Development of a practical and scalable synthesis of the side chain for ASP9726, a successor of micafungin

Article abstract of DOI:10.1021/op400211t

Here, we describe a practical and scalable synthesis of 1, which is a useful side chain of ASP9726 (2), a successor of Micafungin. For large-scale synthesis of 1, reaction conditions were optimized to control impurities and increase yield. In particular, we utilized a high-yield thiadiazole ring formation to prepare thiadiazole 12, a step which was improved by optimization of reaction conditions and isolation method. Further, the number of steps was reduced from 10 to 9, and hazardous reactions were also avoided. Consequently, this process was scaled to produce 21.7 kg of 1 with overall yield improvement from 36.7% to 56.6%.

Full text of DOI:10.1021/op400211t

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Full Paper
Development of a Practical and Scalable Synthesis of
the side chain for ASP9726, a Successor of Micafungin
Shinya Yoshida, ATSUSHI OHIGASHI, Yasuhiro Morinaga, Norio
Hashimoto, Takumi Takahashi, Shigeru Ieda, and Minoru Okada
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/op400211t • Publication Date (Web): 20 Sep 2013
Downloaded from http://pubs.acs.org on October 6, 2013
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Development of a Practical and Scalable Synthesis of
the side chain for ASP9726, a Successor of
Micafungin
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Shinya Yoshida,*,† Atsushi Ohigashi,† Yasuhiro Morinaga,† Norio Hashimoto,† Takumi Takahashi,‡
Shigeru Ieda † and Minoru Okada †
†Process Chemistry Labs., Astellas Pharma Inc., 160-2 Akahama, Takahagi-shi, Ibaraki 318-0001,
Japan
‡Technology Product Management, Astellas Pharma Inc., 2-5-1, Nihonbashi-Honcho, Chuo-ku,
Tokyo, 103-8411, Japan
ABSTRACT
Here, we describe a practical and scalable synthesis of 1, which is a useful side chain of ASP9726
(2), a successor of Micafungin. For large-scale synthesis of 1, reaction conditions were optimized
to control impurities and increase yield. In particular, we utilized a high-yield thiadiazole ring
formation to prepare thiadiazole 12, a step which was improved by optimization of reaction
conditions and isolation method. Further, the number of steps was reduced from 10 to 9, and
hazardous reactions were also avoided. Consequently, this process was scaled to produce 21.7 kg of
1 with overall yield improvement from 36.7% to 56.6%.
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INTRODUCTION
ASP9726 (2, Figure 1) is expected to be a novel echinocandin with potent Aspergillus hyphal
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growth inhibition properties and significantly improved MIC (minimum inhibitory concentration)
against Candida parapsilosis and echinocandin-resistant Candida. Developed as a potential
successor of Micafungin, which was launched by Astellas Pharma Inc. (Fujisawa Pharmaceutical
Co.) in 2002, ASP9726 has shown potent efficacy in treating systemic Candidiasis and
Aspergillosis with no concerns of side effects.1
Trans-4-{5-[4-(4-cyclohexyl-4-methoxypiperidin-1-yl)phenyl]-1,3,4-thiadiazol-2-yl}cyclohexane-1
-carbaldehyde (1, Figure 2) was identified as a potentially useful side chain of ASP9726,2 and the
original medicinal chemistry-based synthetic route of 1 is outlined in Scheme 1.
2HCl
OH
O
O
OH
S
N
H
N
N
N N
N
H
O
HO
O
OH
HO
HO
HN
N
H
N
NH
O
O
H
N
HO
O
OH
HO
O
2
Figure 1. Structures of ASP9726 (2)
H
O
S
O
N
N N
1
Figure 2. Side chain aldehyde (1)
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Scheme 1. Medicinal synthetic method of 1 a)
9
HCl
HN
O
O
O
O
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OH
c
b
O
a
O
O
O
N
N
N
O
3
4
5
6
O
O
O O
O
O
O
O
O
O
10
7
d
e
N
N
N
N N
H H
H2N N
f
H
8
9
11
HO
O
g
h
S
O
O
S
O
N
N
N N
13
N N
O
12
H
O
j
S
O
i
N
S
O
N
O
N
N N
N N
O
1
14
a) Reagents and conditions: (a) chloro(cyclohexyl)magnesium, CeCl3, THF, 95%; (b) MeI, NaH,
DMF, 89%; (c) TFA, anisole, CH2Cl2; (d) ethyl 4-fluorobenzoate (7), K2CO3, DMSO, 76% (2
steps); (e) hydrazine monohydrate, EtOH, THF, 97%; (f)
trans-4-(methoxycarbonyl)cyclohexanecarboxylic acid (10), EDC∙HCl, HOBt, TEA, DMF, quant.;
(g) P2S5, THF, 81%; (h) KOH, THF, EtOH, 86%; (i) N, O-dimethylhydroxylamine hydrochloride,
HBTU, i-Pr2NEt, DMF, 88%; (j) LiAlH4, THF, 96%, overall yield: 36.7%.
