Identification of Thiazoyl Guanidine Derivatives as Novel Antifungal Agents Inhibiting Ergosterol Biosynthesis for Treatment of Invasive Fungal Infections

Article abstract of DOI:10.1021/acs.jmedchem.1c00883

Invasive fungal infections (IFIs) are fatal infections, but treatment options are limited. The clinical efficacies of existing drugs are unsatisfactory because of side effects, drug-drug interaction, unfavorable pharmacokinetic profiles, and emerging drug-resistant fungi. Therefore, the development of antifungal drugs with a new mechanism is an urgent issue. Herein, we report novel aryl guanidine antifungal agents, which inhibit a novel target enzyme in the ergosterol biosynthesis pathway. Structure-activity relationship development and property optimization by reducing lipophilicity led to the discovery of 6h , which showed potent antifungal activity againstAspergillus fumigatusin the presence of serum, improved metabolic stability, and PK properties. In the murine systemicA. fumigatusinfection model, 6h exhibited antifungal efficacy equivalent to voriconazole ( 1e ). Furthermore, owing to the inhibition of a novel target in the ergosterol biosynthesis pathway, 6h showed antifungal activity against azole-resistantA. fumigatus.

Full text of DOI:10.1021/acs.jmedchem.1c00883

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Article
Identification of Thiazoyl Guanidine Derivatives as Novel Antifungal
Agents Inhibiting Ergosterol Biosynthesis for Treatment of Invasive
Fungal Infections
Issei Kato,* Yuuta Ukai, Noriyasu Kondo, Kohei Nozu, Chiaki Kimura, Kumi Hashimoto, Eri Mizusawa,
Hideki Maki, Akira Naito, and Makoto Kawai
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ABSTRACT: Invasive fungal infections (IFIs) are fatal infections, but treatment options are limited. The clinical efficacies of
existing drugs are unsatisfactory because of side effects, drug−drug interaction, unfavorable pharmacokinetic profiles, and emerging
drug-resistant fungi. Therefore, the development of antifungal drugs with a new mechanism is an urgent issue. Herein, we report
novel aryl guanidine antifungal agents, which inhibit a novel target enzyme in the ergosterol biosynthesis pathway. Structure−activity
relationship development and property optimization by reducing lipophilicity led to the discovery of 6h, which showed potent
antifungal activity against Aspergillus fumigatus in the presence of serum, improved metabolic stability, and PK properties. In the
murine systemic A. fumigatus infection model, 6h exhibited antifungal efficacy equivalent to voriconazole (1e). Furthermore, owing
to the inhibition of a novel target in the ergosterol biosynthesis pathway, 6h showed antifungal activity against azole-resistant A.
fumigatus.
(VRCZ, 1e), isavuconazole (ISCZ, 1f), itraconazole (ITCZ,
1g), and posaconazole (PSCZ, 1h)) have fungicidal activity
against Aspergillus but pose drug−drug interaction risks due to
inhibitory activities against human CYPs.13 In this decade,
antifungal resistance to echinocandins and azoles has been
recognized as a serious medical problem.14−17 Thus, there is a
great need to develop a new antifungal drug that is safe, has no
drug interaction concerns, and functions with a new
mechanism of action different from that of existing drugs.18
Abafungin (ABFG, 2), first reported by Bayer in 2008, was
tested in clinical trials for the treatment of dermatomycosis and
showed strong antifungal activity against not only the
Trichophyton species but also Aspergillus, Candida, and
Cryptococcus species that cause IFIs.19 In the case of C.
1. INTRODUCTION
Invasive fungal infections (IFIs), mainly caused by Aspergillus
species (e.g., Aspergillus fumigatus, Aspergillus flavus and
Aspergillus terreus), Candida species (e.g., Candida albicans,
Cornus glabrata, Candida krusei, and Candida parapsilosis), and
Cryptococcus species (e.g., Cryptococcus neoformans and C.
gattii) are life-threatening infections.1−5 It is estimated that
IFIs are responsible for approximately 1.5−2 million deaths
every year.6,7 IFIs are particularly problematic in immunocom-
promised patients such as those who have received organ
transplants, patients with acquired immune deficiency
syndrome (AIDS), and cancer patients receiving immunosup-
pressants.8 Currently, only three structurally distinct classes of
antifungal drugs are clinically available to treat IFIs9,10 (Figure
1). Polyenes (amphotericin B (1a) and lipid formulations)
display strong fungicidal activity against a wide range of
pathogenic fungi but have moderate to severe nephrotoxicity.11
Echinocandins (micafungin (1b), caspofungin (1c)) have
fungicidal activities against Candida and higher safety profiles
compared to other antifungals but do not have fungicidal
activities against Aspergillus, the most frequent pathogen in
fungal pneumoniae.12 Azoles (fluconazole (1d), voriconazole
Received: May 17, 2021
Published: June 30, 2021
https://doi.org/10.1021/acs.jmedchem.1c00883
© 2021 American Chemical Society
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Figure 1. Chemical structures of antifungal agents.
albicans, ABFG (2) is thought to inhibit sterol 24-C-
methyltransferase (SMT), which is a fungi-specific enzyme in
the ergosterol biosynthesis pathway.19 We decided to start
from ABFG (2) to explore for a novel class of antifungal agents
that exert antifungal activity against antifungal-resistant fungi
but have no drug interaction and toxicity concerns unlike
existing azole drugs. However, considering the repositioning of
ABFG (2) as a drug for deep-seated mycosis raises concerns
about some of its undesirable properties (Figure 2): (i) a very
high PPB limits the partitioning of drugs from the blood into
the tissues where they exert activity. Also, increasing the
lipophilicity of a compound enhances the affinity of metabolic
enzymes, particularly to the cytochrome P450 family, leading
to a decrease in metabolic stability. Consideration of these
factors led us to focus our drug design toward reducing
lipophilicity to overcome the high PPB and low metabolic
stability of ABFG (2). Since, to the best of our knowledge,
there was no information about structure−activity relation-
ships (SARs) of the derivatives of ABFG (2), we had to find
which part of the structure of ABFG (2) could be changed but
allow it to retain its antifungal activity. Initially, we focused on
the characteristic six-membered ring guanidine in the structure
of ABFG (2) and verified the tolerability of replacing this
moiety with simple amines or bioisosteres of guanidine (Table
1). However, amines 3a,b, 2-aminopyridine 3c, and 2-
aminopyrimidine 3d, which are known as bioisosteres of
guanidine,20 displayed significant loss of antifungal activity.
Since the basicity of these compounds is lower than that of the
six-membered guanidine of ABFG (2), we thought that the
antifungal activity would diminish as the basicity decreased.
Therefore, we next tested guanidine derivatives (3e−h) that
maintained their basicity. However, dimethylguanidine 3e
showed markedly reduced antifungal activity. On the other
hand, five-membered ring guanidine 3f, seven-membered ring
guanidine 3g, and N-methyl six-membered ring guanidine 3h
showed relatively good retainment of their antifungal activity
against C. albicans, but significant activity loss against A.
fumigatus. These SAR observations indicated that the size of
the six-membered guanidine ring is crucial for antifungal
activity, suggesting that it interacts with the target through a
hydrogen-bond donor (HBD) and might tightly fit into the
lipophilic binding pocket.
Figure 2. Structure of Abafungin (ABFG, 2). A. fumigatus, MIC: 2
(μg/mL); A. fumigatus, MIC in the presence of bovine serum (BS):
>16 (μg/mL); metabolic stability (see Table 3): 64% (human), 0.24%
(rat); fraction of unbound (rat): <0.1%; clearance (rat): 51.4 mL/
(min kg); calculated log P (see Table 3): 5.94.
small fraction unbound in plasma (in rat) can lead to a
significant decrease of antifungal activity in the presence of
bovine serum (BS) and (ii) it is rapidly metabolized in human
and rat liver microsomes and shows a very high clearance in
rat. These undesirable properties are thought to be due to the
high lipophilicity of ABFG (2).
Herein, we report our efforts to identify a new class of
antifungal agent 6h, which exhibited high antifungal activity in
the presence of bovine serum (BS), improved metabolic
stability and a marked in vivo antifungal effect in the A.
fumigatus systemic infection model in mice. Also, 6h showed
antifungal activity against azole-resistant A. fumigatus.
We next examined the tolerability of replacing the thiazole
ring with other heteroaromatic rings while fixing the six-
membered ring guanidine structure, which is crucial for the
activity (Table 2). The 2,5-substituted thiazole 4a was found to
be inactive. This could be explained by the intramolecular
hydrogen bond (IMHB),21 shown in Figure 3. Since the
conformation of ABFG (2) formed by IMHB, which might be
a bioactive conformation, is very different from that of 4a, we
2. RESULTS AND DISCUSSION
2.1. In Vitro SAR. Plasma protein binding (PPB) is known
to be correlated with lipophilicity. In general, as compounds
become more lipophilic, PPB becomes more significant and
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Table 1. SAR of 3a−e and pKa Values
a
b
c
A. fumigatus ATCC 204305 minimum inhibitory concentration (MIC). C. albicans ATCC 90028 MIC. pKa of the conjugate acid of guanidine
group for 2, 3e−h, thiazole group for 3a,b, pyridine group for 3c, and pyrimidine group for 3d, calculated as the strongest base by ACD Percepta
2020.1.17 (GALAS).
presumed that this was the reason for the loss of antifungal
activity of 4a. Based on the above “IMHB hypothesis”, we
designed thiadiazole 4b, which has an IMHB conformation
similar to that of ABFG (2). However, thiadiazole 4b also
showed no antifungal activity, indicating that the 5-position of
the thiazole ring should be lipophilic for antifungal activity.
Next, we designed pyridine 4c, which has an IMHB
conformation similar to that of ABFG (2) and shows lipophilic
properties at the 4−5 positions in its pyridine ring. As
expected, pyridine 4c retained its antifungal activity. However,
as the antifungal activity of 4c was inferior to that of ABFG
(2), we concluded that the combination of a six-membered
ring guanidine and a 2,4-substituted thiazole ring is important
for antifungal activity.
