Improved Scale-up Synthesis and Purification of Clinical Asthma Candidate MIDD0301

Article abstract of DOI:10.1021/acs.oprd.0c00200

We report an improved and scalable synthesis of MIDD0301, a positive GABAA receptor modulator that is under development as oral and inhaled treatments for asthma. In contrast to other benzodiazepines in clinical use, MIDD0301 is a chiral compound that has limited brain absorption. The starting material to generate MIDD0301 is 2-amino-5-bromo-2′-fluorobenzophenone, which has a nonbasic nitrogen due to electron-withdrawing substituents in the ortho and para positions, reducing its reactivity toward activated carboxylic acids. Investigations of peptide coupling reagents on a multigram scale resulted in moderate yields due to incomplete conversions. Second, the basic conditions used for the formation of the seven-membered 1,4-diazepine ring resulted in racemization of the chiral center. We found that neutral conditions comparable to the pKa of the primary amine were sufficient to support the formation of the intramolecular imine but did not enable the simultaneous removal of the protecting group. Both difficulties were overcome with the application of the N-carboxyanhydride of d-alanine. Activated in the presence of an acid, this compound reacted with nonbasic 2-amino-5-bromo-2′-fluorobenzophenone and formed the 1,4-diazepine upon neutralization with triethylamine. Carefully designed workup procedures and divergent solubility of the synthesic intermediates in solvents and solvent combinations were utilized to eliminate the need for column chromatography. To improve compatibility with large-scale reactors, temperature-controlled slow addition of reagents generated the imidazodiazepine at -20 °C. All intermediates were isolated with a purity of >97% and impurities were identified and quantified. After the final hydrolysis step, MIDD0301 was isolated in a 44% overall yield and a purity of 98.9% after recrystallization. The enantiomeric excess was greater than 99.0%.

Full text of DOI:10.1021/acs.oprd.0c00200

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Full Paper
Improved scale-up synthesis and purification
of clinical asthma candidate MIDD0301
Daniel E. Knutson, M. S. RASHID RONI, Md Yeunus Mian,
James M. Cook, Douglas C. Stafford, and Leggy A. Arnold
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.0c00200 • Publication Date (Web): 29 Jul 2020
Downloaded from pubs.acs.org on July 31, 2020
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Improved scale-up synthesis and purification of clinical
asthma candidate MIDD0301
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Daniel E. Knutson,† M.S. Rashid Roni,† Md Yeunus Mian,† James M. Cook,† Douglas C.
Stafford,†,∥ and Leggy A. Arnold*,†,∥
†Department of Chemistry and Biochemistry and the Milwaukee Institute for Drug Discovery,
University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201, United States
∥Pantherics Incorporated, La Jolla, California 92037, United States
*Corresponding Author, Department of Chemistry and Biochemistry, University of Wisconsin-
Milwaukee, Milwaukee, Wisconsin 53201, arnold2@uwm.edu
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Abstract
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We report an improved and scalable synthesis of MIDD0301, a positive GABAA receptor
modulator that is under development as oral and inhaled treatments for asthma. In contrast to other
benzodiazepines in clinical use, MIDD0301 is a chiral compound that has limited brain absorption.
The starting material to generate MIDD0301 is 2-amino-5-bromo-2'-fluorobenzophenone, which
has a non-basic nitrogen due to electron withdrawing substituents in ortho and para positions,
reducing its reactivity towards activated carboxylic acids. Investigations of peptide coupling
reagents on multigram scale resulted in moderate yields due to incomplete conversions. Secondly,
basic conditions used for the formation of the seven-member 1,4-diazepine ring resulted in
racemization of the chiral center. We found that neutral conditions comparable to the pKa of the
primary amine were sufficient to support the formation of the intramolecular imine but did not
enable the simultaneous removal of the protecting group. Both difficulties were overcome with the
application of the N-carboxyanhydride of D-alanine. Activated in the presence of acid, this
compound reacted with non-basic 2-amino-5-bromo-2'-fluorobenzophenone and formed the 1,4-
diazepine upon neutralization with triethylamine. Carefully designed workup procedures and
divergent solubility of the synthesis intermediates in solvents and solvent combinations were
utilized to eliminate the need for column chromatography. To improve compatibility with large
scale reactors, temperature-controlled slow addition of reagents generated the imidazodiazepine at
-20 °C. All intermediates were isolated with a purity of >97% and impurities were identified and
quantified. After the final hydrolysis step, MIDD0301 was isolated with a 44% overall yield and
purity of 98.9% after recrystallization. The enantiomeric excess was higher than 99.0%.
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Keywords
GABAA receptor, asthma, imidazodiazepine, amino acid N-carboxyanhydride.
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Introduction
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The synthesis of 6-phenyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylic acid derivatives
was patented in 19761 and two years later published by Walser et al.2 Three different
benzodiazepines were used as starting material with the following leaving groups in the 2 position:
N-nitrosomethylamino,3 dimorpholinylphosphinyloxy4 and diethyl phosphate. However, the
synthesis included 5-6 steps with an overall yield of 10-14%. A significant improvement was
accomplished in the same year using a phenyl stabilized nitrone5 under basic conditions that
generated only 1-phenyl substituted ethyl 8-chloro-6-(2-fluorophenyl)-4H-benzo[f]imidazo[1,5-
a][1,4]diazepine-3-carboxylates in one step with yields as high as 95%.6 An alternative route was
developed starting from quinazoline 3-oxides using carbanions that gave substituted 6-phenyl-4H-
benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylates in 45-55% yield using four sequential
reactions.7 The first general one-pot procedure to convert readily accessible 5-(2-fluorophenyl)-7-
nitro-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one to ethyl 6-(2-fluorophenyl)-8-nitro-4H-
benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate using diethyl chlorophosphate and ethyl
isocyanoacetate was published in 1978 but no yields were reported.8 The reaction was carried out
with the starting material in tetrahydrofuran (THF) at 0 °C and the addition of potassium t-butoxide
(t-BuOK) followed by sequential addition of diethyl chlorophosphate and a THF solution of ethyl
isocyanate and t-BuOK. More than a decade later Watjen et al.9 used a similar method by reacting
diazepines with sodium hydride (NaH) in dimethylformamide (DMF), followed by the sequential
addition of diethyl chlorophosphate at -20 °C and a THF solution of lithium isopropyl amide and
ethyl isocyanate at -78 °C. The only yield reported was 47% for ethyl 8-chloro-6-oxo-5,6-dihydro-
4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate. A 44% yield was reported for ethyl 8-
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nitro-6-(4-chlorophenyl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate
when
the
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starting material and t-BuOK were stirred in THF at 0 °C followed by the sequential addition of
diethyl chlorophosphate, ethyl isocyanate and t-BuOK.10 Using ethyl (E)-2-
(((dimethylamino)methylene)amino)acetate instead of ethyl isocyanoacetate did not improve the
yield of this reaction.11 An improved yield of 58% for ethyl 8-bromo-6-(2-fluorophenyl)-4H-
benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate was achieved when the starting material was
treated with NaH in THF, followed by the dropwise addition of diethyl chlorophosphate and a
slow addition of ethyl isocyanate and NaH.12 A systematic analysis of this reaction identified t-
BuOK as the best base used at 1.1 equiv. for the reaction of the starting material and diethyl
chlorophosphate (1.3 equiv.) at 0 °C followed by the slow sequential addition of ethyl isocyanate
(1.1 equiv.) and NaH (1.1 equiv.) at -35 °C or below.13 A combined yield of 87% was reported for
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ethyl
8-chloro-6-(2-phenyl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate
when
precipitated in diethyl ether and isolated from mother liquid using column chromatography.
