Improved synthesis of anxiolytic, anticonvulsant, and antinociceptive α2/α3-GABA(A)-ergic receptor subtype selective ligands as promising agents to treat anxiety, epilepsy, and neuropathic pain

Article abstract of DOI:10.1055/s-0037-1610211

An improved synthesis of the anxiolytic, anticonvulsant, and antinociceptive compounds: Hz-166, and its bioisosteres 1,2,4-oxadiazole (MP-III-080) and 1,3-oxazole (KRM-II-81) were synthesized in higher yields and with more facile purification methods (crystallization, etc.) in multigram quantities without column chromatography. In the synthesis of KRM-II-81, an alternative procedure was employed using the selective reducing reagent, potassium diisobutyl-tert-butoxyaluminum hydride (PDBBA), to prepare the desired C(3)-aldehyde in the absence of N(5)-C(6) imine reduction in good yield on a 20 gram scale.

Full text of DOI:10.1055/s-0037-1610211

SYNTHESIS
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©
Georg Thieme Verlag Stuttgart · New York
2018, 50, A–I
paper
A
en
Synthesis
G. Li et al.
Paper
Improved Synthesis of Anxiolytic, Anticonvulsant, and Antinoci-
ceptive α2/α3-GABA(A)-ergic Receptor Subtype Selective Ligands
as Promising Agents to Treat Anxiety, Epilepsy, and Neuropathic
Pain
Guanguan Li
0
0
0
0
0-
0
0
2
2-
2
8
4
0-
3
4
8
O
O
N
N
H
Al
O
Lalit K. Golani
Rajwana Jahan
Farjana Rashid
N
O
K
N
H
N
N
James M. Cook*
N
selective reducing agent PDBBA
N
Department of Chemistry and Biochemistry, Milwaukee Insti-
tute for Drug Discovery, University of Wisconsin-Milwaukee,
Milwaukee, WI 53201, USA
ester 1
aldehyde 4
8
0% (20 g)
8
8%
89%
O
O
N
N
N
capncook@uwm.edu
N
N
N
N
N
N
N
N
1
,2,4-oxadiazole 2
1,3-oxazole 3
Received: 04.06.2018
Accepted after revision: 06.06.2018
Published online: 24.07.2018
Accumulating evidence suggests that α2β2/3γ2 and/or
α3β2/3γ2 GABA R subtype selective ligands exhibit agonist
A
DOI: 10.1055/s-0037-1610211; Art ID: ss-2018-m0272-op
efficacy and are responsible for the anxiolytic, antinocicep-
tive, and much of the anticonvulsant actions when there is
Abstract An improved synthesis of the anxiolytic, anticonvulsant, and
antinociceptive compounds: Hz-166, and its bioisosteres 1,2,4-oxadi-
azole (MP-III-080) and 1,3-oxazole (KRM-II-81) were synthesized in
higher yields and with more facile purification methods (crystallization,
etc.) in multigram quantities without column chromatography. In the
synthesis of KRM-II-81, an alternative procedure was employed using
the selective reducing reagent, potassium diisobutyl-tert-butoxyalumi-
num hydride (PDBBA), to prepare the desired C(3)-aldehyde in the ab-
sence of N(5)–C(6) imine reduction in good yield on a 20 gram scale.
5,8–16
little or no efficacy at the α1-subtypes.
These results
indicated that GABA α2 or α3 agonists can be used for the
A
treatment of anxiety, epilepsy as well as neuropathic pain
with reduced side effects.
Previously, based on our BZD/GABAA pharmacophore
3,17
model,
a series of imidazobenzodiazepine (IBZ) agonists
that are selective at GABA α2/3-subunits was synthesized
A
and investigated. Ligand Hz-166 (1) was one of the first and
the initial lead for IBZs,8 which contained an acetylene
group at the C(8) position in order to reduce α1-subtype ef-
ficacy and/or the binding affinity.3 In both mice and rats,
the lead compound 1 (Figure 1) exhibited antiseizure activ-
ity in a subcutaneous metrazole seizure (scMET) test in the
animal model with both oral and intraperitoneal adminis-
tration.18 Later in the Vogel conflict assay, 1 showed a non-
Key words GABA R, benzodiazepines, anxiety, epilepsy, neuropathic
pain, large scale, imine, selective reduction
A
,17
In the central nervous system (CNS), gamma-aminobu-
tyric acid type A receptors (GABA Rs) are transmembrane
A
1
pentameric ligand-gated ionotropic channels. They are the
major inhibitory neurotransmitter receptors in the CNS and
affect a wide variety of brain functions depending on the
modulation of the GABA-ergic ligands at different subunits
12
sedating anxiolytic effect at 1 mg/kg in rhesus monkeys.
Moreover, 1 was also active in models of inflammatory and
neuropathic pain exerting a dose-dependent antihyperalge-
sic effect without sedation, motor impairment or develop-
ment of tolerance, as compared to the commonly used drug
2
of the protein complex. Benzodiazepines (BZDs), which
3
bind at the α+γ – extracellular interface of GABA Rs, are
2
A
the classical psychoactive drugs prescribed for various CNS
disorders including anxiety, convulsions, as well as insom-
19
gabapentin for the management of neuropathic pain. Al-
though 1 was quite stable in human liver microsomes
(HLMs), it was degraded very rapidly in mouse liver micro-
somes (MLMs) due to hydrolysis of the ester function to its
carboxylic acid by enzymes in the liver.20 The C(3) acid me-
tabolite was not able to penetrate the blood-brain barrier
and achieve the desired effects. This limited the ability to
4–7
nia. They have been employed to treat patients for over
0 years. However, these drugs, for example, diazepam, are
5
not effective in all patients, and moreover, produce serious
adverse effects including sedation, ataxia, amnesia, and ad-
4
diction. Consequently, there is an urgent need for im-
proved treatments, which are devoid of the above side ef-
fects.
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–I

B
Synthesis
G. Li et al.
Paper
do ADME toxicity studies in rodents. The pharmacokinetic
profile of a ligand is one of the major concerns that can re-
sult in failure of promising candidates in clinical trials.