Since it is clear that the 2,4-dimethyl-1-phenoxybenzene
(DMPB) group is responsible for the high lipophilicity of
ABFG (2), we searched for a less lipophilic structure other
than the DMPB group to overcome high PPB and low
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Table 2. SAR of 4a−c
a
b
A. fumigatus ATCC 204305 MIC. C. albicans ATCC 90028 MIC.
Figure 3. Conformation formed by IMHB of ABFG (2) and that of compounds 4a−c.
metabolic stability while retaining antifungal activity (Table 3).
Early SAR revealed that biphenyl compound 5a exhibited
antifungal activity similar to ABFG (2). We tried to convert the
biphenyl group with a phenyl heteroaromatic group to
diminish lipophilicity. Since thiophene is known as a
bioisostere of benzene,22 we designed the thiophene analogue
5b, which showed acceptable antifungal activity against both A.
fumigatus and C. albicans with decreasing lipophilicity. Next,
we tried further reduction of lipophilicity from compound 5b.
To our delight, thiazole analogue 5c showed stronger
antifungal activity than ABFG (2) with increased polarity. In
addition, the metabolic stability of 5c was significantly
improved compared to ABFG (2) presumably due to the
lower lipophilicity. However, pyrazole analogue 5d and oxazole
analogue 5e, with lower lipophilicity than 5c, showed
diminished antifungal activity. Hence, we selected compound
5c as a lead compound for further optimization.
We tried further improvement of antifungal activity in the
presence of BS, which is an important parameter for in vivo
efficacy, and metabolic stability by introduction of a polar
functional group to the terminal phenyl group of 5c (Table 4).
The carboxylic acid analogue 6a showed complete loss of
antifungal activity. In the phenol analogues 6b−d, 2-
substituted phenol 6d showed similar antifungal activity to
that of 5c, but the metabolic stability in humans was not
sufficient. In the pyridine analogues 6e−g, though compounds
6e,f showed improved antifungal activity in the presence of BS
compared to 5c, the metabolic stability in rat was very low. On
the other hand, methanesulfonyl analogue 6h showed 2-fold
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Table 3. SAR of 5a−e
a
b
c
A. fumigatus ATCC 204305 MIC. C. albicans ATCC 90028 MIC. Percent remaining in human liver microsomes after 30 min incubation.
d
e
f
Percent remaining in rat liver microsomes after 30 min incubation. “Classic” log P calculated in ACD Percepta Jan 17, 2020. Not tested.
stronger antifungal activity in the presence of BS than 5c with
improved metabolic stability.
of BS, and 4, >16, and 2 μg/mL in the presence of 50% BS,
respectively. As a result, intravenous administration of 6h at 30
mg/kg/dose exhibited a potent antifungal effect with 4-log
reduction of fungal burden in the kidney and significant
reduction of serum galactomannan, a surrogate marker for
efficacy against invasive aspergillosis,23 compared to the
untreated control (Figure 4). The AUC of 6h at 30 mg/kg
was 48.2 μg·h/mL, which was almost equal to the AUC of the
clinical dose of VRCZ (1e). On the other hand, intravenous
administration of ABFG (2) at 30 mg/kg/dose did not show
an antifungal effect with both efficacious biomarkers probably
due to its poor in vitro activity in BS and higher clearance in
vivo. VRCZ (1e), a first-line antifungal drug for aspergillosis,24
2.2. In Vivo Efficacy. Next, we investigated the in vivo
therapeutic effect in the murine systemic aspergillosis model.
For the in vivo efficacy test, 6h was selected because in vitro
activity in the presence of BS was >4-fold and 2-fold higher
than those of ABFG (2) and 5c, respectively, owing to
increases of free units in plasma (Tables 3−5). In addition, the
in vivo clearance of 6h was approximately 4-fold and 3-fold
lower than those of ABFG (2) and 5c as shown in Table 5
owing to increases in the metabolic stability in rat (Table 4).
The MICs of 6h, ABFG (2) and VRCZ, (1e) for the infected
strain A. fumigatus MA were 2, 2, and 2 μg/mL in the absence
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Table 4. SAR of 6a−i
a
b
c
A. fumigatus ATCC 204305 MIC. A. fumigatus ATCC 204305 MIC + bovine serum (BS). Percent remaining in human liver microsomes after
d
e
30 min incubation. Percent remaining in rat liver microsomes after 30 min incubation. “Classic” log P calculated in ACD Percepta Jan 17, 2020.
f
g
Not tested. Isolated as HBr salt for purification.
showed antifungal effects by intravenous administration at 30
mg/kg/dose both with efficacious biomarkers. As repeated
dosing of VRCZ (1e) in rodents causes a decrease of exposure
due to autoinduction of liver CYP3A4,25,26 VRCZ (1e) was
dosed at 30 mg/kg, where the AUC of 222 μg·h/mL in a single
administration was approximately 4-fold higher than that of a
clinical dose of VRCZ (1e), to maintain enough exposure to
exhibit efficacy. These results indicated that the increase of in
vitro activity in the presence of BS and in vivo exposure of 6h
Table 5. Rat PK Parameters
a
compound
free unit (%)
clearance (mL/(min kg))
Vdss (L/kg)
ABFG (2)
5c
6h
<0.1
1.04
6.7
51.4
33.1
12.3
4.92
7.25
4.21
a
Volume of distribution at steady state.
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Figure 5. Possible ergosterol biosynthesis pathways in C. albicans and
A. fumigatus. Blocking of Cyp51A by VRCZ (1e) causes the
accumulation of eburicol, and blocking of SMT by 6h causes the
accumulation of lanosterol in A. fumigatus.
Figure 4. Therapeutic efficacy in the murine A. fumigatus systemic
infection model. Antifungals were intravenously administered at 2, 16,
21, 26, 40, 45, and 50 h after inoculation. Kidneys and serum samples
were collected at 60 h after inoculation. Each bar represents the
average fungal burden with 18S rRNA relative to the start of
204305 (Table 7). This result also corroborated that 6h
inhibited a novel target enzyme in A. fumigatus ergosterol
biosynthesis.
treatment, and serum galacotomannan amounts
the standard
deviation from five mice. Data were analyzed using Dunnett’s test
with each treatment group compared to the untreated group. *p <
0.05 and **p < 0.01.
Table 7. Antifungal Activity against Azole-Resistant A.
fumigatus
MIC
A. fumigatus
A. fumigatus SR47013 Cyp51A;
led to an antifungal efficacy equivalent to VRCZ (1e) in the
murine systemic A. fumigatus infection model.
μg/mL
ATCC204305
Y121F
VRCZ (1e)
ISCZ (1f)
ITCZ (1g)
PSCZ (1h)
ABFG (2)
6h
0.5
0.25
0.25
0.063
2
>16
>16
>16
4
2
2
2.3. Mode of Action. As ABFG (2) inhibits SMT in the
ergosterol biosynthesis pathway in C. albicans,19 we inves-
tigated whether 6h inhibited the ergosterol biosynthesis in A.
fumigatus using sterol analysis with liquid chromatography/
mass spectrometry (LC/MS). First, we confirmed that
incubation of A. fumigatus ATCC 204305 with VRCZ (1e)
at 0.5 μg/mL caused a marked accumulation of eburicol, which
is the substrate of Cyp51A,27 and a decrease of ergosterol
(Table 6). Next, we observed the decrease of ergosterol when
2
2.4. Chemistry. Replacements of the six-membered ring
guanidine of ABFG (2) were performed as shown in Scheme 1.
The SNAr reaction of 7 with 2,4-dimethylphenol, followed by
bromination afforded 9. Compounds 3a−d were obtained by
reaction of 9 with appropriate thiourea derivatives. Dimethyl
carbonimidodithioate 10 was prepared from compound 3a by
treatment with CS2 and MeI in the presence of NaH.
Compounds 3e−h were synthesized from 10 with methyl-
amine or diamine derivatives.
a
Table 6. Sterol Analysis
untreated
VRCZ (1e) 0.5 μg/mL
6h 2 μg/mL
lanosterol
eburicol
zymosterol
ergosterol
0
0
0
1
34
0
40
1
0
144
99
102
a
Scheme 2 shows the synthetic route of 4a−c. The SNAr
reaction of arylbromide 11 with 2,4-dimethylphenol and
subsequent borylation gave boronic acid 13. Suzuki coupling
of 13 with commercially available heteroarylhalides afforded
14a−c. Six-membered ring guanidine analogues 4a−c were
synthesized from dimethyl carbonimidodithioate 16a−c with
1,3-propanediamine.
Scheme 3 shows the synthetic route of 5a−e. The six-
membered ring guanidine 18 was obtained from the reaction of
guanidine HCl salt 17 with 1,3-propanediamine. Reaction of
guanidine 18 with tert-butyl isothiocyanate was followed by
deprotection of the tert-butyl group by concentrated HCl to
obtain thiourea 20. The thiazole ring was formed by the
reaction of 20 and appropriate haloketones (22a−e) to give
23a,b and 5c−e. Suzuki coupling of 23a,b with phenylboronic
acid gave 5a,b.
Sterol amounts are shown as nmol per 100 mg of mycelia.
incubated with 6h at 2 μg/mL in A. fumigatus ATCC 204305
(Table 6). We also observed a marked accumulation of
lanosterol, with no accumulation of zymosterol (Table 6),
which is the substrate of SMT in C. albicans28 (Figure 5). As
lanosterol has been reported to be the substrate of purified A.
fumigatus SMT in an enzyme assay,29 6h might be an
ergosterol biosynthesis inhibitor against SMT in A. fumigatus
(Figure 5).