Several subsequent publications have used these conditions but the necessity for column
chromatography to achieve moderate to good yields preclude multigram scale production.14-18
Herein, we describe an improved four step synthesis of (R)-8-bromo-6-(2-fluorophenyl)-4-methyl-
4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylic acid or MIDD0301 that enabled us to
manufacture 88 g of this chiral compound without racemization. MIDD0301 is a clinical drug
candidate for asthma that binds allosterically to the gamma amino butyric acid type A receptor
(GABAAR).19 Several in vivo studies have shown that inhaled and orally administered MIDD0301
reduced airway smooth muscle constriction and lung inflammation without toxicity.20-22
Results and Discussion
The synthesis of many benzodiazepines used clinically involves the reaction of substituted 2-
aminobenzophenones and activated carboxylic acids or acid halides. Our published procedure for
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MIDD0301 involved the addition of N,N'-dicyclohexylcarbodiimide (DCC) as coupling reagent to
react 1 and Boc-D-alanine (Table 1, entry 1).12 Incomplete conversion of 1 was observed for a 204
mmol scale reaction resulting in 58.6% yield of 2. A longer reaction time (48 h) did not appreciably
increase the yield (Table 1, entry 2), nor did a reaction temperature of 40 oC (Table 1, entry 3) or
more DCC (2 equiv.) and Boc-D-alanine (2 equiv.) (Table 1, entry 4). The addition of Boc-D-
alanine to a solution of 1 and DCC in dichloromethane resulted in a yield similar to adding DCC
to a solution of Boc-D-alanine and 1 (Table 1, entry 5).
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Table 1: Reaction between 1 and Boc-D-alanine.
O
O
HO
HN
NH2
O
O
N
H
O
NH
Br
Coupling reagents
solvent
+
F
O
O
O
Br
F
2
1
Boc-D-
Alanine
Yield
(%)
59
Entry Coupling Reagent
Additive
Conditions Solvent
1a
2b
DCC, 1.2 eq
DCC, 1.2 eq
1.0 eq
1.0 eq
None
None
24 hr, rtg
48 hr, rtg
CH2Cl2
CH2Cl2
60
24 hrg, 40 CH2Cl2
ºC
3b
DCC, 1.2 eq
1.0 eq
None
55
4 b
5b,c
6b,d
7b
DCC, 2.0 eq
DCC, 1.2 eq
DCC, 1.2 eq
DCC, 1.2 eq
DCC, 1.2 eq
DCC, 1.2 eq
EDCI, 1.2 eq
2.0 eq
1.0 eq
1.0 eq
1.0 eq
1.0 eq
1.0 eq
1.0 eq
None
None
None
24 hr, rtg
24 hr, rtg
24 hr, rtg
24 hr, rtg
24 hr, rtg
24 hr, rtg
24 hr, rtg
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH3CN
CH2Cl2
62
57
10
53
50
9
DMAP, 0.1 eq
DMAP, 1.0 eq
None
TEA, 1.2 eq
TEA, 1.2 eq
DMAP, 1.2 eq
8b
9b
10e
10
11e
EDCI, 1.2 eq
1.0 eq
24 hr, rtg
CH2Cl2
39
Methyl
chloroformate
Ethyl chloroformate
12f
13f
14f
1.5 eq
1.5 eq
1.5 eq
NMM, 3 eq
NMM, 3 eq
NMM, 3 eq
24 hr, rtg
24 hr, rtg
24 hr, rtg
THF
THF
THF
50
25
22
Isobutyl
chloroformate
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a204 mmol scale, b68 mmol scale, cAddition of Boc-D-alanine to a solution of 1 and DCC, dAddition of 1 to a solution
of DCC and Boc-D-alanine, e22.1 mmol scale, f21.6 mmol scale, grt = room temperature
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However, adding 1 to a solution of Boc-D-alanine and DCC reduced the yield of 2 to 9.6% (Table
1, entry 6), probably due to the formation of an N-acylurea species proposed by Valuer and
Bradlely.23 Additives such as 4-dimethylaminopyridine (DMAP) were first reported by Neises and
Steglich24 for amide coupling reactions as an acyl transfer agent to circumvent the 1,3-
rearrangement problem of the O-acylisourea intermediate by rapidly converting it into a more
stable DMAP complex. However, the addition of DMAP in catalytic or stoichiometric amounts
did not improve the yield of 2 (Table 1, entries 7 and 8). The application of acetonitrile instead of
dichloromethane (CH2Cl2) reduced the yield to 8.7% for this reaction (Table 1, entry 9). 1-Ethyl-
3-(3-dimethylaminopropyl)carbodiimide (EDCI) was investigated instead of DCC as coupling
reagent in the presence of triethylamine (TEA) (Table 1, entry 10) or DMAP (Table 1, entry 11).
Both reactions gave lower yields in comparison with the DCC mediated reaction. Next, the mixed
carbonic anhydride approach was explored to form the amide bond25 using methyl-, ethyl-, and
isobutylchloroformates (Table 1, entries 12-14). These conditions were reported by Anderson, et
al.26 and later used in the synthesis of similar benzodiazepine analogs by Reddy, et al.27 The
reaction using methyl chloroformate in the presence of N-methylmorpholine (NMM) gave the best
yield for 2 with 50.0%. From our observation of similar yields using different activation reagents
for Boc-D-alanine, it can be concluded that the nucleophilicity of 1 is the controlling aspect of this
reaction. The pKa of 1 is -1.12 in comparison to 4.61 for aniline.28 This significant change is based
on the electron withdrawing characters of the acetophenone and bromine substituents in the ortho
and para position, respectively. Although the pKa is a measurement of basicity, it is directly related
to nucleophilicity as both parameters define the electronic nature of the nitrogen. Another
important parameter of an amide bond formation reaction is steric hindrance. Therefore, we
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employed highly reactive acyl chlorides for our next approach as reported for synthesis of achiral
benzodiazepines (Scheme 1).29
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Scheme 1. Synthesis of 5 using (9H-fluoren-9-yl)methyl (R)-(1-chloro-1-oxopropan-2-
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yl)carbamate.