(RLM), and dog liver microsomes (DLM).13 This permits fur-
ther investigation of ligands 2 and 3 in models of anxiety,
epilepsy, neuropathic pain, and respiration in rodents as
well as primates.12 Consequently, multiple gram quantities
of both ligands are required for more critical evaluation in
rodent models, as well as further important tests for ADME
toxicity before human trials. As previously reported, the di-
rect conversion from the ethyl ester 1 to the ethyl oxadi-
O
N
N
O
N
azole 2 earlier was only achieved in moderate yield from
N
0% to 71%8
,11,13
on milligram scale. However, it was not
4
possible to obtain a similar yield on gram scale. The major
Hz-166 (1)
roadblock was the synthesis of the oxazole 3, the overall
O
O
13,26
N
N
N
yield of which via the previous three-step procedure
N
13
N
N
N
was only 28% on a maximum scale of 3 grams. The most
critical problem in the total synthesis of these two ligands
was the low overall yield (17%) for the five-step route to the
parent compound 1, which was used directly as the start-
N
N
8
N
N
ing material for both 2 and 3. Therefore, the modification of
the published routes or other efficient and inexpensive pro-
cedures were needed. Herein, the optimization of the previ-
ous methods for the synthesis of 1 and 2, and an improved
route for the synthesis of 3 are reported. They are applica-
ble for the scale-up process with improved yields, which is
key for further studies of clinical potential.
As demonstrated above, the improvement of the syn-
thesis of Hz-166 (1) would play a critical role in the devel-
opment of GABAA α2/3-subtype selective ligands since it
was the parent compound for all analogues in this series.
After many changes and the modifications in each step in
the synthetic route for Hz-166 (1), this provided a better
five-step overall yield of 56% (Scheme 1), as compared to
the previous yield of 17%.8
MP-III-080 (2)
KRM-II-81 (3)
Figure 1 Structures of Hz-166 (1), MP-III-080 (2), and KRM-II-81 (3)
Currently, ester bioisosteres have been commonly used
in drug development to increase the metabolic stability and
achieve better pharmacological effects.
vent hydrolysis of the liable ester moiety and maintain the
anxiolytic-like properties of lead ligand 1, a number of bio-
isosteres to replace the C(3) ester functionality were de-
21–24
In order to pre-
11,25,26
signed, synthesized, and evaluated.
The most potent
analogues among these ligands were the 1,2,4-oxadiazole
MP-III-080 (2) and the 1,3-oxazole KRM-II-81 (3) (Figure 1),
both of which markedly improved the pharmacokinetic
properties in human, mouse, and rat liver microsomes, as
compared to the parent compound 1. More recent results
were reported indicating oxazole 3 exhibited anxiolytic ef-
First, the commercially available amine, 2-(2-amino-5-
bromobenzoyl)pyridine (5), was acylated with bromoacetyl
bromide in the presence of sodium bicarbonate. The inter-
mediate 6, which formed, without further purification, was
directly treated with a saturated solution of ammonia (gas)
in methanol by gradually increasing the temperature from
0 °C to reflux to provide the 1,4-benzodiazepine 7 in 78%
overall yield (two steps) on a 120 gram scale. The amide 7
can be purified by recrystallization from methanol and di-
chloromethane while avoiding any column chromatography
on a large scale. The imidazole ring was introduced by ap-
1
3
fects in a rat Vogel conflict assay at 10 mg/kg when given
intraperitoneally. Furthermore, in a mouse marble-burying
1
3,14,27
assay,
as a model of anxiety, ligand 2 significantly re-
duced the marble-burying activity at 10 mg/kg as an indi-
cation of anxiolytic-like effects, and both of the bioisosteres
2
and 3 exerted anxiolytic activity at 30 mg/kg.13 All three
ligands were shown to be α2/3 subtype selective ligands, as
positive allosteric modulators (PAMs), with little or no ago-
nist efficacy at α1-containing GABA Rs.
12,14,28
29
In addition, li-
plying the modified procedure by treating the amide 7
A
gand 3 exhibited potent antinociceptive activity in a model
of acetic acid- and lactic acid-induced pain in mice.28 Later
in the motor function and anticonvulsant efficacy assays, 2
with potassium tert-butoxide and diethyl chlorophosphate
at –50 °C for 2 hours, after which the solution was allowed
to warm up to 0 °C. Ethyl isocyanoacetate was then added
to the reaction mixture, which was executed at –78 °C, and
this was followed by addition of another equivalent of po-
tassium tert-butoxide to provide IBZ 8 in 81% yield. The
yield of the imidazole ring formation by a previous method
was an effective anticonvulsant versus the GABA negative
A
modulator pentylenetetrazole (PTZ)-induced seizures with
no observation of motor impairment at 30 mg/kg on intra-
14
peritoneal administration.
Taken together all these results suggest that instead of
the rapidly metabolized parent compound 1 in rodents, li-
gands 2 and 3, which are novel α2/3-subtype selective GAB-
using sodium hydride as the base by Fryer at –10 °C was
This process was improved to 81% by replac-
ing sodium hydride with potassium tert-butoxide at a lower
only 37%.3
0–32
A Rs PAMs, are stable in HLM, MLM, rat liver microsomes
temperature.
A
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–I

C
Synthesis
G. Li et al.
Paper
O
Br
H
O
NH2
N
NH
THF, t-BuOK, –50 oC to 0 °C;
ClPO(OEt)2, –50 °C to 0 °C;
sat. MeOH/NH3
0 °C to reflux, 10 h
O
B
r
C
O
C
H
B
r
,
N
a
H
C
O
2
3
O
N
Br
N
Br
Br
dry DCM, 0 °C to rt, 8–10 h
78% (over two steps)
2 2
CNCH CO Et, t-BuOK
–78 °C to rt, 14 h
N
N
81%
5
(120 g scale)
6
7
O
O
N
O
N
N
N
O
TMS-acetylene
N
O
N
O
Pd(OAc)2(PPh3)2
Et3N/ACN
reflux, 10 h
TBAF·xH2O, THF
–78 °C, 0.5 h
Br
N
N
N
N
Si
92%
96%
N
N
8
9
Hz-166 (1) (56% overall yield)
Scheme 1 Optimized total synthesis of Hz-166 (1)
The purification of 8 was easily executed by washing
away the impurities with 10% ethyl acetate in diethyl ether.
The scale of this reaction was increased to 80 grams as
compared to the previous one of 15 grams in the literature.