In addition, we investigated the antifungal activity of 6h
against a pan-azole-resistant A. fumigatus strain SR47013
harboring Cyp51A Y121F substitution. As a result, 6h showed
potent antifungal activity against the pan-azole-resistant strain
as well as the azole-susceptible strain of A. fumigatus ATCC
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a
Scheme 1. Synthesis of Compounds 3a−h
a
Reagents and conditions: (i) 2,4-dimethylphenol, K2CO3, DMA, 170 °C, 35%; (ii) TMSOTf, Et3N, NBS, CH2Cl2, 0 °C, 100%; (iii) appropriate
thiourea, EtOH, reflux, 62−82%; (iv) CS2, NaOH, MeI, DMF 0 °C, 68%; (v) methylamine, DMF, 140 °C, 47% for 3e, appropriate diamine, 29−
59% for 3f−h, dioxane, 100 °C, 47%
a
Scheme 2. Synthesis of Compounds 4a−c
a
Reagents and conditions: (i) 2,4-dimethylphenol, NaH, DMF, 150 °C, 23%; (ii) n-BuLi, triisopropyl borate, THF, −78 °C, 90%; (iii)
commercially available heteroaryl halide, Pd(PPh3)4, Na2CO3, dioxane, water, 90 °C, 26−95%; (iv) TFA, CH2Cl2; (v) CS2, NaOH, MeI, DMF 0
°C, 7−68% (2 steps); (vi) 1,3-propanediamine, DMF, 85 °C, 62−78%
the internal standard. Chemical shifts are expressed as δ (ppm) values
Scheme 4 shows the synthetic route of 6a−i. Bromoketone
for protons relative to the internal standard. The purity of the test
26 was prepared from 24 by thiazole formation with 20 and
compounds was confirmed by ultraperformance liquid chromatog-
subsequent bromination. Compound 26 with commercially
raphy-mass spectrometry (UPLC-MS) with UV diode array detection
available thioamides gave 6a−i.
to determine purity and by MS to confirm the molecular weight.
In summary, we discovered the novel, highly potent thiazoyl
guanidine antifungal reagent 6h that showed in vivo efficacy
Analytical LC/MS was performed on a Shimadzu Shim-pack XR-ODS
(C18, 2.2 μm, 3.0 mm × 50 mm, a linear gradient from 10 to 100% B
equivalent to VRCZ (1e). As ABFG (2), the starting
compound in this study has very high lipophilicity, leading
to poor properties such as a significant decrease of antifungal
activity in the presence of BS, low metabolic stability, and high
clearance, we focused on reducing the lipophilicity to develop
an antifungal agent for treating deep-seated mycoses. Sterol
analysis indicated that 6h inhibits ergosterol biosynthesis via
inhibition of SMT in A. fumigatus. This means that 6h has a
different mode of action from existing antifungal agents for
treatment of IFIs, and owing to its novel target, 6h has
antifungal activity against azole-resistant A. fumigatus. Our
findings show that 6h is a promising lead compound for the
development of a new generation of antifungal drugs.
over 3 min and then 100% B for 0.5 min (A = water + 0.1% formic
acid; B = MeCN + 0.1% formic acid), and a flow rate of 1.6 mL/min)
using a Shimadzu UFLC system equipped with an LCMS-2020 mass
spectrometer, an LC-20AD binary gradient module, an SPD-M20A
photodiode array detector (detection at 254 nm), and an SIL-20AC
sample manager. The purity of all compounds used in the bioassays
was determined by this method to be >95%. Preparative high-
performance liquid chromatography (HPLC) was performed on a
Phenomenex Gemini NX (5 μm, i.d. 100 mm × 300 mm, 110 Å, a
linear gradient from 10 to 100% B over 10 min (A = water + 0.1%
formic acid; B = MeCN + 0.1% formic acid), and a flow rate of 25
mL/min) using the Waters Acquity HPLC system comprising a
binary solvent manager, a sample manager, a PDA detector, a Waters
ZQ or SQD mass spectrometer, and a Waters Acquity evaporative
light scattering detector (detection at 254 nm).
3.1.1. 1-(2-(2,4-Dimethylphenoxy)phenyl)ethan-1-one (8). A
mixture of 7 (12.12 mL, 100 mmol), 2,4-dimethylphenol (14.37
mL, 120 mmol), and potassium carbonate (16.58 g, 129 mmol) in
DMA (200 mL) was stirred at 170 °C for 5 h. After being cooled to
room temperature, water, and EtOAc were added to the mixture. The
3. EXPERIMENTAL SECTION
3.1. Compound Purity and Identity. 1H NMR (400 MHz)
spectra were measured on a Varian MERCURY or Bruker instrument
in a solution of either CDCl3 or DMSO-d6 using tetramethylsilane as
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a
Scheme 3. Synthesis of Compounds 5a−e
a
Reagents and conditions: (i) 1,3-propanediamine, 140 °C, 64%; (ii) (a) NaOH, water, rt; (b) 2-isothiocyanato-2-methylpropane, THF, rt, 59% (2
steps); (iii) concentrated HCl, reflux, 43%; (iv) Br2, CHCl3, 0 °C; (v) 20, EtOH, rt, 16−67% (2 steps); (vi) PhB(OH)2, PdCl2(PPh3)2 K2CO3,
EtOH, water, 100 °C, 24% for 5a, PhB(OH)2, PdCl2(dppf)·CH2Cl2, Na2CO3, THF, water, 100 °C, 17% for 5b.
a
Scheme 4. Synthesis of Compounds 6a−i
a
Reagents and conditions: (i) 20, EtOH, rt, 48%; (ii) 25% HBr in AcOH, Br2, rt, 65%; (iii) commercially available thioamide, EtOH, reflux, 16−
79%.
organic layer was washed with brine, dried over anhydrous
magnesium sulfate, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (hexane−
EtOAc = 100:0 to 95:5) to give 8 (8.4 g, 35%) as a pale yellow oil. 1H
NMR (CDCl3) δ 2.20 (3H, s), 2.34 (3H, s), 2.71 (3H, s), 6.66 (1H,
dd, J = 8.3, 0.8 Hz), 6.81 (1H, d, J = 8.2 Hz), 6.99−7.10 (3H, m),
7.34 (1H, ddd, J = 8.7, 6.9, 1.5 Hz), 7.83 (1H, dd, J = 7.8, 1.8 Hz).
3.1.2. 2-Bromo-1-(2-(2,4-dimethylphenoxy)phenyl)ethan-1-one
(9). To a solution of 8 (500 mg, 2.08 mmol) and Et3N (0.346 mL,
2.50 mmol) in CH2Cl2 (5 mL) was added TMSOTf (0.451 mL, 2.50
mmol) at 0 °C. The mixture was allowed to reach room temperature,
and then stirred for 1 h. NBS (370 mg, 2.08 mmol) was added to the
reaction at 0 °C, with stirring for 1 h. Water and EtOAc were added to
the mixture. The organic layer was washed brine, dried over sodium
sulfate, and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (hexane−EtOAc =
90:10 to 85:15) to give 9 (664 mg, 100%) as a pale yellow oil. 1H
NMR (CDCl3) δ 2.20 (3H, s), 2.35 (3H, s), 4.72 (2H, s), 6.62 (1H,
dd, J = 8.4, 0.8 Hz), 6.87 (1H, d, J = 8.1 Hz), 7.03−7.10 (3H, m),
7.36−7.39 (1H, m), 7.91 (1H, dd, J = 7.9, 1.8 Hz).
3.1.3. 4-(2-(2,4-Dimethylphenoxy)phenyl)thiazol-2-amine (3a).
To a solution 9 (2.00 g, 6.27 mmol) in EtOH (20 mL) was added
thiourea (572 mg, 7.52 mmol). The mixture was heated to reflux for 2
h. After being cooled to room temperature, saturated aqueous sodium
hydrogen carbonate was added and the mixture was extracted with
EtOAc. The extract was washed with brine, dried over MgSO4, and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (hexane−EtOAc = 9:1 to 1:1) to
1
give 3a (1.52 g, 82%) as a colorless oil. H NMR (CDCl3) δ 2.20
(3H, s), 2.32 (3H, s), 4.89 (2H, brs), 6.66 (1H, dd, J = 7.9, 1.5 Hz),
6.80 (1H, d, J = 8.2 Hz), 6.95−6.98 (1H, m), 7.06−7.15 (3H, m),
7.29 (1H, s), 8.16 (1H, dd, J = 7.5, 2.1 Hz). LC-MS (M + H) 297.
3.1.4. 4-(2-(2,4-Dimethylphenoxy)phenyl)-N,N-dimethylthiazol-
2-amine (3b). Compound 3b was obtained (62%) from compound
9 via reaction with 1,1-dimethylthiourea in a manner similar to that
1
described for compound 3a. H NMR (DMSO-d6) δ 2.15 (3H, s),
2.27 (3H, s), 3.09 (6H, s), 6.62 (1H, dd, J = 8.0, 1.1 Hz), 6.74 (1H, d,
J = 8.2 Hz), 7.00 (1H, dd, J = 8.2, 1.9 Hz), 7.10−7.15 (2H, m), 7.17−
7.22 (1H, m), 7.23 (1H, s), 8.18 (1H, dd, J = 7.7, 1.8 Hz). LC-MS (M
+ H) 325.
3.1.5. 4-(2-(2,4-Dimethylphenoxy)phenyl)-N-(pyridin-2-yl)-
thiazol-2-amine (3c). Compound 3c was obtained (64%) from
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compound 9 via reaction with 1-(pyridin-2-yl)thiourea in a manner
similar to that described for compound 3a. H NMR (DMSO-d6) δ
was added dropwise to a suspension of sodium hydride (10.21 g, 255
mmol, 60% in mineral oil) in DMF (150 mL), and the resulting
mixture was stirred at 100 °C for 30 min. To this mixture was added
11 (35 g, 200 mmol) in dry DMF (40 mL), and the resulting mixture
was stirred at 150 °C for 6 h. The mixture was poured onto an ice−
water mixture, and the aqueous phase was extracted with diethyl
ether. The combined organic phase was washed with 2 N sodium
hydroxide, dried over MgSO4, filtered, and concentrated in vacuo.
The residue was purified by column chromatography with petroleum
1
2.17 (3H, s), 2.28 (3H, s), 6.65 (1H, d, J = 8.4 Hz), 6.80 (1H, d, J =
8.2 Hz), 6.92−6.94 (1H, m), 7.02−7.04 (1H, m), 7.11−7.25 (4H, m),
7.48 (1H, s), 7.70−7.73 (1H, m), 8.20 (1H, dd, J = 7.8, 1.8 Hz),
8.30−8.31 (1H, m), 11.37 (1H, s). LC-MS (M + H) 374.