O
NH2
O
H
NH2
O
F
N
NH
1.DCM, room temperature
30 min
O
Br
conditions
a) or b)
+
O
N
F
Br
Cl
Br
HN
Fmoc
2.piperidine, rt, 2h
F
4
5
3
1
a) MeOH:H2O (9:1), NaOH (1 eq), rt, 12h
Yield = 68 % (97.3 g) ee = 0 %
b) MeOH, 60°C, 4h
Yield = 65 % (2.8 g) ee = 76.9 %
The procedure reported by Carpino et al.30 and Prabhu et al.31 was followed to synthesize 147.5 g
of 3 with 93% yield. The reaction between 1 and 3 was carried out at room temperature and
complete conversion was observed after 30 min on a 0.4 M scale. Six equiv. of piperidine were
added slowly to the reaction mixture to form compound 4. The crude product was dissolved in
methanol, followed by the addition of 1.6 M aqueous sodium hydroxide to enable the formation
of 5 after 12 h at room temperature with 68% yield. Despite the increased yield, racemization was
observed for this process. For similar achiral compounds such as nitrazepam32 and nordiazepam,28
the amide hydrogen is relative acidic with pKa of 10.8 and 11.6, respectively. Resonance structures
can result in racemization of α-hydrogen under strong basic conditions. This is supported by the
fact that the same reaction in the absence of the aqueous sodium hydroxide produced 5 with an
enantiomeric excess of 76.9% when performed on a 0.01 M scale. Further analysis confirmed that
traces of piperidine were still present during the cyclization step due to insufficient removal by
multiple washes with water and aqueous bicarbonate during the workup. Because basic conditions
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induce racemization, acid labile protection groups such as t-butyl carbamates were investigated
for the formation of 5 on multigram scale.
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In contrast to Fmoc, Boc groups can be removed in the presence of acid, which has been reported
for 2 using HCl gas. Alternatively, HCl in dioxane was used for multigram scale (Table, 2, entries
2-4). After neutralizing aqueous workup, cyclization of the crude product 4 was achieved in
methanol:water at pH 8.5 using 0.1 equiv. of sodium hydroxide. The product formed for this
reaction was optically pure (Table 2, entry 1). 12
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Table 2: 1,4-Diazepine formation reaction with 2.
O
Boc
O
O
F
H
N
H
NH2
N
NH
NH
a)
CH2Cl2
b)
O
N
Br
O
Br
Br
F
F
4
5
2
Entry a)
b)
Yield % % ee
MeOH / H2O (9 : 1), 0.1 eq NaOH,
1a
2b
3c
4c
8212
99.512
53.9
0
HCl (g), saturated, rtd, 18 h
rtd, 10 h
HCl in dioxane, 4 eq, rtd, 4 MeOH / H2O (9 : 1), 0.1 eq NaOH,
rtd, 48 h
HCl in dioxane, 4 eq, rtd, 4 MeOH / H2O (9 : 1), 1 eq NaOH, rtd,
84
h
50
h
12 h
HCl in dioxane, 4 eq, rtd, 4
h
Toluene, 60 °C, 4 h
48
99.2
a60 mM scale, b116 mM scale, c122 mM scale, drt = room temperature
Longer reaction times were required for complete conversion of 4 and scaling the reaction from
60 mM to 116 mM led to racemization resulting in an enantiomeric excess of 53.9% for 5 (Error!
Reference source not found., entry 2). To reduce the reaction time, one equiv. of sodium
hydroxide was used instead of 0.1 equiv. Full conversion was achieved in 12 h but a racemic
product was obtained (Table 2, entries 3). In an attempt to circumvent the use of base, crude
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product 4 was heated in toluene at 60 °C for 4 h, which gave 5 in a yield of 50% and an
5
6
enantiomeric excess of 99.2% (Table 2, entry 4).
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8
9
To overcome the moderate yield of 2 mentioned earlier and racemization encountered in both
approaches, we followed a procedure developed by Fier and Whitakker33 using amino acid N-
carboxyanhydrides (NCAs) to synthesize optically pure benzodiazepines. Compound 6 was
synthesized using triphosgene and D-alanine, as reported,34, 35 however a semisolid product was
obtained at less than 50% yield. Employing Boc-D-alanine instead D-alanine, 6 was synthesized
using triphosgene and triethylamine using a procedure developed by Wilder and Mobashery.36 On
a 0.53M scale, 35 g of pure crystalline 6 (58% yield) was obtained from a dichloromethane solution
triturated with hexanes.
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Scheme 2. Improved scale-up synthesis of MIDD0301.
O
NH3
H
O
F
O
N
NH
1
, TFA 2 eq
TEA 2 eq
50 °C, 2 h
Triphosgene, TEA
EtOAc, 70 °C, 4 h
O
Boc
O
NH
N
O
N
Br
toluene, 50 °C, 1 h
H
Br
OH
O
4-H+
6
F
5
yield: 58%
yield: 77%
purity: 97.3%
ee: 98.8%
no workup
O
N
O
N
t-BuOK, THF, -20 °C, 1 h
followed by ClPO(OEt)2, -20 °C, 2 h
O
N
OH
N
1. THF, H2O,
NaOH, 55 °C, 4 h
N
F
Br
N
then CNCH2CO2Et, -20 °C, 15 min
followed by t-BuOK in THF 30 min, -20 °C
Br
2. AcOH, H2O
F
7
MIDD0301
yield: 94%
yield: 61%
purity: 97.2%
ee: 99.0%
purity: 98.9%
ee: >99.0%
As reported for natural NCAs, 6 reacted with 1 in the presence of 2 equiv. of trifluoroacetic acid
(TFA) releasing carbon dioxide and forming the salt form of 4 (Scheme 2). Two equiv. of
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trimethylamine were added to the reaction vessel to neutralize the acid and to liberate amine 4
enabling the formation of imine 5 and water. In contrast to the reported procedure, the reaction
was carried out at 50 °C with longer reactions times. Upon reaction completion, an aqueous work-
up removed triethylammonium trifluoroacetate and 5 was triturated in 10% ethyl acetate in heptane
to remove traces of 1 and oligomeric impurities providing 127 g (77% yield). The purity (absolute
area %) of 5 was 97.3% and the enantiomeric excess was 98.8%. Impurities found were compound
5a + 1 (0.7 %) and 5b (2.0 %) (Figure 1 and Supporting Information).