A Heck–Cassar–Sonogashira type reaction was conducted
with IBZ 8 and trimethylsilylacetylene to provide the
trimethylsilyl analogue 9 in 92% yield. Lead ligand Hz-166
starting materials.34 This was deemed not suitable for the
present case with ligands containing multiple functional-
ities. Therefore, many attempts were made to improve the
yield of the oxadiazole formation for the synthesis of grams
of ligand 2.
For this purpose several experiments were conducted
under different conditions. It was found that the equiva-
lents of sodium hydride, the reaction time for the formation
of amidoxime anion, dry reaction conditions, as well as the
reaction temperature played a critical role for good yields.
Initially, the ratio of sodium hydride to the ethyl ester 1 was
2.5:1 on a milligram scale and the yield of the by-product,
whose imine bond had been reduced, was comparable to
(
1) was obtained by the deprotection of the analogue 9 us-
ing tetrabutylammonium fluoride in 96% yield. After work-
up (see Experimental Section) the pure material 9 was re-
crystallized from methanol, which had been heated to 50 °C
and allowed to cool.
With the optimized synthesis of the parent compound
1
1
, the overall yield was improved from 17% to 56%. The pu-
the desired oxadiazole 2, as confirmed by analysis of the H
rification process in case of ligands such as 1 was more dif-
ficult than the other ligands in the IBZ series in the 2′-F, 2′-
H, etc. due to the 2′-pyridine nitrogen, which can be a prob-
lem when using column chromatography on a large scale. In
this new procedure, all the products can be isolated by ei-
ther washing with specific solvents or by crystallization to
avoid column chromatography. This is a more practical, ef-
ficient and scalable synthesis of 2′-pyridine-containing
substituted IBZ 1 as the starting material for other import-
ant analogues.
NMR spectrum. The formation of the major by-product re-
sulted from the extra equivalent of sodium hydride, which
reduced the N(5)–C(6) imine bond of the parent compound
35
1. Hence, the amount of sodium hydride was kept at 1.1
equivalents to avoid the unwanted imine reduction. Since
esters are less reactive than the corresponding acyl chlo-
ride, the formation of the amidoxime anion was the key and
the only driving force for acylation; the first step toward
oxadiazole formation. Therefore, the reaction time of the
amidoxime with sodium hydride should be adequate and
1
The 1,2,4-oxadiazoles are well-known ester bioisosteres
H NMR spectroscopy was required to monitor the reaction
that ameliorate the problems of metabolism in the drug
to completion due to the inability of analysis by TLC. Com-
discovery process.2
0,21,23,24,33
The oxadiazole MP-III-080 (2)
monly, the thermal cyclization of O-acylamidoximes is the
36
is a very promising ligand in this series, which exhibited
anxiolytic, anticonvulsant, and antinociceptive effects in
many animal models as described above. The problems of
inconsistent yields of 2 from 1 required a solution of scale-
up. The development of efficient methods for the synthesis
of 1,2,4-oxadiazoles has been ongoing for decades, however,
most of the existing procedures either required harsh con-
ditions or multiple steps from ester to the corresponding
second step required to prepare the 1,2,4-oxadiazoles;
however, the higher temperature in the IBZ series usually
led to low yields (40–47%),8 along with formation of by-
products. Consequently, instead of using the usual condi-
tions of reflux, the reactions were performed at room tem-
perature. In addition, 3Å molecular sieves were added, the
amount of which depended on the reaction scale. Efficient
removal of water as an alternative method helped push the
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Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–I

D
Synthesis
G. Li et al.
Paper
reaction at room temperature to completion. To date, with
these optimizations, the yield of the one-step conversion
from ester to oxadiazole was improved to 88%, as depicted
in Scheme 2. It is consistent for gram-scale processes.
with activated manganese dioxide, or pyridinium dichro-
mate. This mixture of alcohols (desired and imine-reduced),
which formed, was then directly oxidized to a mixture of al-
dehydes with activated manganese dioxide in an overall
two-step yield of 39%, as reported previously.13 Even though
the next step (the oxazole formation) was executed in a rea-
sonable yield (73%), the three-step overall yield of 3 had
fallen to 29%, which was not acceptable.
In order to overcome this problem, a number of weaker
reducing agents were employed, such as lithium borohy-
dride, sodium borohydride, potassium borohydride, or the
bulkier lithium tri-tert-butoxyaluminum hydride, however,
none of these agents provided the desired aldehyde in good
yield, as shown in Scheme 3. The solution to this issue was
to find a bulkier reducing agent but also a selective reducing
agent to prevent imine reduction and remain at the alde-
hyde stage in the absence of alcohol formation.
O
O
N
N
N
N
O
N
N
OH
N
N
N
NH2
N
N
NaH, 3Å MS,
THF, rt, 3 h
1
2
88%
Scheme 2 Synthesis of MP-III-080 (2) from Hz-166 (1)
Beside 1,2,4-oxadiazoles as bioisosteres, 1,3-oxazoles
are unique bioisosteres with an electron density map simi-
lar to that of an ester function. Oxazole-containing com-
pounds have been reported with desired pharmacological
effects and promising therapeutic profiles in the past.3
Accordingly, the 1,3-oxazole analogues of the parent com-
pound 1, which can be used as an alternative ester bio-
isostere, appeared to be a useful complement to 2. The de-
velopment of the synthesis of oxazoles has been employed
for years, and numerous methods have been reported.40 In
the case under study here, a one- or two-step synthetic
route would be appropriate in order to avoid low overall
yields from multi-step synthesis. As compared to other ap-
(1) LiAlH4, dry THF
0
°C to rt , 1 h
7–39
(2) MnO2, dry DCM, 10 h
39% for 2 steps
O
O
N
N
DIBAL
X
N
OEt
N
H
NaBH4
X
N
N
KBH4
X
N
N
LiBH4
X
1
4
41
LiAlH(Ot-Bu)3
proaches, the Van Leusen oxazole synthesis, which can be
used to prepare 1,3-oxazoles by the direct conversion from
the corresponding aldehyde, seemed advantageous because
of the one-pot process and mild conditions. Indeed, this
transformation provided good yields for oxazole formation
in other series. However, the synthesis of the most potent
oxazole bioisostere, KRM-II-81 (3), as described above (2′-N
analogue), was the most difficult bioisostere in the series of
IBZ analogues (i.e., 2′-F, 2′-H, 2′-Cl, etc.) to prepare. The
problem was not from the formation of the oxazole, but the
reduction reaction from the particular ester 1 to its corre-
sponding aldehyde 4.