3.1.6. 4-(2-(2,4-Dimethylphenoxy)phenyl)-N-(pyrimidin-2-yl)-
thiazol-2-amine (3d). Compound 3d was obtained (63%) from
compound 9 via reaction with 1-(pyrimidin-2-yl)thiourea in a manner
1
1
similar to that described for compound 3a. H NMR (DMSO-d6) δ
ether as the eluent to afford 12 (12.5 g, 23%) as a colorless oil. H
2.16 (3H, s), 2.28 (3H, s), 6.65 (1H, dd, J = 8.0, 0.9 Hz), 6.80 (1H, d,
J = 8.2 Hz), 7.04 (2H, q, J = 4.9 Hz), 7.14−7.25 (3H, m), 7.57 (1H,
s), 8.22 (1H, dd, J = 7.7, 1.8 Hz), 8.65 (2H, d, J = 8.8 Hz), 11.79 (1H,
s). LC-MS (M + H) 375.
NMR (CDCl3) δ 2.19 (3H, s), 2.31 (3H, s), 6.68 (1H, dd, J = 8.2, 1.4
Hz), 6.75 (1H, d, J = 8.2 Hz), 6.91 (1H, td, J = 7.7, 1.3 Hz), 6.95−
6.97 (1H, m), 7.05−7.07 (1H, m), 7.15−7.17 (1H, m), 7.60 (1H, dd,
J = 8.0, 1.6 Hz).
3.1.7. Dimethyl (4-(2-(2,4-dimethylphenoxy)phenyl)thiazol-2-yl)-
carbonimidodithioate (10). To a solution of 3a (434 mg, 1.37
mmol) and CS2 in DMF (2 mL) was added 10 N NaOH aq (0.29 mL,
2.9 mmol), and the mixture was stirred at room temperature for 30
min. MeI (616 μL, 9.86 mmol) was added to the mixture at 0 °C and
stirred at the same temperature for 4 h. Water was added, and the
mixture was extracted with EtOAc. The extract was washed with
brine, dried over Na2SO4, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
(hexane−EtOAc = 50:1 to 10:1) to give 10 (274 mg, 68%) as a
3.1.13. (2-(2,4-Dimethylphenoxy)phenyl)boronic Acid (13). n-
BuLi (24 mL, 58.7 mmol) was added in one portion to a solution of
12 (10 g, 36.7 mmol) in THF (100 mL) at −78 °C, and the mixture
was stirred at −78 °C for 20 min. Triisopropyl borate (25.6 mL, 110
mmol) was added at −78 °C and stirred for 20 min, then warmed to
room temperature, and stirred overnight. After cooling to 0 °C, 2 N
HCl aq. (20 mL) was added and diluted with water (200 mL),
extracted with EtOAc, dried over Na2SO4, purified by column
chromatography (EtOAc−petroleum ether = 1:20) to afford 13 (8.0
g, yield 90%) as a colorless oil. 1H NMR (DMSO-d6) δ 2.12 (s, 3H),
2.27 (s, 3H), 6.48 (d, 1H, J = 7.8 Hz), 6.80 (d, 1H, J = 7.8 Hz), 6.81−
7.05 (m, 2H), 7.11 (s, 1H), 7.25−7.31 (m, 1H), 7.58 (dd, 1H, J = 1.5
Hz, 7.5 Hz).
3.1.14. tert-Butyl (5-(2-(2,4-Dimethylphenoxy)phenyl)thiazol-2-
yl)carbamate (14a). A mixture of compound 13 (317 mg, 1.31
mmol), tert-butyl (5-bromothiazol-2-yl)carbamate (300 mg, 1.08
mmol), Pd(PPh3)4 (62 mg, 0.054 mmol), and 2 M sodium carbonate
aqueous solution (2.15 mL, 4.30 mmol) in dioxane (7 mL) was
heated at 90 °C and stirred overnight. After the reaction mixture was
cooled to room temperature, water and EtOAc were added to the
mixture. The organic layer was washed brine, dried over sodium
sulfate, and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (hexane−EtOAc =
90:10 to 70:30) to give 14a (113 mg, yield 26%) as a white solid. 1H
NMR (CDCl3) δ 1.52 (9H, s), 2.18 (3H, s), 2.33 (3H, s), 6.64 (1H,
d, J = 8.1 Hz), 6.83 (1H, d, J = 8.1 Hz), 6.97−7.15 (4H, m), 7.60
(1H, dd, J = 7.6, 1.7 Hz), 7.90 (1H, s).
1
colorless solid. H NMR (CDCl3) δ 2.21 (3H, s), 2.33 (3H, s), 2.63
(6H, brs), 6.65−6.69 (1H, m), 6.82 (1H, d, J = 8.1 Hz), 6.97 (1H, d, J
= 8.2 Hz), 7.07−7.17 (3H, m), 7.81 (1H, s), 8.31−8.34 (1H, m).
3.1.8. 2-(4-(2-(2,4-Dimethylphenoxy)phenyl)thiazol-2-yl)-1,3-di-
methylguanidine (3e). To a solution of 10 (30 mg, 0.075 mmol)
in DMF (2 mL) was added a solution of 2 N methylamine in THF
(0.374 mL, 0.749 mmol), and the mixture was stirred at 140 °C for 10
h. After being cooled to room temperature, water was added, and the
mixture was extracted with EtOAc. The extract was washed with
brine, dried over MgSO4, and concentrated under reduced pressure.
The residue was purified by amino silica gel column chromatography
1
(hexane−EtOAc = 9:1 to 1:1) to give 3e (13 mg, 47%). H NMR
(CDCl3) δ 2.21 (3H, s), 2.31 (3H, s), 2.93 (6H, d, J = 3.8 Hz), 6.69
(1H, dd, J = 7.8, 1.6 Hz), 6.80 (1H, d, J = 8.2 Hz), 6.94−6.96 (1H,
m), 7.05−7.15 (3H, m), 7.30 (1H, s), 8.01 (1H, dd, J = 7.4, 2.1 Hz).
LC-MS (M + H) 367.
3.1.9. N-(4-(2-(2,4-Dimethylphenoxy)phenyl)thiazol-2-yl)-
imidazolidin-2-imine (3f). To a solution of 10 (30 mg, 0.075
mmol) in dioxane (2 mL) was added ethylenediamine (45 mg, 0.749
mmol). The mixture was stirred at 100 °C for 2 h. After being cooled
to room temperature, the reaction was concentrated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy (hexane−EtOAc = 9:1 to 1:1) to give 3f (16 mg, 59%) as a
white solid. 1H NMR (CDCl3) δ 2.22 (3H, s), 2.31 (3H, s), 3.69 (4H,
brs), 6.71 (1H, dd, J = 7.8, 1.4 Hz), 6.79 (1H, d, J = 8.2 Hz), 6.95−
6.97 (1H, m), 7.05−7.17 (3H, m), 7.30 (1H, s), 8.04 (1H, dd, J = 7.5,
2.1 Hz). LC-MS (M + H) 365.
3.1.15. Dimethyl(5-(2-(2,4-dimethylphenoxy)phenyl)thiazol-2-yl)
carbonimidodithioate (16a). To a solution of 14a in CH2Cl2 (0.9
mL) was added TFA (0.9 mL) at 0 °C. The reaction was warmed to
room temperature and then stirred for 2 h. The reaction was poured
into sat NaHCO3 aq. and extracted with EtOAc. The extract was
washed with brine, dried over Na2SO4, and concentrated under
reduced pressure to give 15a (74 mg, 93%) as a slightly reddish solid.
To a solution of 15a in DMF (0.5 mL) was added 10 N NaOH
(0.049 mL, 0.49 mmol) and CS2 (0.046 mL, 0.764 mmol) at room
temperature. The mixture was stirred for 30 min at room temperature.
MeI (0.104 mL, 1.67 mmol) was added to the reaction at 0 °C, and
the mixture was stirred at 0 °C for 4 h. Water was added to the
reaction. The mixture was extracted with EtOAc. The organic layer
was washed brine, dried over sodium sulfate, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (hexane−EtOAc = 95:5 to 3:1) to give 63 mg (yield
3.1.10. N-(4-(2-(2,4-Dimethylphenoxy)phenyl)thiazol-2-yl)-1,3-
diazepan-2-imine (3g). Compound 3g was obtained (35%) from
compound 10 via reaction with 1,4-diaminobutane in a manner
1
similar to that described for compound 3f. H NMR (DMSO-d6) δ
1.24 (2H, br s), 1.62 (4H, br s), 2.16 (3H, s), 2.28 (3H, s), 3.13 (4H,
br s), 6.65 (1H, d, J = 8.1 Hz), 6.75 (1H, d, J = 8.2 Hz), 7.01−7.03
(1H, m), 7.12−7.18 (2H, m), 7.19−7.25 (1H, m), 7.26 (1H, s), 8.01
(1H, dd, J = 7.7, 1.6 Hz). LC-MS (M + H) 393.
3.1.11. (E)-N-(4-(2-(2,4-Dimethylphenoxy)phenyl)thiazol-2-yl)-1-
methyltetrahydropyrimidin-2(1H)-imine (3h). Compound 3h was
obtained (29%) from compound 10 via reaction with N-methyl-1,3-
diaminopropane in a manner similar to that described for compound
3f. 1H NMR (DMSO-d6) δ 1.88−1.93 (2H, m), 2.15 (3H, s), 2.27
(3H, s), 2.99 (3H, s), 3.27−3.39 (4H, m), 6.64 (1H, dd, J = 8.1, 1.2
Hz), 6.73 (1H, d, J = 8.2 Hz), 7.00 (1H, d, J = 4.1 Hz), 7.11−7.16
(3H, m), 7.20 (1H, dt, J = 10.8, 3.9 Hz), 7.98 (1H, dd, J = 7.7, 1.9
Hz), 9.64 (1H, s). LC-MS (M + H) 393.
1
68%) of compound 16a as a yellow oil. H NMR (CDCl3) δ 2.22
(3H, s), 2.32 (3H, s), 2.61 (6H, s), 6.71 (1H, d, J = 8.2 Hz), 6.78
(1H, d, J = 8.1 Hz), 6.93−7.19 (4H, m), 7.07 (4H, tt, J = 27.3, 9.9
Hz), 7.68 (1H, dd, J = 7.7, 1.6 Hz), 8.05 (1H, s).