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Figure 1. Purity determination of each synthesis intermediate for manufactured MIDD0301
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The third step of the MIDD0301 synthesis installs the imidazole ring to produce imidazodiazepine
7. The established procedure13 involves formation of an iminophosphate in the presence of base
(potassium t-butoxide) followed by addition of diethylchlorophosphate. The subsequent reaction
with the enolate of ethyl isocyanoacetate yields the desired imidazobenzodiazepine. Earlier trial
reactions monitored by thin layer chromatography (TLC) showed that the iminophosphate was not
formed until a reaction temperature of -20 °C was reached. Similarly, it was observed that the
reaction onset temperature of the iminophosphate with the enolate of ethyl isocyanoacetate was
above -30 °C. Therefore, 5 in THF was cooled to -20 °C and reagents adjusted to room temperature
were added dropwise sequentially ensuring a reaction temperature of -20 °C throughout the process
with the use of a dry ice/isopropanol bath. After a 30 min addition of t-BuOK (1.3 equiv.) in THF
(1.56M), the reaction mixture was stirred for an additional 30 min to ensure complete
deprotonation of the amide starting material. Next, diethylchlorophosphate (1.4 equiv.) was added
dropwise over 15 minutes, followed by 2 h of stirring at -20 °C to complete the formation of
iminophosphate as indicated by TLC. Then, ethyl isocyanoacetate (1.3 equiv.) was added dropwise
over 15 min followed directly by a 30 min addition of t-BuOK (1.3 equiv.) in THF (1.56M) at -20
°C. After the addition, the cooling bath was removed and the reaction mixture stirred for an
additional 1 h once room temperature was reached, at which point full conversion was observed
by TLC. After aqueous workup, the crude product was treated with t-butyl methyl ether at 55 °C
for 30 min to dissolve impurities. After stirring at room temperature for 2 h, 7 was collected by
filtration and washed with t-butyl methyl ether. The synthesis provided 96.6 g (61% yield) of 7
with a purity of 97.2%. Identified impurities were the t-butyl ester of MIDD0301 (7a, 1.4 %) and
6H isomer 7b (1.4 %) (Figure 1 and Supporting Information). The transesterification with t-BuOK
and formation of the 6H isomer of imidazobenzodiazepines under these reaction conditions have
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been reported.37, 38 The enantiomeric excess of 7 was 99.0 %. Using our published hydrolysis
conditions to scale-up synthesis of MIDD0301 (8 equiv. of sodium hydroxide in ethanol) an
insoluble and gel-like MIDD0301 sodium salt was observed. To overcome this problem a 1:4 ratio
of water and THF was employed, which remained as a biphasic solution throughout the process.
The initial 8 equiv. of sodium hydroxide were reduced to 4 equiv. and the reaction temperature
lowered from 78 °C to 50 °C, achieving full conversion of the starting material within 4 h. Our
group recently reported the pH dependent equilibrium between a closed seven-membered ring and
the open-ring acyclic benzophenone structure of MIDD0301.20 Although both compounds are
interconvertible without racemization, they exhibited different aqueous solubility. The lowest
aqueous solubility of MIDD0301 was observed a pH 3-6. Therefore, acetic acid was used to adjust
the pH to 5.0 after dilution with water. The isolated yield was 98% with a purity of 97.2%. One
impurity of this product was 7a (Figure 1 and Supporting Information), which was removed by
recrystallization in ethanol. The final yield after recrystallization was 94% with a purity of 98.9
%. The 6H isomer MIDD0301a (1.14%) (Figure 1 and Supporting Information) was the only
impurity. The enantiomeric excess of MIDD0301 was >99.0%.
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Conclusion
We conclude that NCAs are superior amino acid derivatives for reaction with non-basic nitrogens.
Furthermore, these compounds obviate the need for protecting groups and therefore enable a one
pot peptide coupling reaction and imine formation without racemization. The careful adjustment
of solvent mixtures can avoid column chromatography by simply dissolving byproducts and
precipitating the product. Furthermore, slow addition of reagents to form the imidazole ring can
significantly reduce that need for low reaction temperatures if products like imidazodiazepines are
thermodynamically stable. Other important achievements reported are multiple reactions carried
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out in the same reaction vessel and reducing the synthesis of MIDD0301 to three steps. Classified
metal salts and class 1 solvents are not employed and optically pure MIDD0301 was reliably
generated at intermediate scale.
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Experimental Procedure
All reactions were performed in round-bottom flasks with overhead mechanical stirrers under an
argon atmosphere. Chemicals were purchased from either Millipore Sigma, Oakwood Chemical,
Alfa Aesar, Matrix Scientific, or Acros Organic and used as received. Reaction progress was
monitored by silica gel TLC (Dynamic Adsorbents Inc.) with fluorescence indicator. 1H, 13C and
19F-NMR spectra were obtained on Bruker 300 MHz and 500 MHz instruments with the chemical
shifts in δ (ppm) reported by reference to the deuterated solvents as an internal standard DMSO-
D6: δ = 2.50 ppm (1H-NMR) and δ = 39.52 ppm (13C-NMR) and CDCl3: δ = 7.20 ppm (1H-NMR)
and δ = 77.00 ppm (13C-NMR) (see Supporting Information for NMR spectra). HRMS spectral
data were recorded using a LCMS-IT-TOF spectrometer (Shimadzu). High performance liquid
chromatography (Shimadzu Nexara series HPLC) coupled with a Photo Diode Array detector
(PDA, Shimidzu SPD-M30A) and a single quadrupole mass analyzer (LCMS 2020, Shimadzu,
Kyoto, Japan) was used for purity analysis (absolute area %). Analytes were separated using a
Restek Pinnacle-C18 (4.6 mm x 50 mm, 5 μm particle size) column with gradient elution of water
and methanol (0.1% formic acid) at a flow rate of 0.8 mL/min. Optical purity was determined with
an Agilent 1100 HPLC system using a DAD detector. The mobile phase consisted of HPLC grade
ethanol and n-hexane and the stationary phase was a Pirkle Whelk-01 column (4.6 mm x 25 cm, 5
μm particle size) to separate 5 and 7 and a Chiralpak IB-N3 column (4.6 mm x 15 cm, 3 μm) for
MIDD0301. Trifluoroacetic acid (0.1%) was used as modifier for the analysis of MIDD0301.
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Tert-Butyl-(R)-(1-((4-bromo-2-(2-fluorobenzoyl)phenyl)amino)-1-oxopropan-2-yl)
5
6
carbamate (2) (Table 1, entries 1-9)
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2-Amino-5-bromo-2’fluorobenzophenone (1) (60 g, 204 mmol) and Boc-D-alanine (38.6 g, 204
mmol) were dissolved in anhydrous dichloromethane (300 mL) and stirred at 0 °C.
Dicyclohexylcarbodiimide (51.4 g, 248.9 mmol) was dissolved in anhydrous dichloromethane
(200 mL) to form a homogenous solution, which was added to the former mixture dropwise over
a 30 min period at 0 °C. The solution formed a white precipitate and was allowed to stir for 22 h
at room temperature, at which point no further conversion of the starting materials was detected
(TLC, 50% ethyl acetate in hexanes, Rf (1) = 0.5 and Rf (Boc-D alanine) = 0.4). The formed
dicyclohexyl urea byproduct was removed by filtration and washed with dichloromethane until the
solid was colorless. The organic layers were combined and concentrated under reduced pressure.
The residue, was triturated with hexanes (200 mL) to yield a pale-yellow solid. The solid was
filtered and washed with hexanes (50 mL x 3) followed by reflux in hexanes (200 mL) for 30 min.