However, this medicinal chemistry method presents several drawbacks: LiAlH4 as a reducing agent
and methylation conditions involving combination of NaH with DMF should be avoided from a
safety point of view.3 In addition, the relatively low yield for synthesis of thiadiazole 12 (typically
81%) should be improved. Further, HBTU as a coupling agent should be avoided due to low
cost-efficiency. Moreover, hydrolysis of ethyl ester to carboxylic acid followed by the amide
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formation, and reduction of amide to aldehyde were required in this sequence, so the number of
steps makes it less efficient.
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Given the above, urgent demand has risen for a practical and scalable method of preparing
the desired side chain aldehyde 1 for GMP campaign. We therefore developed a more efficient
method of synthesizing 1 than the original protocol that avoids the aforementioned shortcomings.
Here, we describe our efforts to obtain the desired side chain aldehyde 1.
RESULTS AND DISCUSSION
Development of Grignard reaction (Scheme 2)
Scheme 2. Preparation of 4.
MgCl
OH
O
O
O
O
N
N
3
O
THF
4
Commercially available tert-butyl 4-oxopiperidine-1-carboxylate 3 was treated with the
organocerium reagent4 derived from cyclohexylmagesium chloride and CeCl3 in THF. Conducting
this reaction without CeCl3 typically results in generation of 30 to 40% of aldol adduct impurity A
(Figure 3) on GC analysis, hampering isolation of the desired compound without using SiO2
5
column chromatography purification. We attempted to use ZnCl2 instead of CeCl3 to reduce the
cost of reagent; however, 10% of aldol adduct impurity A was observed on GC analysis.
Consequently, the synthetic method using CeCl3 was demonstrated in the large-scale synthesis, and
28.4 kg of the desired 4 was prepared in a quantitative yield (GC assay) without forming impurity
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A.
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O
O
N
O
HO
N
O
O
Figure 3. Structure of aldol adduct impurity A.
Development of methylation (Scheme 3)
Scheme 3. Development of methylation.
O
O
O
O
OH
methylation
O
N
N
4
5
In the medicinal chemistry synthetic method, methylation is conducted with methyl iodide and
NaH as a base in DMF, conditions which should be avoided in large-scale synthesis due to hazards
associated with the use of NaH in DMF; indeed, plant-scale incidents involving these reagents have
been reported, with onset of exotherm at 40 °C followed by rapid self-heating and eventual
explosion.3 To resolve this issue, a number of candidate bases and solvents were screened for this
reaction (Table 1), with the combination of NaH and THF ultimately providing the best reaction
profile (entry 1). Yield of desired compound 5 was improved from 89% to 95%, and scale-up
synthesis was successfully accomplished, yielding 28.4 kg of 5.
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Table 1. Preparation of 5a
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Entry
Base (equiv.)
NaH
Solvent
THF
GC yield (%)b
1
2
3
4
5
6
95
77
64
78
40
23
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LiOH
THF
KOH
THF
Bu4NOH
t-BuOK
t-BuOK
THF
THF
DMF
a Reaction temp: 0 to 50 °C.
b Determined by GC method A (see the Experimental Section).
Development of deprotection and SNAr reaction (Scheme 4)
Scheme 4. Development of deprotection and SNAr reaction.
O
F
O
7
HCl
HN
4M HCl / EtOAc
K2CO3
O
O
O
O
O
O
O
N
N
DMSO
8
6
5
Deprotection of the N-Boc group was successfully accomplished with HCl/EtOAc at 20 °C, and the
desired amine HCl salt 6 was isolated as a crystalline solid at >90% yield with an overall 87% yield
from 3, yielding 20.4 kg upon scale-up. The subsequent SNAr reaction also achieved good yield
with potassium carbonate in DMSO heated to approximately 110 °C. The target intermediate 8 was
isolated in 90.7% yield simply by adding water to the reaction mixture, and consequently, 27.3 kg
of desired 8 was prepared in large-scale synthesis.
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Development of acid hydrazide formation and amidation (Scheme 5)
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Scheme 5. Development of acid hydrazide formation and amidation.