X
Scheme 3 Attempts to prepare aldehyde 4 from Hz-166 (1)
Because of work in another series, potassium diisobu-
tyl-tert-butoxyaluminum hydride (PDBBA)42 seemed to be a
suitable reducing agent for this series. PDBBA can be easily
prepared by addition of DIBAL-H in hexane dropwise into a
solution of the same equivalents of potassium tert-butoxide
in THF at 0 °C for 2 hours.42 In theory, the PDBBA should not
attack the imine double bond due to the three bulky
groups; second, it can only offer one hydride and might be
easier to stop the reaction at the aldehyde stage, as com-
After many attempts with many reducing agents, which
either reduced the N(5)–C(6) imine bond or reduced the al-
dehyde, so formed, at C(3) to the alcohol, diisobutylalumi-
num hydride (DIBAL-H), a commonly used reducing agent,
was employed efficiently in good yield (75–80%) for the 2′-F
pared to four hydride units provided by LiAlH . The report-
4
ed mechanism is straightforward.4
3,44
The hydride from
PDBBA attacks the carbonyl carbon forming a tetrahedral
intermediate, and the elimination of the ethoxy group
yields the desired aldehyde.
13,26
and 2′-H series.
The reduction with DIBAL-H for the eth-
yl ester 1, however, failed to achieve the desired aldehyde in
a temperature range of –78 °C to 25 °C due to the reduction
to the corresponding alcohol and/or imine bond as ob-
served in the case of the oxadiazole synthesis. Moreover,
the purification of this crude reaction mixture was difficult.
After many attempts, it was difficult to stop the reduction
in the 2′-N case at the aldehyde stage. The next strategy was
to employ lithium aluminum hydride (LAH) to reduce the
ester to the alcohol and then reoxidize it to the aldehyde
However, the first attempts were not successful under
the reported conditions42 or at lower temperatures (Table 1,
entries 1–4). The reactions were stopped at 3 hours due to
more alcohol formation. The reactions were then conduct-
ed by using different equivalents of PDBBA from 1.5 to 2.5
at 0 °C at three time points 5, 30, 180 minutes, respectively
(entries 5–14). The use of higher equivalents of PDBBA at 0
°C not only increased the yield of aldehyde, but also the rate
of reduction to the alcohol at the first 5-minute time point.
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–I

E
Synthesis
G. Li et al.
Paper
Along with longer reaction times, the yields of aldehyde
were not significantly improved and began to drop at 30
minutes for 2 and 2.5 equivalents in trials of PDBBA (entries
Table 1 Optimization of Reduction Conditionsa
H
O
N
O
N
Al
O
K
9, 10, 12, and 13). The aldehyde, which formed, was imme-
N
OEt
N
H
diately converted into the alcohol in this series of IBZ, as ob-
served at 0 °C. If the reactivity of the ester could be in-
creased, then it could compete with the aldehyde and as a
result, additional aldehyde and less alcohol could be pre-
dicted. With this in mind, a modification was made to in-
crease the reaction temperature. As expected, more alde-
hyde and less alcohol formation were observed within the
first 5 minutes at room temperature for all equivalents of
PDBBA; the yields of the aldehyde quickly dropped to pro-
vide more alcohol after reaching the maximum yield of al-
dehyde in 5 minutes (entries 14–23), as compared to the
condition at 0 °C. The best yield of aldehyde 4 from all the
trials was 79% (entry 18) with 1.5 equivalents of PDBBA at
N
N
DCM, conditions
N
N
1
4
Entry
PDBBA
equiv)
Temp (°C) Time (min) Yield (%)
CHO
Yield (%)
CH OH
(
2
1
2
3
4
5
6
7
8
9
1.3
1.3
1.3
1.3
1.5
1.5
1.5
2.0
2.0
2.0
2.5
2.5
2.5
1.3
1.3
1.3
1.5
1.5
1.5
2.0
2.0
2.5
2.5
1.3
1.3
–78
0
720
5
0
22
31
38
45
57
60
56
62
51
58
65
40
30
56
11
62
79
18
69
61
70
56
36
40
0
12
14
17
20
21
29
23
31
40
22
29
51
5
0
30
180
5
25 °C; only 12% of the alcohol had formed. At 40 °C, the
0
yield of aldehyde was worse, and the process rapidly
formed the alcohol instead (entries 24, 25).
0
0
30
180
5
From the results, the best condition to obtain a good
yield (79%) of aldehyde was with 1.5 equivalents of PDBBA,
0
42
which was a bit higher than reported, at room tempera-
ture. The reaction process had to be controlled carefully and
monitored by TLC analysis for the first 5–10 minutes to
avoid alcohol formation due to the very short reaction time.
Most importantly, the imine bond was not reduced by PDB-
BA at all; the spectroscopy and properties of the aldehyde 4
match with the reported values as confirmed by NMR and
LCMS. At lower temperature such as at 0 °C, the ester was
much less reactive than at 25 °C, the aldehyde, which
formed at 0 °C, would favorably be reduced and resulted in
a mixture of unreacted ester, desired aldehyde, and the al-
cohol by-product. Moreover, longer reaction times should
be avoided due to the reactive aldehyde, as expected.
0
0
30
180
5
10
11
12
13
0
0
0
30
180
5
0
14
15
25
25
25
25
25
25
25
25
25
25
40
40
30
180
1
28
67
8
16
17
18
19
5
12
68
20
28
25
38
10
33
30
1
Even though the reaction time was very short, this pro-
cedure can be applied on large scale if monitored every 5–
20
1
0 minutes by TLC. To date, the best scale for this reduction
reaction under the optimized reaction conditions was up to
0 grams and in 80% yield, as illustrated in Scheme 4. The
2
2
2
2
1
2
3
4
5
5
1
2
5
van Leusen reaction for the synthesis of the 1,3-oxazoles
occurred smoothly. The oxazole 3 can be purified via crys-
tallization from ethyl acetate to replace the earlier em-
ployed column chromatography, that was reported previ-
1
2
5
a
All reactions were conducted with 0.07 mmol of starting material (the
13
ethyl ester 1) in anhyd DCM as the solvent of the reaction under different
conditions listed above. The yields of aldehyde and alcohol were calculated
after the isolation of the products.
ously.