After cooling to room temperature, the white solid 2 was filtered and washed with hexanes (50 mL
x 3) and dried under vacuum at 40 °C (55.3 g, 59% yield). 1H NMR (300 MHz, CDCl3) δ 11.68
(s, 1H), 8.71 (d, J = 9.0 Hz, 1H), 7.69 (dd, J = 9.0, 2.3 Hz, 1H), 7.64 – 7.53 (m, 2H), 7.51 – 7.42
(m, 1H), 7.35 – 7.27 (m, 1H), 7.21 (t, J = 9.1 Hz, 1H), 5.13 (s, 1H), 4.37 (s, 1H), 1.52 (d, J = 7.2
Hz, 3H), 1.45 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 195.47 (s), 172.42 (s), 159.56 (d, J = 253.2
Hz), 155.28 (s), 139.68 (s), 137.97 (s), 135.97 (d, J = 1.9 Hz), 133.62 (d, J = 8.4 Hz), 130.31 (d, J
= 2.4 Hz), 126.77 (d, J = 14.5 Hz), 124.51 (s), 124.46 (s), 122.63 (s), 116.55 (d, J = 21.4 Hz),
114.95 (s), 80.26 (s), 51.86 (s), 28.26 (s), 18.58 (s); HRMS (ESI/IT-TOF) m/z: [M + H]+ Calcd
for C21H23BrFN2O4 465.0820; found 465.0845.
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Tert-Butyl-(R)-(1-((4-bromo-2-(2-fluorobenzoyl)phenyl)amino)-1-oxopropan-2-yl)
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carbamate (2) (Table 1, entries 10-11)
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2-Amino-5-bromo-2'-fluorobenzophenone (6.5 g, 22.1 mmol), Boc-D-alanine (4.6 g, 24.3 mmol),
triethylamine (2.9 g, 28.7 mmol) in anhydrous dichloromethane (50 mL) was stirred at room
temperature for 30 min. The solution was cooled to 0 °C and N-(3-dimethylaminopropyl)-N'-
ethylcarbodimide hydrochloride (EDC, 5.5 g, 28.7 mmol) added in one portion. The yellow
solution was allowed to warm to room temperature and stirred for 24 h, at which point TLC
analysis (50% ethyl acetate in hexanes, Rf (1) = 0.5, Rf (Boc-D-alanine) = 0.4) showed no further
conversion. The reaction mixture was diluted in water (50 mL) and the biphasic mixture was
separated and washed with 25% aqueous potassium carbonate (50 mL) and 10% aqueous sodium
chloride (50 mL). The organic layer was dried over sodium sulfate, evaporated under reduced
pressure, and triturated with 10% ethyl acetate in hexanes (100 mL). The white solid 2 was
collected by filtration, washed with hexanes (10 mL x 2), and dried under vacuum at 40 °C (1.0 g,
10% yield).
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Tert-Butyl-(R)-(1-((4-bromo-2-(2-fluorobenzoyl)phenyl)amino)-1-oxopropan-2-yl)
carbamate (2) (Table 1, entries 12-14)
Methyl chloroformate (1.7 mL, 32.3 mmol) was added dropwise over 20 min to a solution of Boc-
D-alanine (4.1 g, 21.6 mmol) and N-methylmorpholine (2.7 mL, 32.3 mmol) in THF (100 mL) at
-20 °C. To the white slurry, a solution of 2-amino-5-bromo-2'-fluorobenzophenone (6.3 g, 21.6
mmol), N-methylmorpholine (2.7 mL, 32.3 mmol) in THF (50 mL) was added dropwise over 15
min at -20 °C. The mixture was allowed to warm to room temperature and stirred for 24 h, at which
point analysis of the reaction progress by TLC indicated a 50/50 mixture of product to starting
material (50% ethyl acetate in hexanes, Rf (1) = 0.5, Rf (Boc-D-alanine) = 0.4). The solvent was
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removed under reduced pressure and the residue dissolved in dichloromethane (100 mL) followed
by the addition of 25% aqueous sodium chloride (100 mL). The biphasic mixture was separated,
washed twice with 25% aqueous potassium carbonate (100 mL x 2), and dried over sodium sulfate.
The solvents were removed under reduced pressure and the residue was triturated with 10% ethyl
acetate in hexanes (40 mL). A white solid 2 was filtered, washed with hexanes (10 mL x 2), and
dried under vacuum at 40 °C (4.9 g, 50% yield).
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(9H-fluoren-9-yl)methyl (R)-(1-chloro-1-oxopropan-2-yl)carbamate (3)
To a mixture of Fmoc-D-alanine (150 g, 481.8 mmol), N,N-dimethylformamide (3.7 mL, 48.2
mmol), and anhydrous dichloromethane (3000 mL) was added thionyl chloride (351.4 mL, 4818.0
mmol) dropwise over 60 min while keeping the temperature between 25 – 30 °C. The resulting
SO2(g) and HCl(g) was trapped with a methanolic scrubber system. The solution was stirred at room
temperature for 1 h followed by 2 h at 40 °C. The completion of the reaction was determined by
converting an aliquot with methanol in the presence of trimethylamine. The starting material was
eluted by TLC using 25% methanol in dichloromethane with 1% trimethylamine. The solvents and
excess thionyl chloride were removed under reduced pressure. The residual thionyl chloride was
removed by drying the solid residue under reduced pressure at 40 °C, followed by dissolution in
dichloromethane (350 mL) and addition of heptane (3150 mL) over 1 h with vigorous stirring to
precipitate the product. The stirring was continued for 2 h. The solid was filtered and washed with
10 % dichloromethane in heptane (250 mL x 2). The solid was dried under vacuum at room
temperature to yield 3 as an off-white solid (147.5 g, 93%): 1H NMR (500 MHz, CDCl3) δ 7.80
(d, J = 7.6 Hz, 2H), 7.65 – 7.53 (m, 2H), 7.48 – 7.40 (m, 2H), 7.35 (td, J = 7.4, 1.2 Hz, 2H), 5.37
– 5.22 (m, 1H), 4.64 (p, J = 7.4 Hz, 1H), 4.53 (dd, J = 10.6, 6.6 Hz, 1H), 4.45 (dd, J = 10.8, 6.9
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Hz, 1H), 4.25 (t, J = 6.8 Hz, 1H), 1.57 (d, J = 7.3 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 175.55,
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155.48, 143.54, 141.36, 127.83, 127.15, 125.02, 124.95, 120.07, 67.34, 58.67, 47.09, 17.28.