O
O
O
OH
10
O
O
O O
O
O
O
H2NNH2.H2O
EtOH
EDC.HCl, HOBt, Et3N
O
O
O
N
N
N
N N
H H
H2N N
H
DMF
9
11
8
The formation of acid hydrazide was accomplished by the reaction of compound 8 and hydrazine
hydrate by heating the reaction mixture to 80 °C in EtOH, the target intermediate 9 was isolated in
95.9% yield simply by adding water to the reaction mixture, ultimately providing 25.1 kg of 9 in
large-scale synthesis. Subsequent amidation with trans-4-(methoxycarbonyl)cyclohexanecarboxylic
acid (10) was accomplished using EDC∙HCl, TEA, and HOBt in DMF. While 1 eq. of HOBt was
used in the medicinal chemistry method, we attempted to reduce the amount of HOBt. The catalytic
amount of HOBt (0.3 equiv.) demonstrated a favorable reaction profile, and the reaction proceeded
as in the medicinal chemistry method, with the target intermediate 11 isolated in 94.2% yield
simply by adding water to the reaction mixture, ultimately providing 34.1 kg of 11 in large-scale
synthesis.
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Development of thiadiazole ring formation (Scheme 6)
Scheme 6. Development of thiadiazole ring formation.
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O
O
O
O
O
O
O
S
O
N
N
N N
H H
N N
11
12
In the medicinal chemistry synthetic method, thiadiazole 12 was prepared from amide 11 with P2S5
but produced rather unsatisfactory yield, typically less than 80%. To improve yield of this reaction,
thiadiazole ring formation was attempted using P2S5 under a range of different reaction conditions
or Lawesson's reagent6 (Table 2).
Table 2. Preparation of thiadiazole 12 a
Temperature/
time
HPLC ratio (%)
11/11a/11b/11c/12 a
medicinal method
25/44/ 23/<1/8
Entry
Reagents/solvents
Comments
1
2
3
P2S5 × 1.2 mol/THF
P2S5 × 0.25 mol/THF
P2S5 × 1.2 mol/THF
P2S5 × 1.2 mol
25 °C/21 h
25 °C/2 h
50 °C/3 h
78% isolated yield
-
0.4/1.5/n.d./<1/97
94% isolated yield
4
5
6
7
25 °C/16 h
50 °C/3 h
97/1/n.d./<1/2
-
Pyridine × 1.2 mol/DME
P2S5 × 1.2mol.
21/53/n.d./<1/26
21/53/n.d./<1/26
-
Na2CO3 × 1.2 mol/THF then 50 °C/3 h
P2S5×1.2mol.
50 °C/6 h
basic Al2O3 ×1 wt./THF
21/4/n.d./<1 /85
n.d./n.d./Tr/2/97
-
Lawesson's reagent × 1.2
60-65 °C/3 h
Isolation difficulty
mol./THF
a Determined by HPLC method C (see the Experimental Section)
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During the reaction, formation of monothioamide 11a and dithioamide 11b was observed by
LC-MS analysis (Figure 4). It was observed that the desired cyclization from dithioamide 11b to 12
was much faster than that of 11a, so at the higher temperature reaction condition such as 50 °C, 11b
was not detected on HPLC (entries 1-3). Further, during the reaction, trace amounts of oxadiazole
11c were detected on LC-MS as an impurity; fortunately, however, this contaminant was purged
into the mother liquid during filtration. Using bases such as pyridine, Na2CO3, and basic Al2CO3
(entries 4-6) prevented performing the desired cyclization, leaving behind large amounts of starting
material 11 and monothioamide 11a in the reaction mixture. Given the above, we hypothesized that
optimum cyclization would require acid to improve the reaction rate because the addition of base
did not work well. To confirm this hypothesis, cyclization of monothioamide 11a and dithioamide
11b were conducted under acidic conditions, such as in the presence of methanesulfonic acid. As
expected, the reaction was indeed accelerated under the acidic condition. Use of Lawesson's
reagent was also attempted (entry 7), and while the desired reaction proceeded well in comparison
with the case of P2S5, removing the residue left by the reagent was difficult without SiO2 column
chromatography purification. As such, using P2S5 will likely prove to be the optimum condition in
this case.
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O
O
O O
P2S5
O
O
O
O
S
O
O
O
O
S
O
N
N
N
N
N
N
-H2O
N
H
N
H
N
N
H
H
11a
12
11
-H2S
faster
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O
O
S
S
N
H
N
H
Accelerated by the addition of MeSOH
3
O
O
O
11b
N
N
N
O
11c
Figure 4. Thiadiazole ring formation.
O
O
O
S
N
N N
cis-12
Figure 5. Structure of cis-12.