In conclusion, modifications of previous procedures for
the synthesis of Hz-166 (1), MP-III-080 (2), and KRM-II-81
(
3) are presented in this work under milder conditions and
with much-improved yields. Moreover, an alternative
method for the synthesis of the aldehyde 4 from ester 1 was
optimized and overcame the long-standing problem of
imine reduction in good yield. These methods can be em-
ployed for larger scale reactions. In addition, there is no
need for column chromatography for most of the com-
pounds, which provided a much more practical route. With
the improved synthesis, large quantities of these three
α2/3-subtype selective GABA Rs PAMs, which exhibited
A
anxiolytic, anticonvulsant, and antinociceptive effects in
many tests, can now be supplied with high-efficiency for
further investigation of ADME toxicity and many behavior
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–I

F
Synthesis
G. Li et al.
Paper
O
O
N
N
O
N
N
N
PDBBA
DCM, 0 °C to rt
TosMIC
K2CO3, MeOH
75 °C, 1 h
O
N
N
H
20 min
N
N
80%
89%
N
N
N
N
1
(20 g)
4
3
Scheme 4 The optimized synthesis of oxazole 3 with a significantly improved overall yield
assays including those in primates or other animal models
for anxiety and epilepsy, as well as for neuropathic pain.
ing material was consumed on analysis by TLC (silica gel) in 12 h. The
mixture was then cooled to r.t. and the solvent was removed under
reduced pressure. The solid which remained was filtered and washed
with H O (3 × 150 mL), cold EtOAc (3 × 50 mL), and DCM (3 × 50 mL).
2
The crude solid was dissolved in MeOH (600 mL) and DCM (100 mL)
at 60 °C, and the solution was concentrated to one quarter of its origi-
nal volume. The amide 7 was recrystallized from MeOH and DCM at
r.t. and filtered, after which it was washed with DCM to obtain the
majority of the pure amide 7 as white crystals. The filtrates were
combined and concentrated under reduced pressure to an oily resi-
due, which was further purified by flash chromatography on silica gel
All reactions were carried out in oven-dried round-bottom flasks and
cooled under an argon atmosphere. All organic solvents and chemi-
cals were purchased from commercial suppliers, then further dried
and purified with the PURESOLV Solvent Purification System (Innova-
tive Technology, Inc.) and KNF LABOPORT diaphragm laboratory vacu-
um pumps. Reactions were analyzed and monitored on TLC plates
from Dynamic Adsorbents Inc. under a fluorescence indicator (UV254).
The purification of some compounds were completed by column
chromatography on silica gel (230–400 mesh, Dynamic Adsorbents)
using a glass column. The melting points were determined on a Stuart
melting point SMP3 apparatus manufactured by Barloworld Scientific
(
EtOAc/hexanes 1:1 and 1% of Et N) to afford additional amide 7;
3
yield: 108.6 g (78% over the two steps); mp 228–229 °C; R = 0.4 (EtO-
f
Ac/hexanes 1:1 and 1% of Et N).
3
1
H NMR (300 MHz, DMSO-d ): δ = 10.63 (s, 1 H), 8.55 (d, J = 4.1 Hz, 1
6
1
13
H), 8.04 (d, J = 7.7 Hz, 1 H), 7.93 (d, J = 7.4 Hz, 1 H), 7.69 (d, J = 8.6 Hz,
US Ltd. The H and C NMR spectra were obtained on Bruker Spectro-
1
2
H), 7.54–7.44 (m, 1 H), 7.42 (s, 1 H), 7.18 (d, J = 8.7 Hz, 1 H), 4.23 (s,
H).
spin 300 MHz or 500 MHz instruments in CDCl or DMSO-d . Chemi-
3
6
cal shifts are reported in δ (ppm) relative to TMS as an internal stan-
dard. Standard abbreviations are used to present multiplicities. The
determination of compound purity (>95%) was performed by HPLC-
MS on a Shimadzu LCMS-2020 and HRMS was performed on a Shi-
madzu LCMS-IT-TOF at the Milwaukee Institute for Drug Discovery in
the Shimadzu Laboratory for Advanced and Applied Analytical Chem-
istry.
13C NMR (75 MHz, DMSO-d
): δ = 170.3, 168.1, 156.3, 148.9, 139.3,
6
137.5, 134.4, 134.1, 127.9, 125.4, 123.9, 123.6, 114.5, 57.6.
HRMS (ESI/IT-TOF): m/z [M + H]+ calcd for C14H11BrN O: 316.0080;
3
found: 316.0076.
Ethyl 8-Bromo-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]di-
azepine-3-carboxylate (8)
7
-Bromo-5-(pyridin-2-yl)-1H-benzo[e][1,4]diazepin-2(3H)-one (7)
The amide 7 (80 g, 253.2 mmol) was suspended in anhyd THF (1.5 L),
and cooled to –50 °C using a dry ice bath, after which t-BuOK (34.1 g,
The 2′-pyridyl ketone 5 (120 g, 433.2 mmol) was dissolved in anhyd
DCM (1.4 L) in a 3-necked round-bottom flask and stirred with an
overhead mechanical stirrer for 15 min to obtain a homogenous solu-
303.8 mmol) was added in one portion. The reaction mixture was
stirred until it reached 0 °C and then stirred for 0.5 h at 0 °C. The mix-
ture was then cooled to –50 °C, after which diethyl chlorophosphate
tion. Then solid NaHCO (72.8 g, 866.4 mmol) was added to the solu-
3
tion with vigorous stirring to avoid clogging of the stirrer, and the
mixture was cooled to 0 °C using an ice bath. A solution of bromoace-
tyl bromide (144.9 g, 714.8 mmol) in anhyd DCM (200 mL) was then
added dropwise at 0 °C with an addition funnel. The reaction mixture
was allowed to warm to r.t. and stirred overnight until the starting
material was consumed as monitored on analysis by TLC (silica gel,
EtOAc/hexanes 1:1). The mixture was quenched with ice-water (500
mL) and stirred at r.t. for 30 min. The organic layer was then separat-
ed, and the aqueous layer was extracted with DCM (3 × 250 mL). The
organic layers were combined, washed (200 mL each) sequentially
with sat. aq NaHCO , H O, 10% HCl, brine, and then dried (Na SO ).