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(R)-7-bromo-5-(2-fluorophenyl)-3-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (5)
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(Scheme 1)
2-Amino-5-bromo-2'-fluorobenzophenone (1) (120 g, 408.0 mmol) was dissolved in anhydrous
dichloromethane (1200 mL) and a solution of (9H-fluoren-9-yl)methyl (R)-(1-chloro-1-
oxopropan-2-yl)carbamate (3) (148 g, 448.8 mmol) in anhydrous dichloromethane (1000 mL)
added dropwise over 60 min and keeping the reaction temperature between 25 – 30 °C. After
continuous stirring at room temperature for 1 h, the reaction was deemed complete by TLC analysis
(silica gel, 50% ethyl acetate in hexanes). Piperidine (241.8 mL, 2448 mmol) was added dropwise
to the reaction mixture over 30 min while maintaining the temperature at 25 – 30 °C. Stirring at
room temperature was continued for 12 h resulting disappearance of the intermediate as analyzed
by TLC (silica gel, 50% ethyl acetate in hexanes). The reaction mixture was quenched with 5%
aqueous sodium bicarbonate (1200 mL) and separated. The aqueous layer was extracted with
dichloromethane (1200 mL) and the combined organic layers washed with 5% aqueous sodium
bicarbonate solution (1200 mL) and then twice with deionized water (1200 mL x 2). The solvents
were removed under reduced pressure and the residue was dissolved in dichloromethane (1200
mL) and methanol (1200 mL). A solution of sodium hydroxide (16.3 g, 408.0 mmol) and deionized
water (240 mL) was added dropwise to the reaction mixture over 15 min and the reaction mixture
was stirred for an additional 12 h at room temperature. Upon completion of conversion (TLC
analysis: silica gel, 50% ethyl acetate in hexanes), solvents were removed under reduced pressure.
The resulting semi-solid was dissolved in dichloromethane (1200 mL) and treated with 10%
aqueous sodium chloride (1200 mL). The biphasic mixture was separated and the aqueous layer
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was extracted with dichloromethane (1200 mL) followed by the combination of the organic layers
and a wash with 10% aqueous sodium chloride (1200 mL). The organic layer was dried over
Na2SO4, evaporated under reduced pressure, and triturated in methanol (1600 mL) at 50 °C for 30
min. After stirring for 3 h at room temperature, the solid byproduct (1-((9H-fluoren-9-
yl)methyl)piperidine) was removed by filtration and washed with methanol (200 mL x 2). The
combined methanol layers were evaporated under reduced pressure and the residue was stripped
with 10% ethyl acetate in heptane (200 mL x 2). The solid was triturated in 10% ethyl acetate in
heptane (1600 mL) at 60 °C for 30 min to dissolve 1. The mixture was cooled to room temperature
and stirred for 2 h. The solid material was collected by filtration and washed with 10% ethyl acetate
in heptane (50 mL x 2) and then with heptane (50 mL x 2). The solid was dried under vacuum at
35 °C to afford 5 as an off-white solid (97.3 g, 69%) with a purity of 99.8%. 1H NMR (500 MHz,
CDCl3) δ 9.69 (s, 1H), 7.63 – 7.59 (m, 1H), 7.59 (dd, J = 8.5, 2.3 Hz, 1H), 7.47 (dddd, J = 8.2, 7.0,
5.0, 1.8 Hz, 1H), 7.36 (d, J = 2.2 Hz, 1H), 7.26 (td, J = 7.5, 1.1 Hz, 1H), 7.12 (d, J = 8.6 Hz, 1H),
7.08 (ddd, J = 10.2, 8.3, 1.1 Hz, 1H), 3.79 (q, J = 6.5 Hz, 1H), 1.78 (d, J = 6.5 Hz, 3H); 13C NMR
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(126 MHz, CDCl3) δ 172.39 (s), 164.53 (s), 160.45 (d, JCF = 251.9 Hz), 136.47 (s), 134.74 (s),
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132.19 (d, JCF = 8.3 Hz), 132.03 (d, JCF = 1.5 Hz, NOE coupling), 131.56 (d, JCF = 2.2 Hz),
130.15 (s), 127.13 (d, 2JCF = 12.4 Hz), 124.46 (d, 4JCF = 3.6 Hz), 122.98 (s), 116.54 (s), 116.29 (d,
2JCF = 21.5 Hz), 58.85 (s), 16.93 (s); HRMS (ESI/IT-TOF): m/z [M + H]+ calcd for
C16H13BrFN2O: 347.0190; found: 347.0181.
(R)-7-bromo-5-(2-fluorophenyl)-3-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (5)
(Table 2, entry 2-4)
Compound 2 (55 g, 118.2 mmol) in anhydrous dichloromethane (500 mL) was cooled to 0 °C. A
solution of 4.0M hydrogen chloride in dioxane (118 mL, 472.8 mmol) was added dropwise to the
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reaction mixture at 0 °C over a period of 60 min. The reaction mixture was stirred for 4 h at 0 °C,
at which point the starting material was converted (TLC analysis, 50% ethyl acetate in hexanes).
The reaction mixture was diluted with 10% aq sodium bicarbonate (500 mL) and the biphasic
mixture was separated. The aqueous layer was extracted with dichloromethane (500 mL) and the
combined organic layers were washed with 5% aqueous sodium bicarbonate (500 mL) and twice
with deionized water (500 mL x 2). The solvents were removed under reduced pressure and the
residue was dissolved in methanol (500 mL). A solution of sodium hydroxide (0.5 g, 11.8 mmol)
and deionized water (50 mL) was added dropwise to the reaction mixture over 15 min and stirred
at room temperature for 48 h, at which point the reaction was deemed complete by TLC analysis
(silica gel and 50% ethyl acetate in hexanes). The solvent was removed under reduced pressure
and the residue was dissolved in dichloromethane (500 mL) and treated with 10% aqueous sodium
chloride (500 mL). The biphasic mixture was separated and the aqueous layer was extracted with
dichloromethane (500 mL). The combined organic layers were washed with 10% aqueous sodium
chloride (500 mL) and dried with Na2SO4. The solvent was removed under reduced pressure and
the residue was stripped with 10% ethyl acetate in heptane (200 mL x 2) and triturated with 10%
ethyl acetate in heptane (500 mL) at 60 °C for 30 min. Upon stirring at room temperature for 2 h,
the solid product was collected by filtration and washed with 10% ethyl acetate in heptane (50 mL
x 2) followed by heptane (50 mL x 2). The solid was dried under vacuum at 40 °C to yield 5 as an
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off-white solid (34.6 g, 84%): H NMR (500 MHz, CDCl3) δ 9.69 (s, 1H), 7.63 – 7.59 (m, 1H),
7.59 (dd, J = 8.5, 2.3 Hz, 1H), 7.47 (dddd, J = 8.2, 7.0, 5.0, 1.8 Hz, 1H), 7.36 (d, J = 2.2 Hz, 1H),
7.26 (td, J = 7.5, 1.1 Hz, 1H), 7.12 (d, J = 8.6 Hz, 1H), 7.08 (ddd, J = 10.2, 8.3, 1.1 Hz, 1H), 3.79
(q, J = 6.5 Hz, 1H), 1.78 (d, J = 6.5 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 172.39 (s), 164.53
(s), 160.45 (d, 1JCF = 251.9 Hz), 136.47 (s), 134.74 (s), 132.19 (d, 3JCF = 8.3 Hz), 132.03 (d, JCF
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= 1.5 Hz, NOE coupling), 131.56 (d, JCF = 2.2 Hz), 130.15 (s), 127.13 (d, JCF = 12.4 Hz), 124.46
(d, JCF = 3.6 Hz), 122.98 (s), 116.54 (s), 116.29 (d, 2JCF = 21.5 Hz), 58.85 (s), 16.93 (s); HRMS
(ESI/IT-TOF): m/z [M + H]+ calcd for C16H13BrFN2O: 347.0190; found: 347.0181; HPLC Purity:
97.1%; Optical Purity: 53.9%.