3.1.16. N-(5-(2-(2,4-Dimethylphenoxy)phenyl)thiazol-2-yl)-
tetrahydropyrimidin-2(1H)-imine (4a). To a solution of 16a (54
mg, 0.135 mmol) in DMF (0.5 mL) was added 1,3-propanediamine
(12 mg, 0.162 mmol), and the mixture was stirred for 7 h at 85 °C.
After being cooled to room temperature, water was added to the
reaction. The obtained insoluble material was collected by filtration.
The obtained material was dissolved in CHCl3, dried over Na2SO4,
and concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (EtOAc−hexane = 45:55 to 65:35)
3.1.12. 1-(2-Bromophenoxy)-2,4-dimethylbenzene (12). A sol-
ution 2,4-dimethylphenol (24 g, 196 mmol) in dry DMF (100 mL)
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to give 4a (32 mg, 62%) as a white solid. H NMR (DMSO-d6) δ
1.72−1.87 (2H, m), 2.19 (3H, s), 2.25 (3H, s), 3.20−3.32 (4H, m),
6.58 (1H, d, J = 8.2 Hz), 6.70 (1H, d, J = 7.6 Hz), 6.95 (1H, d, J = 8.2
Hz), 7.08−7.17 (3H, m), 7.56−7.66 (2H, m), 8.21 (2H, brs). LC-MS
(M + H) 379.
(1H, m), 7.22−7.26 (2H, m), 7.59−7.67 (2H, m), 7.80−7.85 (2H,
m).
3.1.21. Dimethyl (6-(2-(2,4-Dimethylphenoxy)phenyl)pyridin-2-
yl)carbonimidodithioate (16c). To a solution of 14c (425 mg, 1.088
mmol) in CH2Cl2 (4.2 mL) was added TFA (4.2 mL) at 0 °C. The
reaction was warmed to room temperature and stirred for 2 h. The
reaction mixture was poured into sat NaHCO3 aq. and extracted with
EtOAc. The extract was washed with brine, dried over Na2SO4, and
concentrated under reduced pressure to give 15c (298 mg, 94%) as a
white solid. To a solution of 15c (296 mg, 1.02 mmol) in DMF (2
mL) was added 10 N NaOH (0.214 mL, 2.14 mmol) and CS2 (0.203
mL, 3.36 mmol) at room temperature. The mixture was stirred for 30
min at room temperature. MeI (0.459 mL, 7.34 mmol) was added to
the reaction at 0 °C, and the mixture was stirred at 0 °C for 4 h. Water
was added to the reaction. The mixture was extracted with EtOAc.
The organic layer was washed with brine, dried over sodium sulfate,
and concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (hexane−EtOAc = 100:0 to 85:15)
3.1.17. tert-Butyl (3-(2-(2,4-Dimethylphenoxy)phenyl)-1,2,4-thia-
diazol-5-yl)carbamate (14b). A mixture of compound 13 (316 mg,
1.31 mmol), tert-butyl (3-bromo-1,2,4-thiadiazol-5-yl)carbamate (300
mg, 1.07 mmol), Pd(PPh3)4 (62 mg, 0.054 mmol), and 2 M sodium
carbonate aqueous solution (2.14 mL, 4.30 mmol) in dioxane (7 mL)
was heated at 90 °C and stirred overnight. After the reaction mixture
was cooled to room temperature, water and EtOAc were added to the
mixture. The organic layer was washed brine, dried over sodium
sulfate, and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (hexane−EtOAc =
90:10 to 70:30) to give 14b (177 mg, yield 42%) as a white
1
amorphous substance. H NMR (DMSO-d6) δ: 1.56 (9H, s), 2.18
(3H, s), 2.28 (3H, s), 6.72 (1H, d, J = 8.2 Hz), 6.81 (1H, dd, J = 8.3,
0.9 Hz), 6.96−6.98 (1H, m), 7.08−7.11 (1H, m), 7.20 (1H, td, J =
7.5, 1.1 Hz), 7.42 (1H, ddd, J = 8.7, 6.9, 1.4 Hz), 7.88 (1H, dd, J =
7.6, 1.8 Hz), 12.06 (1H, br s).
1
to give 16c (29 mg, 7.2%). H NMR (CDCl3) δ 2.20 (3H, s), 2.29
(3H, s), 2.56 (6H, s), 6.73−6.75 (2H, m), 6.90−6.92 (2H, m), 7.03
(1H, brs), 7.15 (1H, td, J = 7.4, 1.3 Hz), 7.20−7.23 (1H, m), 7.65
(1H, t, J = 7.7 Hz), 7.75 (1H, dd, J = 7.7, 0.8 Hz), 8.06 (1H, dd, J =
7.5, 1.9 Hz).
3.1.18. Dimethyl (3-(2-(2,4-Dimethylphenoxy)phenyl)-1,2,4-thia-
diazol-5-yl)carbonimidodithioate (16b). To a solution of 14b (166
mg, 0.418 mmol) in CH2Cl2 (1.6 mL) was added TFA (1.6 mL) at 0
°C. The reaction was warmed to room temperature and stirred for 2
h. The reaction was poured into sat NaHCO3 aq. and extracted with
EtOAc. The extract was washed with brine, dried over Na2SO4, and
concentrated under reduced pressure to give 15b (114 mg, 92%) as a
white solid. To a solution of 15b (111 mg, 0.373 mmol) in DMF
(0.75 mL) was added 10 N NaOH (0.078 mL, 0.784 mmol) and CS2
(0.074 mL, 1.23 mmol) at room temperature. The mixture was stirred
for 30 min at room temperature. MeI (0.168 mL, 2.69 mmol) was
added to the reaction at 0 °C, and the mixture was stirred at 0 °C for
4 h. Water was added to the reaction. The mixture was extracted with
EtOAc. The organic layer was washed brine, dried over sodium
sulfate, and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (hexane−EtOAc = 95:5
to 3:1) to give 16b (111 mg, 74%). 1H NMR (CDCl3) δ 2.21 (3H, s),
2.29 (3H, s), 2.62 (6H, s), 6.75−6.85 (2H, m), 6.88−6.95 (1H, m),
6.98−7.06 (1H, m), 7.08−7.16 (1H, m), 7.26−7.34 (1H, m), 8.09
(1H, dd, J = 7.7, 1.8 Hz).
3.1.22. N-(6-(2-(2,4-Dimethylphenoxy)phenyl)pyridin-2-yl)-
tetrahydropyrimidin-2(1H)-imine (4c). To a solution of 16c (25
mg, 0.269 mmol) in DMF (0.25 mL) was added 1,3-propanediamine
(0.0079 mL, 0.095 mmol), and the mixture was stirred at 85 °C for 8
h. After being cooled to room temperature, the reaction was diluted
with EtOAc, washed with brine, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by silica gel column
chromatography (CHCl3−MeOH = 95:5) to give 4c (18 mg, 78%) as
a white solid. 1H NMR (CDCl3) δ 1.89−1.97 (2H, m), 2.17 (3H, s),
2.30 (3H, s), 3.28−3.40 (4H, m), 6.71−6.80 (3H, m), 6.92−6.95
(1H, m), 7.01−7.05 (1H, m), 7.11−7.13 (1H, m), 7.19−7.21 (2H,
m), 7.47 (1H, t, J = 7.9 Hz), 7.70 (1H, dd, J = 7.5, 1.8 Hz). LC-MS
(M + H) 373.
3.1.23. Tetrahydropyrimidin-2(1H)-imine Hydrochloride (18). A
mixture of 17 (309 g, 3.24 mol) and 1,3-propanediamine (300 g, 4.05
mol) was stirred at 140 °C for 20 h. After being cooled to room
temperature, 2-propanol was added to the mixture. The obtained solid
was collected by filtration, washed with methyl tert-butyl ether, and
dried under high vacuum to give 18 (279 g, 64%) as a white solid. 1H
NMR (DMSO-d6) δ 1.74−1.80 (m, 2H), 3.13−3.23 (m, 4H), 7.40
(br s, 4H).
3.1.19. N-(3-(2-(2,4-Dimethylphenoxy)phenyl)-1,2,4-thiadiazol-
5-yl)tetrahydropyrimidin-2(1H)-imine (4b). To a solution of 16b
(108 mg, 0.269 mmol) in DMF (1 mL) was added 1,3-propanedi-
amine (0.027 mL, 0.323 mmol), and the mixture was stirred at 85 °C
for 4 h. After being cooled to room temperature, water was added to
the reaction. The obtained insoluble material was collected by
filtration. The obtained material was dissolved in CHCl3, dried over
Na2SO4, and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (EtOAc−hexane =
3.1.24. 1-(tert-Butyl)-3-(tetrahydropyrimidin-2(1H)-ylidene)-
thiourea (19). To a solution of 18 in water (700 mL) was added
NaOH (55.8 g, 1.4 mol), and the mixture was stirred at rt for 30 min.
The mixture was concentrated under reduced pressure. EtOH was
added to the residue, the resulting mixture was filtered, and the
obtained filtrate was concentrated under reduced pressure. The
obtained residue was dissolved in THF (600 mL), and then 2-
isothiocyanato-2-methylpropane (115 g, 0.98 mol) was added to the
solution at 0 °C. The mixture was allowed to reach room temperature
and stirred for 16 h. The mixture was concentrated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy (CH2Cl2−MeOH, gradient) to give crude 19. The obtained
crude 19 was washed with petroleum ether−EtOAc = 9:1 to give 19
1
45:55 to 70:30) to give 4b (70 mg, 68%) as a white solid. H NMR
(DMSO-d6) δ 1.69−1.75 (2H, m), 2.17 (3H, s), 2.26 (3H, s), 3.08−
3.09 (4H, m), 6.71 (1H, d, J = 8.2 Hz), 6.76 (1H, d, J = 8.3 Hz), 6.99
(1H, dd, J = 8.0, 1.6 Hz), 7.11 (1H, s), 7.16−7.20 (1H, m), 7.37−
7.41 (1H, m), 7.91 (1H, dd, J = 7.8, 1.6 Hz), 8.37 (2H, s). LC-MS (M
+ H) 380.