(
61.2 g, 354.5 mmol) was added dropwise with an addition funnel.
The dry ice bath was removed to allow the temperature to rise to 0 °C,
after which it was allowed to stir for 2 h on an ice-water bath. The
solution was then cooled to –78 °C with a dry-ice bath, and ethyl iso-
cyanoacetate (40.1 g, 354.5 mmol) was added, immediately followed
by a second portion of t-BuOK (34.1 g, 303.8 mmol). This solution was
allowed to stir overnight during which period it was allowed to warm
to r.t. The reaction was completed after 14 h on analysis by TLC (silica
gel, EtOAc/hexanes/DCM, 2:2:1, and 1% Et N). The mixture was
quenched by addition of a cold sat. aq NaHCO (500 mL) solution and
extracted with EtOAc. The organic layers were combined and washed
with brine (2 × 200 mL) and dried (Na SO ). The solvent was removed
under reduced pressure to obtain a dark brown solid residue. The sol-
3
3
3
2
2
4
The solvent was then concentrated to one quarter of its original vol-
ume under reduced pressure. The intermediate 6, which was pre-
pared, was used in the next step without further purification.
2
4
id was washed with Et O/EtOAc (9:1) to remove most of the impuri-
2
MeOH (1.4 L) was cooled to 0 °C using an ice-water cooling bath and
saturated with anhyd ammonia gas. The DCM solution of the above
prepared intermediate 6 was added to the solution of saturated
ties and the solid was further recrystallized from EtOAc and hexane
(1:4), followed by washing the solid with cold Et O to afford the ma-
2
jority of the pure ethyl ester 8. The remaining filtrates were com-
MeOH/NH at 0 °C. The mixture was allowed to warm to r.t. and slow-
bined, concentrated, and purified by flash chromatography to obtain
3
ly heated to reflux (Caution! mild exotherm observed) until the start-
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–I

G
Synthesis
G. Li et al.
Paper
additional ethyl ester 8 (silica gel, EtOAc/hexanes/DCM 2:2:1 and 1%
Et N) as an off-white solid; yield: 84.2 g (81%); mp 212–213 °C; R =
MeOH (400 mL) at 50 °C and the solution was concentrated to one
third of its original volume. The majority of the ethyl ester 1 was re-
crystallized at r.t. with addition of a seed crystal, and the solid was
further washed with cold MeOH. The filtrates were combined, con-
centrated, and purified by a wash column (silica gel, EtOAc/hex-
3
f
0
.3 (EtOAc/hexanes/DCM, 2:2:1 and 1% of Et N).
3
1
H NMR (300 MHz, CDCl ): δ = 8.60 (d, J = 4.4 Hz, 1 H), 8.11 (d, J = 7.9
3
Hz, 1 H), 8.00 (s, 1 H), 7.86 (td, J = 8.0, 1.7 Hz, 1 H), 7.80 (dd, J = 8.6, 2.2
Hz, 1 H), 7.60 (d, J = 2.1 Hz, 1 H), 7.51 (d, J = 8.6 Hz, 1 H), 7.41 (dd, J =
anes/DCM 8:1:2, and 1% Et N) to afford additional ethyl ester 1 as a
3
white powder; yield: 37.8 g (96%); mp 243–244 °C; R = 0.4 (EtOAc/
7.1, 5.2 Hz, 1 H), 6.13 (d, J = 10.4 Hz, 1 H), 4.51–4.33 (m, 2 H), 4.17 (d,
f
hexanes/DCM, 8:1:2 and 1% of Et N).
J = 11.6 Hz, 1 H), 1.44 (t, J = 7.1 Hz, 3 H).
3
1H NMR (300 MHz, CDCl
): δ = 8.59 (d, J = 4.6 Hz, 1 H), 8.08 (d, J = 7.9
13
3
C NMR (75 MHz, CDCl ): δ = 167.0, 162.9, 156.2, 148.7, 138.4, 136.9,
3
Hz, 1 H), 7.96 (s, 1 H), 7.83 (td, J = 7.8, 1.6 Hz, 1 H), 7.77 (dd, J = 8.4, 1.7
Hz, 1 H), 7.61–7.54 (m, 2 H), 7.38 (dd, J = 7.0, 5.3 Hz, 1 H), 6.12 (d, J =
1
6
35.3, 135.0, 134.5, 134.4, 129.3, 128.5, 124.9, 124.3, 123.9, 120.5,
0.7, 45.0, 14.4.
10.6 Hz, 1 H), 4.56–4.27 (m, 2 H), 4.16 (d, J = 10.2 Hz, 1 H), 3.17 (s, 1
HRMS (ESI/IT-TOF): m/z [M + H]+ calcd for C19H16BrN O : 411.0451;
4
2
H), 1.44 (t, J = 7.1 Hz, 3 H).
found: 411.0454.
13
C NMR (75 MHz, CDCl ): δ = 167.6, 162.8, 156.2, 148.6, 138.4, 137.1,
3
1
8
36.2, 135.4, 135.4, 134.6, 129.2, 127.0, 124.9, 124.1, 122.9, 121.3,
1.6, 79.6, 60.9, 45.0, 14.4.
Ethyl 6-(Pyridin-2-yl)-8-[(trimethylsilyl)ethynyl]-4H-benzo[f]im-
idazo[1,5a][1,4]diazepine-3-carboxylate (9)
HRMS (ESI/IT-TOF): m/z [M + H]+ calcd for C21H17N O : 357.1346;
found: 357.1344.