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(R)-4-methyloxazolidine-2,5-dione (6)
To a solution of Boc-D-Alanine (100 g, 528.5 mmol), triphosgene (62.7g, 211.4 mmol) in
anhydrous ethyl acetate (4000 mL) was added triethylamine (81.0 mL, 581.4 mmol) dropwise over
60 min while keeping the temperature below 30 °C. The formed CO2, HCl, and residual phosgene
was trapped by a methanolic scrubber system. The reaction mixture was stirred for 1 h at room
temperature followed by 2 h at reflux (65-70 °C), at which point the gas evolution ceased and the
Boc-D-alanine was converted (TLC, 50% ethyl acetate in hexanes, Rf = 0.5). The reaction mixture
was cooled to room temperature and a white solid (TEA-HCl and residual D-Alanine) was
removed by filtration to yield a pale-yellow solution. The solid was washed with ethyl acetate (2
x 100 mL) and the organic layers were combined and evaporated under reduced pressure. The
semi-solid was dissolved in dichloromethane (400 mL) and hexanes was added dropwise (400mL)
over 1 h with vigorous stirring to precipitate the product. After 12 h at -20 °C, the solid was
filtered, washed with hexanes (250 mL x 2), and dried under vacuum at room temperature to afford
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6 as an off-white solid (35.2 g, 58%): H NMR (500 MHz, D6-DMSO) δ 9.01 (s, 1H), 4.48 (qd, J
= 7.0, 1.1 Hz, 1H), 1.34 (d, J = 7.1 Hz, 3H); 13C NMR (126 MHz,D6-DMSO) δ 172.88, 152.15,
53.28, 17.23.
(R)-7-bromo-5-(2-fluorophenyl)-3-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (5)
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A mixture of 2-amino-5-bromo-2'-fluorobenzophenone (1) (140.7 g, 478.4 mmol) and
trifluoroacetic acid (73.3 mL, 956.7 mmol) in anhydrous toluene (2200 mL) was stirred at room
temperature for 30 min to form a solution. N-carboxy-D-alanine anhydride (66.0 g, 574.0 mmol)
was added and the reaction mixture was heated for 1 h at 50 °C. After disappearance of 1 (TLC,
50% ethyl acetate in hexanes), triethylamine (133.3 mL, 956.7 mmol) was added dropwise to the
reaction mixture over 30 min, while maintaining the temperature at 50 °C. After 2 h at 50 °C, the
intermediate 4 was not detected (TLC, 50% ethyl acetate in hexanes). Upon cooling the reaction
mixture to room temperature, solvents were removed under reduced pressure and the residue was
dissolved in ethyl acetate (1500 mL) and water (1500 mL). The resulting biphasic mixture was
separated and the organic layer was washed with 5% aqueous sodium bicarbonate solution (1500
mL) followed by 10% aqueous sodium chloride solution (1500 mL). The organic layer was dried
over Na2SO4 and evaporated under reduced pressure. The residue was stripped with 10% ethyl
acetate/heptane (25 mL x 2) and slurried with 10% ethyl acetate in heptane (1700 mL) at 60 °C
for 30 min to dissolve unreacted starting material 1. The reaction mixture was cooled to room
temperature and stirred for an additional 2 h before the product was collected by filtration and
washed with 10% ethyl acetate in heptane (50 mL x 2) followed by heptane (50 mL x 2). The
solid was dried under vacuum at 40 °C to afford 5 as an off-white solid (127.0 g, 77%): 1H NMR
(500 MHz, CDCl3) δ 9.69 (s, 1H), 7.63 – 7.59 (m, 1H), 7.59 (dd, J = 8.5, 2.3 Hz, 1H), 7.47 (dddd,
J = 8.2, 7.0, 5.0, 1.8 Hz, 1H), 7.36 (d, J = 2.2 Hz, 1H), 7.26 (td, J = 7.5, 1.1 Hz, 1H), 7.12 (d, J =
8.6 Hz, 1H), 7.08 (ddd, J = 10.2, 8.3, 1.1 Hz, 1H), 3.79 (q, J = 6.5 Hz, 1H), 1.78 (d, J = 6.5 Hz,
3H); 13C NMR (126 MHz, CDCl3) δ 172.39 (s), 164.53 (s), 160.45 (d, 1JCF = 251.9 Hz), 136.47
(s), 134.74 (s), 132.19 (d, 3JCF = 8.3 Hz), 132.03 (d, JCF = 1.5 Hz, NOE coupling), 131.56 (d, 3JCF
= 2.2 Hz), 130.15 (s), 127.13 (d, 2JCF = 12.4 Hz), 124.46 (d, 4JCF = 3.6 Hz), 122.98 (s), 116.54 (s),
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116.29 (d, 2JCF = 21.5 Hz), 58.85 (s), 16.93 (s); 19F NMR (471 MHz, CDCl3) δ -112.53; HRMS
(ESI/IT-TOF): m/z [M + H]+ calcd for C16H13BrFN2O: 347.0190; found: 347.0181; HPLC Purity:
97.3%; Optical Purity: 98.8% ee.
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Ethyl (R)-8-bromo-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a] [1,4]diazepine-
3-carboxylate (7)
Compound 5 (125.0 g, 360.0 mmol) in anhydrous tetrahydrofuran (2000 mL) was cooled to -20
°C using a dry ice/IPA bath. A solution of t-BuOK (52.5 g, 468.0 mmol) in tetrahydrofuran (300
mL) was added dropwise to the reaction mixture over 30 min, while maintaining a temperature of
-20 °C. Upon completion of the addition, the reaction mixture was allowed to stir for an additional
60 min at -20 °C. Diethyl chlorophosphate (72.8 mL, 504.1 mmol) was then added dropwise to the
reaction mixture over 15 min at -20 °C. After 2 h at -20 °C the starting material was converted
(TLC, 100% ethyl acetate). Ethyl isocyanoacetate (51.2 mL, 468.0 mmol) was added dropwise
over 15 min while maintaining -20 °C, followed by the dropwise addition of a solution of t-BuOK
(52.5 g, 468.0 mmol) in tetrahydrofuran (300 mL) over 30 min at -20 °C. Upon completion of the
addition, the reaction mixture was allowed to warm to room temperature and stir for an additional
1 h, at which point the intermediate was fully converted (TLC, 100% ethyl acetate). The reaction
mixture was diluted with 5% aqueous sodium bicarbonate (2000 mL) and ethyl acetate (2000 mL).