1
3.1.20. tert-Butyl (6-(2-(2,4-Dimethylphenoxy)phenyl)pyridin-2-
yl)carbamate (14c). A mixture of compound 13 (336 mg, 1.39
mmol), tert-butyl (6-bromopyridin-2-yl)carbamate (400 mg, 1.16
mmol), Pd(PPh3)4 (67 mg, 0.058 mmol), and 2 M sodium carbonate
aqueous solution (2.31 mL, 4.63 mmol) in dioxane (8 mL) was
heated at 90 °C and stirred for 3 h. After the reaction mixture was
cooled to room temperature, water and EtOAc were added to the
mixture. The organic layer was washed brine, dried over sodium
sulfate, and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (hexane−EtOAc =
100:0 to 90:10) to give 14c (430 mg, 95%) as a white amorphous
substance. 1H NMR (CDCl3) δ 1.53 (9H, s), 2.18 (3H, s), 2.30 (3H,
s), 6.73−6.75 (2H, m), 6.91−6.94 (1H, m), 7.03 (1H, s), 7.11−7.14
(124 g, 59%) as a white solid. H NMR (DMSO-d6) δ 1.29 (s, 9H),
1.78 (m, 2H), 3.25 (m, 4H), 6.28 (s, 1H), 8.99 (s, 2H).
3.1.25. 1-(Tetrahydropyrimidin-2(1H)-ylidene)thiourea (20).
Compound 19 (145 g, 0.69 mol) was added to concentrated HCl
(220 mL). The mixture was heated to reflux for 2 h. The mixture was
concentrated under reduced pressure. Saturated aqueous sodium
hydrogen carbonate was added to the residue, and the obtained solid
was collected by filtration, washed with ice water, and dried under
1
high vacuum to give 20 (46 g, 43%). H NMR (DMSO-d6) δ 1.76−
1.79 (m, 2H), 3.22−3.26 (m, 4H), 6.79 (br, 2H), 8.90 (br, 2H).
3.1.26. N-(4-(3-Iodophenyl)thiazol-2-yl)tetrahydropyrimidin-
2(1H)-imine (23a). To a solution of 21a (514 mg, 2.09 mmol) in
CHCl3 (10 mL) was added Br2 (0.118 mL, 2.30 mmol) at 0 °C, and
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the mixture was warmed to room temperature and stirred for 7 h.
Saturated aqueous sodium hydrogen carbonate was added, and the
mixture was extracted with CHCl3. The extract was washed with
brine, dried over anhydrous magnesium sulfate, and concentrated
under reduced pressure. EtOH (8 mL) and 20 (305 mg, 1.93 mmol)
were added to the obtained residue, and the mixture was stirred for
1.5 h. Saturated aqueous sodium hydrogen carbonate and CHCl3
were added to the mixture, and the obtained solid was collected by
filtration, washed with water, and dried under high vacuum to give
7.24 (s, 1H), 7.49−7.57 (m, 3H), 7.98−8.03 (m, 2H), 8.15 (s, 1H),
8.41 (br s, 2H). LC-MS (M + H) 342.
3.1.31. N-(4-(1-Phenyl-1H-pyrazol-3-yl)thiazol-2-yl)-
tetrahydropyrimidin-2(1H)-imine (5d). Compound 5d was prepared
from compound 21d in a manner similar to that described for
1
compound 5c as an off-white solid. Yield 49%. H NMR (CDCl3) δ
1.97−2.03 (2H, m), 3.47−3.48 (4H, m), 6.74 (1H, d, J = 2.4 Hz),
6.98 (1H, s), 7.29 (1H, d, J = 7.7 Hz), 7.46 (2H, t, J = 7.8 Hz), 7.76
(2H, d, J = 7.3 Hz), 7.92 (1H, d, J = 2.5 Hz). LC-MS (M + H) 325.
3. 1. 32. N-(4-(2-Phenyloxazol-4-yl)thiazol-2-yl)-
tetrahydropyrimidin-2(1H)-imine (5e). Compound 5e was prepared
from compound 21e in a manner similar to that described for
compound 5c as an off-white solid. Yield 16%. 1H NMR (DMSO-d6)
δ 1.92−1.97 (2H, m), 3.47−3.48 (4H, m), 7.53 (1H, s), 7.58−7.59
(3H, m), 8.04−8.07 (2H, m), 8.87−8.90 (3H, m). LC-MS (M + H)
326.
3.1.33. 1-(2-((Tetrahydropyrimidin-2(1H)-ylidene)amino)thiazol-
4-yl)ethan-1-one (25). To a solution of 24 (4.01 g, 24.31 mmol) in
EtOH (50 mL) was added 20 (5.77 g, 36.50 mmol) at 0 °C, and the
reaction was warmed to room temperature and stirred for 23 h. The
mixture was concentrated under reduced pressure. Saturated aqueous
sodium hydrogen carbonate was added to the residue, and the mixture
was extracted with CHCl3. The extract was washed with brine and
dried over anhydrous magnesium sulfate, then concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (CHCl3−MeOH = 100:0 to 95:1) to give 25 (2.59 g,
48%) as a white solid. 1H NMR (CDCl3) δ 1.95−2.03 (m, 2H), 2.51
(s, 3H), 3.44−3.48 (m, 4H), 7.39 (s,1H).
1
23a (278 mg, 34%) as a white solid. H NMR (DMSO-d6) δ 1.77−
1.89 (2H, m), 3.30−3.36 (4H, m), 7.14−7.21 (2H, m), 7.61 (1H, d, J
= 7.6 Hz), 7.86 (1H, d, J = 7.8 Hz), 8.14 (1H, s), 8.20 (2H, br s).
3. 1. 27. N-(4-([1, 1′ -Biphe nyl]-3-yl)thiaz ol-2-yl)-
tetrahydropyrimidin-2(1H)-imine (5a). A mixture of 23a (100 mg,
0.26 mmol), phenylboronic acid (38 mg, 0.312 mmol), PdCl2(PPh3)2
(18 mg, 0.026 mmol), and 2 M potassium carbonate aqueous solution
(0.26 mL, 0.52 mmol) in EtOH(1.5 mL) was stirred at 100 °C in a
sealed tube for 2 h. After cooling to room temperature, water and
CHCl3 were added to the mixture. The organic layer was washed with
brine and dried over anhydrous magnesium sulfate. The filtrate was
concentrated, and the residue was purified by amino silica gel column
chromatography (hexane−EtOAc gradient) to give 5a (21 mg, 24%),
1H NMR (DMSO-d6) δ 1.76−1.91 (2H, m), 3.31−3.37 (4H, m), 7.22
(1H, s), 7.39 (1H, t, J = 7.3 Hz), 7.45−7.52 (3H, m), 7.56 (1H, d, J =
7.8 Hz), 7.73 (2H, d, J = 7.4 Hz), 7.84 (1H, d, J = 7.7 Hz), 8.05 (1H,
s), 8.30 (2H, br s). LC-MS (M + H) 335.
3.1.28. N-(4-(5-Bromothiophen-3-yl)thiazol-2-yl)-
tetrahydropyrimidin-2(1H)-imine (23b). To a solution of 21b (5.0
g, 24,4 mmol) in Et2O was added AlCl3 (0.163 g, 1.22 mmol) at 0 °C.
Br2 (3.90 g, 24.4 mmol) was added dropwise to the mixture at 0 °C,
and the mixture was stirred at 10 °C for 3 h. Saturated aqueous
sodium hydrogen carbonate was added, and the mixture was extracted
with EtOAc. The extract was washed with brine and dried over
anhydrous magnesium sulfate, then concentrated under reduced
pressure to give crude 22b (4.7 g, 68%). A mixture of 22b (4.87 g,
17.2 mmol) and 20 (1.1 g, 6.86 mmol) in EtOH (18 mL) was stirred
at 110 °C for 10 min in a sealed tube. After being cooled to room
temperature, saturated aqueous sodium hydrogen carbonate and
EtOAc were added to the mixture. The extract was washed with brine
and dried over anhydrous magnesium sulfate, then concentrated
under reduced pressure. The residue was purified by column
chromatography (hexane−EtOAc = 50:50 to 0:100) to give 23b
3.1.34. 2-Bromo-1-(2-((tetrahydropyrimidin-2(1H)-ylidene)-
amino)thiazol-4-l)ethan-1-one (26). To 25% HBr (3.85 g, 17.2
mmol) in AcOH (77 mL) was added 25 (3.85 g, 17.2 mmol). Br2
(0.884 mL, 17.2 mol) was added to the mixture and stirred at room
temperature for 23 h. The mixture was concentrated under reduced
pressure. Saturated aqueous sodium hydrogen carbonate was added to
the residue, and the mixture was extracted with CHCl3. The extract
was washed with brine, dried over anhydrous magnesium sulfate, and
then concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (hexane−EtOAc = 50:50 to
0:100) to give 26 (4.20 g, 65%) as a yellow solid. 1H NMR (CDCl3) δ
1.97−2.04 (m, 2H), 3.45−3.47 (m, 4H), 4.40 (s, 2H), 7.55 (s,1H).
3.1.35. 4-(2′-((Tetrahydropyrimidin-2(1H)-ylidene)amino)-[4,4′-
bithiazol]-2-yl)benzoic Acid (6a). To the mixture of 26 (30 mg,
0.099 mmol) in EtOH (1 mL) was added 4-carbamothioylbenzoic
acid (18.8 mg, 0.104 mmol), followed by heating to reflux for 2 h.