4
2
The ethyl ester 8 (63.8 g, 155.1 mmol) was dissolved in Et N (400 mL)
3
and anhyd MeCN (600 mL) and placed in a 3-necked round-bottom
flask with a reflux condenser attached. The solution was then de-
gassed three times under vacuum and argon. Trimethylsilylacetylene
3
-Ethyl-5-[8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-
a][1,4]diazepin-3-yl]-1,2,4-oxadiazole (MP-III-080, 2)
(
22.9 g, 232.7 mmol) and Pd(OAc) (PPh ) (6.4 g, 8.53 mmol) were
2 3 2
added to the solution under argon, and the mixture was degassed four
times (as above). The mixture was then heated to reflux under argon
and allowed to stir overnight. The reaction process was completed in
The ethyl ester 1 (4.4 g, 12.4 mmol) was dissolved in anhyd THF (200
mL) at r.t. under argon. In a separate flask that contained 3Å molecu-
lar sieves, N-hydroxypropionimidamide (4.35 g, 19.4 mmol) was dis-
solved in anhyd THF (50 mL) under argon and then treated with NaH
(60% dispersion in mineral oil, 360 mg, 14.8 mmol). The mixture was
allowed to stir for 1.5 h and was then added dropwise to the solution
of ethyl ester 1. The reaction was completed in 3 h as analyzed by TLC
(silica gel). The reaction mixture was quenched with a sat. aq NaHCO3
solution (10 mL) and extracted with EtOAc (3 × 250 mL). The organic
layers were combined, washed with brine, and dried (Na SO ). The
15 h as monitored on analysis by TLC (silica gel, EtOAc/hexanes 7:3,
and 1% Et N). The mixture was then cooled to 0 °C and filtered
3
through Celite. This was followed by washing with EtOAc and drying
(
Na SO ). The filtrate was concentrated under reduced pressure. The
2 4
black residue, which resulted, was loaded onto a silica plug (4 g of sil-
ica gel/1 g of the product) and washed with a mixture of EtOAc and
hexanes (1:1 with 1% Et N) to remove the baseline impurities and the
3
2
4
material was recrystallized from EtOAc. The crystals were filtered and
solvent was removed under reduced pressure. The solid, which result-
ed, was purified by flash chromatography (silica gel, EtOAc/hexanes
washed with Et O to afford pure trimethylsilyl ethyl ester 9. The fil-
2
trate was concentrated and purified by flash chromatography to af-
4:1 and 1% Et N) to afford pure 1,2,4-oxadiazole 2 as a white powder;
3
ford additional ester 9 as an off-white solid; yield: 61.1 g (92%); mp
yield: 4.1 g (88%); mp 204–205 °C; R = 0.6 (EtOAc/hexanes, 4:1 and
f
13
2
03–204 °C; R = 0.5 (EtOAc/hexanes 1:1 and 1% of Et N).
1% of Et N).
f
3
3
1
1
H NMR (300 MHz, CDCl ): δ = 8.56 (d, J = 4.1 Hz, 1 H), 8.02 (d, J = 7.8
H NMR (500 MHz, CDCl ): δ = 8.58 (d, J = 4.3 Hz, 1 H), 8.07 (d, J = 8.0
3
3
Hz, 1 H), 7.90 (s, 1 H), 7.78 (t, J = 7.7 Hz, 1 H), 7.70 (d, J = 8.3 Hz, 1 H),
Hz, 1 H), 8.05 (s, 1 H), 7.83 (dd, J = 11.8, 4.4 Hz, 1 H), 7.78 (dd, J = 8.4,
1.3 Hz, 1 H), 7.61 (d, J = 8.3 Hz, 1 H), 7.57 (d, J = 1.0 Hz, 1 H), 7.38 (dd,
J = 6.7, 5.3 Hz, 1 H), 6.16 (d, J = 11.4 Hz, 1 H), 4.29 (d, J = 11.2 Hz, 1 H),
3.19 (s, 1 H), 2.85 (q, J = 7.6 Hz, 2 H), 1.44 (t, J = 7.6 Hz, 3 H).
13
7.51 (d, J = 9.2 Hz, 2 H), 7.34 (t, J = 5.9 Hz, 1 H), 6.08 (d, J = 9.2 Hz, 1 H),
4.47–4.31 (m, 2 H), 4.10 (d, J = 6.9 Hz, 1 H), 1.41 (t, J = 7.0 Hz, 3 H),
0.21 (s, 9 H).
13
C NMR (75 MHz, CDCl ): δ = 167.8, 162.9, 156.6, 148.8, 138.5, 136.8,
C NMR (126 MHz, CDCl ): δ = 171.9, 170.8, 167.9, 156.3, 148.7,
3
3
1
1
35.7, 135.2, 135.1, 134.5, 129.3, 127.0, 124.7, 124.0, 122.7, 122.2,
02.8, 97.0, 60.7, 45.0, 14.4, –0.2.
137.1, 136.3, 136.2, 135.9, 135.5, 135.2, 127.1, 125.0, 124.8, 124.1,
122.9, 121.4, 81.6, 79.7, 44.9, 19.8, 11.6.
HRMS (ESI/IT-TOF): m/z [M + H]+ calcd for C24H25N O Si: 429.1741;
HRMS (ESI/IT-TOF): m/z [M + H]+ calcd for C22H17N O: 381.1458;
4
2
6
found: 429.1747.
found: 381.1461.
Ethyl 8-Ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-
a][1,4]diazepine-3-carboxylate (HZ-166, 1)
8-Ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diaze-
pine-3-carbaldehyde (4)
The trimethylsilyl ethyl ester 9 (50 g, 116.8 mmol) was dissolved in
Preparation of PDBBA: t-BuOK (29.3 g, 261 mmol) was suspended in
anhyd THF (261 mL). DIBAL-H (261 mL, 1.0 M in hexane, 261 mmol)
was added dropwise to the solution of t-BuOK cooled at 0 °C using an
ice-water bath. The mixture was allowed to stir at r.t. for 2 h to
achieve a colorless homogeneous solution. The concentration of PDB-
BA solution in THF/hexanes was measured by GC analysis by hydroly-
sis of the solution with a mixture of t-BuOH/THF (1:1) at 0 °C.39
THF (1 L) and cooled to –78 °C. Bu NF·xH O (1 M solution in THF,
4
2
1
75.1 mmol) was added to the solution, and this was followed by H O
2
(50 mL). The reaction mixture was stirred at –78 °C until the starting
material was consumed in 0.5 h, as indicated by TLC (silica gel). The
mixture was allowed to warm to 0 °C and quenched by a slow addi-
tion of H O (500 mL). The organic layer was separated and the aque-
2
ous layer was extracted with EtOAc (5 × 300 mL). The combined or-
ganic layers were washed with brine (2 × 400 mL) and dried (Na SO ).