The resulting emulsion was cleared by filtration and separated after 30 min. The aqueous layer
was extracted with ethyl acetate (2000 mL) and the combined organic layers were washed with
10% aqueous sodium bicarbonate solution (2000 mL) and 20% aqueous sodium chloride solution
(2000 mL). The organic layer was dried over Na2SO4 and evaporated under reduced pressure. The
residue was stripped with t-butyl methyl ether (200 mL x 2) and slurried with t-butyl methyl ether
(1000 mL) at 55 °C for 30 min. The mixture was stirred for 12 h at room temperature followed by
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filtration and washed with t-butyl methyl ether (100 mL x 4). The solid was dried under vacuum
at 40 °C to yield 7 as a white powder (96.6 g, 61%): 1H NMR (500 MHz, CDCl3) δ 7.92 (s, 1H),
7.73 (dd, J = 8.5, 2.2 Hz, 1H), 7.60 (dt, J = 7.3, 3.9 Hz, 1H), 7.48 (d, J = 8.6 Hz, 1H), 7.50 – 7.42
(m, 1H), 7.42 (d, J = 2.2 Hz, 1H), 7.26 (td, J = 7.5, 1.1 Hz, 1H), 7.10 – 7.02 (m, 1H), 6.71 (q, J =
7.3 Hz, 1H), 4.54 – 4.28 (m, 2H), 1.42 (t, J = 7.1 Hz, 3H), 1.29 (d, J = 7.4 Hz, 3H); 13C NMR (126
MHz, CDCl3) δ 162.93 (s), 162.67 (s), 160.10 (d, 1JCF = 250.7 Hz), 141.59 (s), 134.85 (s), 134.75
(s), 133.68 (s), 133.05 (s), 132.12 (d, 3JCF = 8.2 Hz), 131.17 (s), 129.59 (s), 128.42 (d, 2JCF = 12.3
Hz), 124.57 (d, 4JCF = 3.3 Hz), 123.65 (s), 120.96 (s), 116.25 (d, 2JCF = 21.4 Hz), 60.82 (s), 50.12
(s), 14.87 (s), 14.43 (s); 19F NMR (471 MHz, CDCl3) δ -112.36; HRMS (ESI/IT-TOF): m/z [M +
H]+ calcd for C21H18BrFN3O2: 442.0561; found: 442.0563; HPLC Purity: 97.2%; Optical Purity:
99.0% ee.
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(R)-8-bromo-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-
carboxylic acid [MIDD0301]
To a solution of 7 (96.0 g, 217.0 mmol) in tetrahydrofuran (1500 mL),500 mL of a 1.74M aqueous
solution of sodium hydroxide was added dropwise over 15 min while keeping the temperature at
30 °C. The reaction mixture was heated for 4 h at 55 °C to convert the starting material (TLC,
100% ethyl acetate). The reaction mixture was then cooled to room temperature followed by the
addition of water (1000 mL) and acetic acid (74.5 mL, 1302.3 mmol) dropwise over 15 min while
maintaining the temperature at 25 °C. Tetrahydrofuran was removed under reduced pressure and
methanol (750 mL) was added to the mixture and heated for 30 min at 60 °C. After cooling to
room temperature, the mixture was stirred for 12 h. The solid was filtered and washed with water
(200 mL x 4). The solid was dried under vacuum at 40 °C to yield MIDD0301 as an off-white
powder (87.7 g, 97.5%). The purity was 97.2% by HPLC. Recrystallization in ethanol gave 94%
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yield with a 98.9% purity. The optical purity: >99.0% ee. H NMR (500 MHz, D6-DMSO) δ
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12.83 (s, 1H), 8.42 (s, 1H), 8.09 – 7.92 (m, 1H), 7.89 (d, J = 8.7 Hz, 1H), 7.59 (t, J = 5.5 Hz, 1H),
7.55 (dtd, J = 7.5, 5.4, 2.5 Hz, 1H), 7.36 – 7.30 (m, 2H), 7.23 (dd, J = 10.7, 8.2 Hz, 1H), 6.52 (q,
J = 7.3 Hz, 1H), 1.17 (d, J = 7.3 Hz, 3H); 13C NMR (126 MHz, D6-DMSO) δ 164.68 (s), 162.37
(s), 159.85 (d, 1JCF = 248.3 Hz), 140.86 (s), 136.64 (s), 135.49 (s), 134.02 (s), 132.74 (d, 3JCF = 7.9
Hz), 132.38 (s), 131.93 (s), 130.89 (s), 129.58 (s), 128.70 (d, 2JCF = 11.7 Hz), 125.64 (s), 125.18
(d, 4JCF = 2.8 Hz), 120.26 (s), 116.43 (d, 2JCF = 21.1 Hz), 49.83 (s), 15.05 (s); 19F NMR (471 MHz,
DMSO) δ -114.15; HRMS (ESI/IT-TOF): m/z [M + H]+ calcd for C19H14BrFN3O2: 414.0248;
found: 414.0246.
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Supporting Information
The Supporting Information is available free of charge at: https://pubs.acs.org
Detailed HPLC methods for purity and optical purity analysis, HPLC, MS, 1H, 13C and 2D NMR
spectra of compounds.
Acknowledgements
This work was supported by the National Institutes of Health (USA) R41HL147658 (L.A.A. and
D.C.S.), R01NS076517 (J.M.C. and L.A.A.) and R01HL118561 (J.M.C., L.A.A., and D.C.S.), as
well as the University of Wisconsin-Milwaukee, University of Wisconsin-Milwaukee Research
Foundation (Catalyst grant), the Lynde and Harry Bradley Foundation, and the Richard and Ethel
Herzfeld Foundation. In addition, this work was supported by grant CHE-1625735 from the
National Science Foundation, Division of Chemistry.
Notes
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The authors declare the following competing financial interest(s): Drs. Stafford, Cook and Arnold
are inventors of patent application WO2018035246A1, Gaba(A) receptor modulators and methods
to control airway hyperresponsiveness and inflammation in asthma. Stafford and Arnold have
equity interests in Pantherics Incorporated, which has certain intellectual property rights to these
patents.
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Abbreviations
THF, tetrahydrofuran; t-BuOK, potassium t-butoxide; DMF, dimethylformamide; NaH, sodium
hydride; GABAAR, gamma amino butyric acid type
dicyclohexylcarbodiimide; Boc-D-alanine, tert-butoxycarbonyl-D-alanine; DMAP, 4-
dimethylaminopyridine; CH2Cl2, dichloromethane; EDCI, 1-ethyl-3-(3-
A
receptor; DCC, N,N'-
dimethylaminopropyl)carbodiimide; TEA, trimethylamine; NMM, N-methyl morpholine; NCA,
N-carboxyanhydride; Fmoc, ((9H-fluoren-9-yl)methoxy)carbonyl); thin layer chromatography
(TLC); NMR, nuclear magnetic resonance; LC-MS, liquid chromatography-mass spectrometry;
DMSO, dimethyl sulfoxide; TLC, thin layer chromatography; HRMS, high resolution mass
spectrometry; ESI, electron spray ionization; IT-TOF, ion trap-time of flight; HSQC, heteronuclear
single quantum coherence.
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