However, we noted a significant issue hampering large-scale synthesis during reaction with
P2S5 in THF: namely, undesired isomerization and an unacceptable level of cis-12 on HPLC
analysis (Figure 5). Further, removing cis-12 via crystallization proved quite difficult. To solve
these issues, the stability of the product 12 was measured and results were shown in Figure 6. As
results, cis-12 was gradually increased after the completion of the reaction at 50 °C after the
completion of the reaction.
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◆cis-12
Time (h)
Figure 6. Stability test of 12 in the reaction mixture.
To minimize the undesired isomerization, qunching methods were investigated as shown in Table
3.
Table 3. Minimization of the formation of cis-12.
additional 1 h at 50°Ca
Entry
conditions
12 / cis-12b
97.9：2.1
98.1：1.9
98.3：1.7
98.3：1.7
1
2
3
4
without quenching
water (× 0.5 v /wt.)
Et3N (×3 mol.)
aqueous solution of Na2CO3 ( ×3 mol.)
a After completion of the reaction (50°C / 3h) 12 / cis-12 = 98.3 / 1.7,
b Determined by HPLC method D (see the Experimental Section)
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Ultimately, the addition of triethylamine (Et3N) and an aqueous solution of Na2CO3 prevented the
isomerization well. After the quenching process with aqueous solution of Na2CO3, desired 12 was
isolated in 93.8% yield simply by filtration of the resulting slurry. Further, this procedure was
demonstrated in a large scale synthesis and yielded 31.9 kg of 12 that contained 0.5 % of cis-12.
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Preparation of aldehyde 1
Direct preparation of aldehyde 1 from ester 12 (Scheme 7).
Scheme 7. Direct reduction pass from methyl ester 12 to aldehyde 1.
O
O
O
S
N
N N
12
O
S
N
HO
N N
15
H
O
O
S
N
N N
1
Direct reduction of methyl ester 12 to aldehyde 1 was investigated as shown in Table 4. The
method using sodium bis (2-methoxyethoxy) aluminum hydride (SMEAH) with pyrrolidine7 did
not work well in this case, with extremely low reaction conversion and an unacceptable level of
unknown impurities observed (entries 2 and 3). Further, DIBAL-H reduction also provided poor
results, as the double-bond on thiadiazole ring was reduced by hydride, an unacceptable level of
over-reduced alcohol 15 was observed on HPLC analysis (entry 1), and controlling formation of 15
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was difficult.
Table 4. Direct reduction of ester 12 to aldehyde 1.
8
9
HPLC area (%)a
12/15/1
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Entry
Reagents (× mol)/solvents
Conditions
DIBAL-H (1.1)
Toluene-CH2Cl2
1
−78 to −70 °C
2/22/ 76
SMEAH 7
1) Red-Al (× 1.49 mol., 65% toluene
solution) pyrrolidine (1.74)/THF
2) t-BuOK (0.1)/THF
74/n.d./8; many unknown
peaks observed on HPLC
2
0 to 25 °C
3) 2) was added to a solution of 12 in THF
SMEAH 7
1) Red-Al (2.9, 65% Toluene solution)
pyrrolidine (3.47)/MTBE
2) t-BuOK (0.2)/THF
52/n.d./2; many unknown
peaks observed on HPLC
3
0 to 25 °C
3) 2) was added to a solution of 12 in THF
a Determined by HPLC method D (see the Experimental Section)
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Development of an alternative method for aldehyde 1.
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Given aldehyde 1, we considered that a step-wise method might be suitable for this compound,
therefore reduction of methyl ester 12 to alcohol 15 was investigated (Table 5). DIBAL-H did not
work well for this reaction (entries 1 and 2), producing large amounts of impurity B produced from
reduction of the double bond on the thiadiazole ring (Figure 7). In contrast, Red-Al worked well,
and the reaction proceeded properly at 0 to 15 °C to produce the desired alcohol 15 in 93% yield. In
addition, no impurity B was observed on HPLC analysis (entry 3), and the large-scale synthesis of
alcohol 15 was successfully accomplished, producing 27.9 kg of alcohol 15 in 93.1% yield.
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Table 5. Preparation of 15.
HPLC area (%)
Entry
Reagents (× mol.)/solvents
Conditions
12/15/1/impurity B
n.d./36/64/n.d.
n.d./83/17/n.d.
n.d./59/ 40/1
DIBAL-H (2), then DIBAL (1)/
Toluene-CH2Cl2
−78 °C, then
−55 °C
1
−75 °C/0.5 h,
then −75 °C/1 h,
then −55 °C/0.5 h
2
3
DIBAL-H (3)/Toluene-CH2Cl2
Red-Al (1.5)/THF
n.d./70/25/5
n.d./56/n.d./44
n.d./> 99/n.d./n.d.
isolated yield: 93%
0 °C then 15 °C
S
O
N
HO
N N
H H
Figure 7. Structure of the over-reduced impurity B.
