling to rt, the reaction mixture was
poured into water (50 mL) and the resulting solid was filtered off. The aqueous
solution was extracted with DCM (30 mL Â 3), the organic layers were
combined and dried over Na2SO4. The product 34d was obtained by column
chromatograph (PE: EA = 1:8) as a white solid (0.30 g, 67%). Compound 34d
was dissolved in 15 mL acetic acid in the presence of 30 mg 10% Pd/C, the
mixture was heated at 50 °C under Ar. for 2 h. Pd/C was removed by filtration
and the solvent was evaporated to dryness, the product 36d was obtained by
pre-TLC (DCM: MeOH = 15:1) as a white solid (0.2 g, 75%). 1H NMR (CDCl3): d
8.35 (s, 1H), 8.22 (s, 1H), 8.16–8.11 (m, 2H), 7.89 (s, 1H), 7.54–7.39 (m, 2H),
6.95–6.79 (m, 6H), 4.71 (s, 1H), 4.41 (m, 1H), 4.01–3.98 (m, 2H), 3.81–3.77 (m,
3H), 3.48–3.46 (m, 1H), 3.40–3.38 (m, 4H), 3.26 (m, 6H), 0.99 (s, 6H). 13C NMR
Acknowledgments
The project described was supported by Key New Drug Creation
and Manufacturing Program, China (Number: 2009ZX09301-001).
References and notes
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2. Girmenia, C. Expert Opin. Invest. Drugs. 2009, 18, 1279.
(CDCl3):
d 163.6 (dd, J1 = 244 Hz, J2 = 11.8 Hz), 160.4 (dd, J1 = 251 Hz,
J2 = 11.8 Hz), 152.7, 152.4, 151.3, 146.0, 145.7, 144.9, 139.5, 136.0, 132.7,
129.2, 125.4, 121.6 (d, J = 10.0 Hz), 118.6 (2C), 115.2 (2C), 113.7, 111.2 (d,
J = 20.5 Hz), 106.7, 105.2 (t, J = 24.7 Hz), 74.9, 68.1, 67.5 (2C), 54.4, 52.1, 50.5
(2C), 48.8 (2C), 37.7, 22.6 (2C). MS (ESI): m/z 704.4 (M+1), 726.4 (M+Na). HRMS
(ESI): m/z calcd for C35H39N9O5F2Na [M+Na]+ 726.2940, found 726.2935.
9. (a) National Committee for Clinical Laboratory Standards. Reference Method for
Broth Dilution Antifungal Susceptibility Testing of Yeast, Approved Standard.
Document M27-A; National Committee for Clinical Laboratory Standards:
Wayne, PA, USA, 1997; (b) National Committee for Clinical Laboratory
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of Conidium-forming Filamentous Fungi: Proposed Standard. Document M38-P;
National Committee for Clinical Laboratory Standards: Wayne, PA, USA, 1998.
10. The mutagenesis assays were conducted using a plate incorporation protocol.
The mixture containing bacterial cells (109) in 1.8 mL culture medium was
treated with 0.1 mL of test compounds in dimethyl sulfoxide without S9 mix
and poured in triplicate onto plates containing hardened agar. The plates were
incubated at 37 °C for 48 h prior to counting colonies. The number of revertant
colonies was given as mean standard deviation.
3. Ueda, Y.; Matiskella, J. D.; Golik, J.; Connolly, T. P.; Hudyma, T. W.; Venkatesh,
S.; Dali, M.; Kang, S.-H.; Barbour, N.; Tejwani, R.; Varia, S.; Knipe, J.; Zheng, M.;
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6. Compound 30c: NaH (93 mg, 2.33 mmol, 40% in oil) was add to the solution of
Compound 8c (0.65 g, 1.44 mmol) in dry 30 mL DMF at 0 °C, the mixture was
stirred for 30 min, then Compound 29 (0.65 g, 1.44 mmol) in 20 mL DMF was
added to the solution. After the addition, the reaction was heated to 50 °C and
kept the temperature for 2 h. The reaction was cooled to rt and poured into ice-
water (50 mL), the aqueous solution was extracted with ethyl acetate
(30 mL Â 3), the organic layers were combined and dried over Na2SO4. The
product 30c was obtained by column chromatograph (DCM:MeOH = 100: 1–
50:1) as a white solid (0.45 g, 79%). 1H NMR (CDCl3): d 8.25 (d, J = 2.7 Hz, 1H),
8.21 (s, 1H), 7.88 (s, 1H), 7.71 (dd, J1 = 9.0 Hz, J2 = 2.7 Hz, 1H), 7.57 (d,
J = 0.6 Hz, 1H), 7.53–7.45 (m, 1H), 6.96–6.75 (m, 7H), 4.70 (m, 2H), 4.54 (m,
J = 6.9 Hz, 1H), 4.41 (m, J = 4.8 Hz, 1H), 4.00–3.95 (m, 1H), 3.82–3.75 (m, 6H),
3.52–3.46 (m, 1H), 3.22–3.17 (m, 4H), 1.41 (d, J = 6.6 Hz, 6H). 13C NMR (CDCl3):
d 163.6 (dd, J1 = 250 Hz, J2 = 11.8 Hz), 160.3 (dd, J1 = 250 Hz, J2 = 11.8 Hz),
158.3, 152.6, 151.5, 151.3, 146.0, 144.8, 142.4, 133.7, 133.0, 129.2, 121.6 (d,
11. ICR mice were randomly divided into eight groups (10 mice/group and female:
male = 1: 1) and fasting the night before the test. 36d was given 100, 144, 207,
299, 430, or 619 mg/kg on day 0 orally. Continuous observation for 10 days to
record the number of deaths and investigate the toxicity of drugs. LD50 was
calculated using a modified Kaber method.