After being cooled to room temperature, the obtained solid was
collected by filtration and washed with EtOH. The crude material was
1
(600 mg, 26%) as a brown solid. H NMR (DMSO-d6) δ 1.74−1.86
(m, 2H), 3.30−3.38 (m, 4H), 6.94 (s, 1H), 7.66 (d, J = 4.0 Hz, 1H),
7.85 (d, J = 4.0 Hz, 1H), 8.15−8.23 (br s, 2H)
1
3.1.29. N-(4-(5-Phenylthiophen-3-yl)thiazol-2-yl)-
tetrahydropyrimidin-2(1H)-imine (5b). A mixture of 23b (30 mg,
0.087 mmol), phenylboronic acid (16 mg, 0.13 mmol), PdCl2(dtbpf)
(5.7 mg, 8.74 μmol), and 2 N Na2CO3 aq (0.131 mL, 0.262 mmol)
was stirred at 130 °C for 30 min in a sealed tube. After being cooled
to room temperature, water and EtOAc were added to the mixture.
The extract was washed with brine, dried over anhydrous magnesium
sulfate, and then concentrated under reduced pressure. The residue
was purified by column chromatography (hexane−EtOAc = 50:50 to
0:100) to give 5b (5.0 mg, 17%) as a white solid. 1H NMR (DMSO-
d6) δ 1.78−1.88 (m, 2H), 3.32−3.36 (m, 4H), 6.97 (s,1H), 7.33 (t, J
= 8.0 Hz, 1H), 7.44 (t, J = 8.0 Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H), 7.84
(s, 1H), 7.93 (s, 1H), 8.10−8.30 (br s, 2H). LC-MS (M + H) 341.
3.1.30. N-(2′-Bromo-[4,4′-bithiazol]-2-yl)tetrahydropyrimidin-
2(1H)-imine (5c). To a solution of 21d (200 mg, 0.984 mmol) in
CHCl3 was added pyridinium tribromide (350 mg, 0.984 mmol), and
the mixture was stirred at room temperature for 1.5 h. The mixture
was concentrated under reduced pressure to give crude 22c. To the
mixture of 22c in EtOH was added 20 (156 mg, 0.984 mmol), and
the mixture was stirred overnight. The reaction was concentrated, and
then saturated aqueous sodium hydrogen carbonate and ether were
added to the residue. The obtained solid was collected by filtration,
washed with water and ether, and dried under high vacuum to give 5c
(225 mg, 67%). 1H NMR (DMSO-d6) δ 1.87 (m, 2H), 3.37 (m, 4H),
purified by preparative HPLC to give 6a (6.2 mg, 16%). H NMR
(DMSO-d6) δ 1.79−1.90 (2H, m), 3.29−3.36 (4H, m), 7.13 (1H, s),
8.06 (2H, d, J = 8.4 Hz), 8.11 (2H, d, J = 7.8 Hz), 8.16 (1H, s), 8.27
(2H, s). LC-MS (M + H) 386.
3.2. General Procedure A. To the mixture of 26 (1.0 equiv) in
EtOH (0.3 M) was added appropriate thioamide (1.5 equiv),
followed by heating to reflux for 2 h. After being cooled to room
temperature, the obtained solid was collected by filtration and washed
with EtOH. Saturated aqueous sodium hydrogen carbonate was
added to the solid, and the mixture was extracted with CHCl3/MeOH
= 9:1. The extract was washed with brine, dried over anhydrous
magnesium sulfate, and then concentrated under reduced pressure.
The obtained solid was washed with EtOAc and dried under high
vacuum to afford the title compound.
3.2.1. 4-(2′-((Tetrahydropyrimidin-2(1H)-ylidene)amino)-[4,4′-bi-
thiazol]-2-yl)phenol (6b). The title compound was prepared
according to general procedure A using 4-hydroxybenzothioamide.
1
Yield 78%. H NMR (DMSO-d6) δ 1.78−1.88 (2H, m), 3.30−3.36
(4H, m), 6.88 (2H, d, J = 8.7 Hz), 7.05 (1H, t, J = 4.5 Hz), 7.82 (2H,
d, J = 8.7 Hz), 7.91 (1H, s), 8.25 (2H, s), 10.05 (1H, s). LC-MS (M +
H) 358.
3.2.2. 3-(2′-((Tetrahydropyrimidin-2(1H)-ylidene)amino)-[4,4′-bi-
thiazol]-2-yl)phenol (6c). The title compound was prepared
according to general procedure A using 3-hydroxybenzothioamide.
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1
Yield 36%. H NMR (DMSO-d6) δ 1.80−1.87 (2H, m), 3.30−3.36
and the cells were incubated for 24 h at 35 °C in YNB with 2%
glucose and a complete mix of amino acids (AAmix), which was
buffered to pH 7.0 with 50 mM MOPS. MIC was defined as the
minimal concentration of antifungal agents that resulted in >50%
reduction of growth compared to the untreated control well with
optical density at 600 nM.
(4H, m), 6.88−6.90 (1H, m), 7.05 (1H, s), 7.31 (1H, t, J = 7.8 Hz),
7.39−7.41 (2H, m), 8.04 (1H, s), 8.25 (2H, s), 9.80 (1H, s). LC-MS
(M + H) 358.
3.2.3. 2-(2′-((Tetrahydropyrimidin-2(1H)-ylidene)amino)-[4,4′-bi-
thiazol]-2-yl)phenol (6d). The title compound was prepared
according to general procedure A using 2-hydroxybenzothioamide.
3.5. Therapeutic Efficacies in the Systemic Aspergillosis
Murine Model. Male ICR mice at 5 weeks of age (CLEA Japan)
were immunosuppressed by intraperitoneal administration of cyclo-
phosphamide at 150 mg/kg on day −4 and at 100 mg/kg on day −1.
Approximately 1.5 × 105 conidia of A. fumigatus in 200 μL of saline
were inoculated intravenously on day 0. ABFG (2), 6h, and VRCZ
(1e) were intravenously administered via tail vein at 2, 16, 21, 26, 40,
45, and 50 h after inoculation to determine prophylactic effects.
ABFG (2) and 6h were dissolved in 5% DMSO, 5% Cremophor EL
and 4% glucose solution for experimental early-stage evaluations.
VRCZ (1e) was dissolved in 5% glucose solution. Kidneys and serum
samples were collected at 60 h after inoculation. Kidneys in lysis
solution were homogenized in a 48-well deep well plate with
ShakeMaster Auto (BMS, Japan) for 5 min at 1100 rpm, and the
resultant homogenates were used for A. fumigatus 18S rRNA
quantification for measuring viable fungal burden as previously
reported.31 Serum samples were subjected to GM quantification. This
animal experiment was approved by the Shionogi Institutional Animal
Care and Use Committee. For 18S rRNA quantification in kidneys,
total RNAs were extracted from the same homogenates of whole
infected kidney with lysis solution containing 4 M guanidine
thiocyanate. The homogenates were filtered through a 96-well
silica-membrane plate (Wizard SV 96 Binding Plates, Promega),
and total RNAs were eluted with distilled water after washing twice
with 70% ethanol in 10 mM Tris buffer. These RNA eluates were
reverse-transcribed with a high-capacity cDNA Reverse Transcription
Kit (Thermo Fisher Scientific) for quantification of the amount of 18S
rRNA. Duplex real-time quantitative PCR was performed using
TaqMan primer and probe sets targeting A. fumigatus 18S rRNA gene
as follows. Forward primer: 5′-GGGCCGCTGGCTTCTTAG-3′,
reverse primer: 5′-TGTTATTGCCGCGCACTTC-3′, and probe: 5′-
(FAM)- ACTATCGGCTCAAGCC MGB-3′. Murine GAPDH was
used as the normalizer for relative quantification with the standard
curve method using Mouse GAPD (GAPDH) Endogenous Control
(VIC/MGB Probe) (Thermo FisherScientific). For serum GM
quantification, PLATELIA ASPERGILLUS Ag kit (BIO-RAD) was
used according to the manufacturer’s protocol. GM amounts are
shown as the values of absorbance at 450 nm in this study.
Nondiluted serum samples were applied to ELISA tests. When the
value of absorbance in the nondiluted serum sample was over 1.0, the
samples were diluted 10-fold and their absorbance was remeasured.
Dunnett’s test was used to compare the untreated control group and
antifungal-treated groups using SAS Ver.9.4.
1
Yield 25%. H NMR (DMSO-d6) δ 1.80−1.90 (2H, m), 3.30−3.36
(4H, m), 6.96 (1H, t, J = 7.5 Hz), 7.04 (1H, d, J = 7.8 Hz), 7.10 (1H,
s), 7.30−7.35 (1H, m), 8.04 (1H, s), 8.13 (1H, dd, J = 7.8, 1.6 Hz),
8.27 (2H, s), 11.39 (1H, s). LC-MS (M + H) 358.
3. 2. 4. N-(2′-(Pyridin-4-yl)-[4, 4′-bithiazol]-2-yl)-
tetrahydropyrimidin-2(1H)-imine (6e). The title compound was
prepared according to general procedure A using pyridine-4-
carbothioamide. Yield 33%. 1H NMR (DMSO-d6) δ 1.83−1.84
(2H, m), 3.34−3.36 (4H, m), 7.15 (1H, s), 7.94 (2H, dd, J = 4.5, 1.6
Hz), 8.24−8.26 (3H, m), 8.73 (2H, dd, J = 4.5, 1.6 Hz). LC-MS (M +
H) 343.
3. 2. 5. N-(2′-(Pyridin-3-yl)-[4, 4′-bithiazol]-2-yl)-
tetrahydropyrimidin-2(1H)-imine (6f). The title compound was
prepared according to general procedure A using pyridine-3-
carbothioamide. Yield 47%. 1H NMR (DMSO-d6) δ 1.79−1.89
(2H, m), 3.31−3.38 (4H, m), 7.14 (1H, s), 7.57 (1H, dd, J = 7.9, 4.8
Hz), 8.17 (1H, s), 8.26 (2H, br s), 8.32 (1H, s), 8.36 (1H, dt, J = 7.9,
1.9 Hz), 8.69 (1H, dd, J = 4.8, 1.6 Hz). LC-MS (M + H) 343.
3. 2. 6. N-(2′-(Pyridin-2-yl)-[4, 4′-bithiazol]-2-yl)-
tetrahydropyrimidin-2(1H)-imine (6g). The title compound was
prepared according to general procedure A using pyridine-2-
carbothioamide. Yield 42%. 1H NMR (DMSO-d6) δ 1.80−1.90
(2H, m), 3.30−3.39 (4H, m), 7.11 (1H, s), 7.51−7.53 (1H, m), 8.00
(1H, td, J = 7.7, 1.7 Hz), 8.15 (1H, s), 8.20 (1H, d, J = 7.9 Hz), 8.27
(2H, s), 8.65 (1H, d, J = 4.3 Hz). LC-MS (M + H) 343.