The solvent was removed under reduced pressure and the residue,
which resulted, was dissolved in a mixture of DCM (100 mL) and
The Reduction of Ethyl Ester 1 to Aldehyde 4: The ethyl ester 1 (20 g,
56.1 mmol) was dissolved in anhyd DCM (500 mL) at 0 °C. The ice-
water bath was removed, and the PDBBA solution (0.42 M in THF/hex-
anes, 200 mL) was added in one portion with vigorous stirring. The
2
4
©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–I

H
Synthesis
G. Li et al.
Paper
reaction was monitored by TLC (silica gel) after each 3 min and was
completed in 20 min, and this was followed by hydrolysis of the mix-
Funding Information
We would like to thank the NIH for generous financial support
ture in 1 N aq HCl at 0 °C. H O (200 mL) was added to the mixture and
2
(MH096463, NS076517).
N
oaitn
a
l
nItusite
of
M
e
n
atl
H
e
hatl
M(
H
0
9
6
4
6
3
N) oaitn
a
l
n
Itsu
i
te
of
N
e
urolgcai
l
Dsoidrers
a
n
d
Srto
k
e
N( S
0
7
6
5
1
7)
stirred for 0.5 h. The aluminum salt formed was filtered off on a Celite
pad. The filtrate was extracted with DCM (3 × 150 mL). The organic
layers were combined, washed with brine, and dried (Na SO ). The
2
4
Acknowledgment
solid residue obtained by concentration was dissolved in a mixture of
DCM (100 mL) and EtOAc (100 mL) at 50 °C. A seed crystal was added
to the mixture and the solvent was reduced to one quarter of its orig-
inal volume. The aldehyde 4 was crystallized from the mixture at r.t.
We acknowledge the Milwaukee Institute for Drug Discovery and the
University of Wisconsin-Milwaukee’s Shimadzu Laboratory for Ad-
vanced and Applied Analytical Chemistry for spectroscopy.
and washed with cold Et O. The filtrates were combined, concentrat-
2
ed, and further purified by flash chromatography (silica gel, EtOAc
and 1% each of Et N and MeOH) to afford additional aldehyde 4 as a
3
Supporting Information
white solid; yield: 14 g (80%); mp 242–243 °C; R = 0.6 (EtOAc and 1%
f
of Et N).
3
Supporting information for this article is available online at
1
H NMR (300 MHz, CDCl ): δ = 10.04 (s, 1 H), 8.54 (d, J = 4.1 Hz, 1 H),
https://doi.org/10.1055/s-0037-1610211.
S
u
p
p
ortioIgnfmr oaitn
S
u
p
p
o
nrtogI
i
f
rm oaitn
3
8
7
.06 (d, J = 7.9 Hz, 1 H), 7.96 (s, 1 H), 7.78 (dd, J = 17.3, 8.8 Hz, 2 H),
.56 (dd, J = 4.6, 3.3 Hz, 2 H), 7.35 (dd, J = 6.6, 5.1 Hz, 1 H), 5.99 (s, 1
H), 4.18 (s, 1 H), 3.18 (s, 1 H).
References
13
C NMR (75 MHz, CDCl ): δ = 186.8, 167.8, 156.3, 148.7, 137.9, 136.9,
3
(1) Richter, L.; de Graaf, C.; Sieghart, W.; Varagic, Z.; Morzinger, M.;
1
8
36.7, 136.3, 135.5, 135.3, 135.0, 127.2, 124.9, 124.0, 122.8, 121.4,
1.6, 79.8, 44.4.
de Esch, I. J.; Ecker, G. F.; Ernst, M. Nat. Chem. Biol. 2012, 8, 455.
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(
HRMS (ESI/IT-TOF): m/z [M + H]+ calcd for C19H13N O: 313.1084;
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4
(3) Clayton, T.; Chen, J. L.; Ernst, M.; Richter, L.; Cromer, B. A.;
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2
3
(
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600 mL) was added to the mixture under an argon atmosphere at r.t.
(
The reaction mixture was heated slowly to reflux for 2 h. After com-
pletion of the reaction on analysis by TLC (silica gel, EtOAc and 1%
(
(
(
(
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3
cold H O (300 mL) and extracted with EtOAc (3 × 100 mL). The organic
2
layers were combined and this was followed by washing with brine.
The solution was then dried (Na SO ) and concentrated under re-
duced pressure to dryness. The solid residue, which formed, was dis-
solved in EtOAc (200 mL) at 60 °C, and the solvent was reduced to one
quarter of its original volume. A seed crystal was then added to the
solution. The majority of oxazole 3 was crystallized at r.t. and filtered.
2
4
(
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T. M.; Li, G.; Arnold, L. A.; Li, J. X.; Schkeryantz, J. M.; Methuku,
K. R.; Cook, J. M.; Poe, M. M. Pharmacol. Biochem. Behav. 2017,
This was followed by washing with cold Et O. The filtrates were com-
bined, concentrated, and further purified by flash chromatography
2
(silica gel, EtOAc and 1% each of Et N and MeOH) to afford additional
3
(
oxazole 3 as a white solid; yield: 14.2 g (89%); mp 241–242 °C; R = 0.3
f
13
(EtOAc and 1% each of MeOH and Et N).
3
(
1
H NMR (500 MHz, CDCl ): δ = 8.55 (d, J = 4.5 Hz, 1 H), 8.00 (d, J = 7.9
3
Hz, 1 H), 7.95 (s, 1 H), 7.92 (s, 1 H), 7.78 (t, J = 7.4 Hz, 1 H), 7.72 (d, J =
.2, 0.8 Hz, 1 H), 7.58–7.49 (m, 2 H), 7.42 (s, 1 H), 7.33 (dd, J = 6.9, 5.3
8
Hz, 1 H), 5.72 (d, J = 11.9 Hz, 1 H), 4.27 (d, J = 12.0 Hz, 1 H), 3.16 (s, 1
H).
(
(
(
13
C NMR (126 MHz, CDCl ): δ = 167.8, 156.6, 149.8, 148.8, 146.6,
3
1
1
36.9, 136.3, 135.7, 135.4, 135.2, 129.8, 127.4, 126.9, 124.8, 123.9,
22.7, 122.5, 120.9, 81.8, 79.5, 45.2.
HRMS (ESI/IT-TOF): m/z [M + H]+ calcd for C21H14N O: 352.1193;
found: 352.1195.
5
157, 35.
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©
Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–I

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©
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