3.2.7. N-(2′-(4-(Methylsulfonyl)phenyl)-[4,4′-bithiazol]-2-yl)-
tetrahydropyrimidin-2(1H)-imine (6h). The title compound was
prepared according to general procedure A using 4-(methylsulfonyl)-
benzothioamide. Yield 69%. 1H NMR (CDCl3) δ 2.00−2.05 (2H, m),
3.09 (3H, s), 3.46−3.52 (4H, m), 7.22 (1H, s), 7.58 (1H, s), 8.03
(2H, d, J = 8.8 Hz), 8.21 (2H, d, J = 8.5 Hz). LC-MS (M + H) 420.
3.2.8. N-(2′-(3-(Methylsulfonyl)phenyl)-[4,4′-bithiazol]-2-yl)-
tetrahydropyrimidin-2(1H)-imine Hydrobromide (6i). To the
mixture of 26 (45.6 mg, 0.15 mmol) in EtOH (1 mL) was added
3-(methylsulfonyl)benzothioamide (32.4 mg, 0.15 mmol), followed
by heating to reflux for 2 h. After being cooled to room temperature,
the obtained solid was collected by filtration and washed with EtOH
1
and then CHCl3 to give 6i (59.5 mg, 79%). H NMR (DMSO-d6,
HBr salt) δ 1.92−1.98 (2H, m), 3.33−3.35 (3H, m), 3.47−3.50 (4H,
m), 7.74 (1H, br s), 7.84 (1H, t, J = 7.6 Hz), 8.09 (1H, d, J = 7.4 Hz),
8.35 (1H, d, J = 7.9 Hz), 8.50−8.50 (1H, m), 8.56 (1H, s), 8.93 (2H,
br s), 11.92 (1H, br s). LC-MS (M + H) 420.
3.3. Strains and Agents. A. fumigatus ATCC 204305 and C.
albicans ATCC 90028 for MIC tests were obtained from the
American Type Culture Collection. A. fumigatus SR47013 (pan-azole
resistance) for the MIC test and MA for in vivo testing were clinical
isolated strains in Japan collected by Shionogi. VRCZ (1e) and ITCZ
(1g) powder were purchased from Tokyo Chemical Industry. ISCZ
(1f) was purchased from AstaTech, Inc., and PSCZ (1h) powder was
purchased from Carbosynth Limited. VRCZ (1e) vials for injection
were purchased from Pfizer (Tokyo, Japan).
3.4. MIC Tests. MIC was determined by the broth microdilution
method, according to the CLSI M38-A2 standard30 with some
modifications as described below. For A. fumigatus, the conidia
concentration was adjusted to approximately 1 × 104 CFU/mL, and
conidia were incubated for 48 h at 35 °C in yeast nitrogen base
(YNB) with 2% glucose and a complete mix of amino acids (AAmix),
which was buffered to pH 7.0 with 50 mM morpholinepropane-
sulfonic acid (MOPS) in the presence or absence of 50% heat-
inactivated bovine serum (Thermo Fisher Scientific). MIC was
defined as the minimal concentration of antifungal agents that
resulted in no visible growth. For C. albicans, the yeast cell
concentration was adjusted to approximately 2 × 103 CFU/mL,
3.6. Metabolic Stability Studies. The metabolic stabilities of the
test compound in rat and human liver microsomes were determined
at one concentration (0.5 μM). The compounds were incubated with
0.5 mg protein/mL in suspension in 50 mM Tris−HCl buffer (pH
7.4), 150 mM KCl, 10 mM MgCl2, and 1 mM NADPH at 37 °C.
Microsomal incubations were initiated by the addition of 100-fold
concentrated solution of the compounds. Incubations were
terminated by addition of 2-fold volume of organic solvent
(MeCN/MeOH = 1:1) after 0 and 30 min of incubation at 37 °C.
The preparation protein was removed by centrifugation. The
supernatants were analyzed by LC/MS/MS. All incubations were
conducted in duplicate, and the percentage of compound remaining at
the end of the incubation was determined from the LC/MS/MS peak
area ratio.
3.7. Pharmacokinetic Studies. For pharmacokinetic tests in rats,
male Sprague−Dawley rats (8 weeks) were purchased from Charles
River Laboratories. Compounds were formulated as solutions in
DMA/propylene glycol (1:1, v/v) and dosed intravenously from the
tail vein at 0.5 mg/1 mL/kg (n = 2) under isoflurane anesthesia under
nonfasted condition. Blood samples were collected with 1 mL syringes
containing anticoagulants (EDTA-2 K and heparin) at 2, 5, 15, 30, 60,
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120, 240, 360, 480, and 1440 min after dosing. For pharmacokinetic
tests in mice, male ICR mice (6 weeks) were purchased from Japan
Clea. 6h, formulated as solutions in 5% DMSO, 5% Cremophor EL,
and 4% glucose, and VRCZ (1e) for injection were used as solutions
in 5% glucose. These agents were dosed intravenously from the tail
vein at 30 mg/10 mL/kg (n = 3). Blood samples were collected with 1
mL syringes containing anticoagulants (EDTA-2 K and heparin) at 5,
30, 60, 180, and 360 min after dosing. Blood samples were centrifuged
to obtain plasma samples, and plasma concentrations were
determined by LC/MS/MS following protein precipitation with
methanol. Pharmacokinetic parameters were calculated using
WinNonlin based on a noncompartment model.
Makoto Kawai − Shionogi Pharmaceutical Research Center,
Toyonaka, Osaka 561-0825, Japan
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00883
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully thank Hiroyuki Nakai for coordination
of the efficacy tests, Yukio Horita for the MIC tests, Mari Izawa
for the pharmacokinetic tests, and Kazuyo Terada for the
purification by preparative HPLC.
3.8. Sterol Analysis. Cultures of 1 × 104 CFU/mL of A. fumigatus
ATCC 204305 in 50 mL of YNB medium with 2% glucose and
AAmix were kept for 24 h at 35 °C, and then VRCZ (1e) and 6h were
added to the culture, followed by incubation for 24 h. The mycelial
pellets were harvested by vacuum filtration and mixed with 25%
ethanolic KOH. The mixtures were incubated at 85 °C for 1 h; then,
sterols were extracted by addition of n-hexane and sterile distilled
water. The upper n-hexane layer was dried under nitrogen at 40 °C
and redissolved in 200 μL of acetone for 100 mg of mycelial dry
weight; then, 2 μL of the extract was subjected to HPLC/APCI-MS
analysis. Ergosterol, lanosterol, and eburicol were individually
analyzed by liquid chromatography/mass spectrometry (LC/MS).
The LC/MS analysis of sterols was performed with an Acquity Ultra
Performance liquid chromatography (Waters) LC system equipped
with an Orbitrap (Thermo Fisher Scientific) FT-MS mass
spectrometer operated in positive atmospheric pressure chemical
ionization (APCI) mode. Chromatographic separations were
performed on a 1.7 μm BEH C18 column (2 mm × 100 mm i.d.)
at 40 °C with gradient elution using acetonitrile.
ABBREVIATIONS USED
■
ABFG, abafungin; BS, bovine serum; Cmpd, compound;
DMPB, 2,4-dimethyl-1-phenoxybenzene; IFIs, invasive fungal
infections; ISCZ, isavuconazole; ITCZ, itraconazole; MIC,
minimum inhibitory concentration; PSCZ, posaconazole;
SMT, sterol 24-C-methyltransferase; VRCZ, voriconazole
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00883.
■
sı
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AUTHOR INFORMATION
Corresponding Author
Issei Kato − Shionogi Pharmaceutical Research Center,
Toyonaka, Osaka 561-0825, Japan; orcid.org/0000-
0002-0234-8639; Phone: +81-6-6331-5428;
Email: issei.katou@shionogi.co.jp; Fax: +81-6-6332-6385
■
Authors
Yuuta Ukai − Shionogi Pharmaceutical Research Center,
Toyonaka, Osaka 561-0825, Japan
Noriyasu Kondo − Shionogi Pharmaceutical Research Center,
Toyonaka, Osaka 561-0825, Japan
Kohei Nozu − Shionogi Pharmaceutical Research Center,
Toyonaka, Osaka 561-0825, Japan
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(13) Bruggemann, R. J. M.; Alffenaar, J. W. C.; Blijlevens, N. M. A.;
̈
Billaud, E. M.; Kosterink, J. G. W.; Verweij, P. E.; Burger, D. M.
Clinical Relevance of the Pharmacokinetic Interactions of Azole
Antifungal Drugs with Other Coadministered Agents. Clin. Infect. Dis.
2009, 48, 1441−1458.
Chiaki Kimura − Shionogi Pharmaceutical Research Center,
Toyonaka, Osaka 561-0825, Japan
Kumi Hashimoto − Shionogi Pharmaceutical Research Center,
Toyonaka, Osaka 561-0825, Japan
Eri Mizusawa − Shionogi Pharmaceutical Research Center,
Toyonaka, Osaka 561-0825, Japan
Hideki Maki − Shionogi Pharmaceutical Research Center,
Toyonaka, Osaka 561-0825, Japan
(14) haabane, F.; Graf, A.; Jequier, L.; Coste, A. T. Review on
Antifungal Resistance Mechanisms in the Emerging Pathogen
Candida Auris. Front. Microbiol. 2019, 10, No. 2788.
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Mechanisms and Clinical Impact. Clin. Infect. Dis. 2008, 46, 120−128.
(16) Perlin, D. S. Resistance to Echinocandin-Class Antifungal
Drugs. Drug Resist. Updates 2007, 10, 121−130.
Akira Naito − Shionogi Pharmaceutical Research Center,
Toyonaka, Osaka 561-0825, Japan
10495
https://doi.org/10.1021/acs.jmedchem.1c00883
J. Med. Chem. 2021, 64, 10482−10496

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