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End-game for preparation of aldehyde 1
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The investigation of the oxidation conditions is shown in Table 6. Oxidizing agents such as
TEMPO-NaOCl (entry 1) and TEMPO-Oxone method (entry 2) did not give a good reaction profile.
On the other hand, the use of SO3-Pyridine-TEA with DMSO have the best reaction profile (entries
3 and 4). The reaction was performed under mild conditions, and the yield of desired aldehyde 1
was over 90%.
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Table 6. Investigation of oxidizing agent.
Reagents (× mol.)/
Entry
Conditions
Results
solvent (v/wt.)
TEMPO (0.01), NaOCl (1.1)
KBr (0.1)/CH2Cl2 (50), water (20).
TEMPO (0.01), Oxone (2.2)
Bu4NBr (0.04)/CH2Cl2 (60)
SO3-Pyridinea (3.4)
−15 °C, then
25 °C, 0.5h
-15 °C, then
25 °C, 6 h
Level of unknown impurities
was unacceptable
1
2
3
4
no reaction
90% yield
91% yield
20 to 25 °C, 3 h
20 to 25 °C, 3 h
DMSO (4.4), TEA (6.7)/CH2Cl2 (50)
SO3-Pyridine a (3.0),
DMSO (4), TEA (6)/CH2Cl2 (20)
a sulfur trioxide pyridine complex
In large-scale synthesis, 21.7 kg of desired aldehyde 1 was obtained, results comparable to those
achieved in lab-scale operations. No undesired isomerization occurred during operations, and the
level of cis-1 was < 0.05 % in aldehyde 1 (Figure 8).
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H
O
O
S
N
N N
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cis-1
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Figure 8. Structure of cis-1.
CONCLUSION
In summary, we have developed a practical and scalable method for synthesizing the side chain of
ASP9726 that may prove useful as a successor of Micafungin 2. This new synthesis method was
deemed to be the optimum large-scale route due to its improved safety and yield over medicinal
chemistry method. Employing our method in large-scale synthesis produced 21.7 kg of desired side
chain aldehyde 1, with overall yield improved from 36.7% to 56.6%.
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EXPERIMENTAL SECTION
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General
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Starting materials, reagents, and solvents were obtained from commercial suppliers and used
1
without further purification. H and 13C NMR spectra were recorded in the specified deuterated
solvent. Chemical shifts of 1H NMR spectra are reported in parts per million on the δ scale from an
internal standard of residual solvent (CHCl3 7.26 ppm; DMSO-d6 2.50 ppm) or TMS. Data are
reported as follows: chemical shift, multiplicity (s, singlet; d, doublet; dd, doublet doublet; t, triplet;
m, multiplet; br, broad), coupling constant (hertz), and integration. Chemical shifts of
proton-decoupled 13C NMR spectra are reported in parts per million from the central peak of CDCl3
(77.0 ppm), DMSO-d6 (39.5 ppm) on the δ scale. Mass spectra were measured using
UPLC/SQD-LC/MS, JEOL JMS-GC mate II, Waters ZQ 2000, and JEOL JMSLX2000 instruments.
KF were measured using the JP method. HPLC was performed using a Hitachi D-2500 or D-7500
system via methods described below. Gas chromatograph model GC-17A with Static-Headspace
and FID detector were obtained from Shimadzu for GC analysis, and GC methods are also
described below. Glass lined reactors were used for all steps.
GC Method A - Column: DB-WAX, 0.25 mm × 30 m, 0.25 μm (GL Sciences), carrier gas: He
1.41mL/min. (37 cm/sec.), column temp.: 150 °C, injection temp.: 200 °C, detector temp.: FID
250 °C, split ratio: 1/30, time program: 150 °C (0 min), 5 °C/min., 250 °C (10 min), injection: 1 μm,
operation time: 30 min; 3: 6.8 min, 4: 19.5 min, 5: 14.8 min
GC Method B - Column: DB-1, 0.25 mm × 30 m, 0.25 μm (GL Sciences), carrier gas: He 1.74
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mL/min. (40.2 cm/sec.), column temp.: 100 °C, injection temp.: 250 °C, detector temp.: FID
300 °C, split ratio: 1/30, time program: 100 °C (0 min), 10 °C/min., 300 °C (10 min.), injection: 1
μm, operation time: 30 min; 5: 13.9 min, 6: 8.8 min, 7: 4.7 min, 8: 24.4 min
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HPLC Method A - Column: YMC-Pack ODS-AM, 5 μm, 4.6 mm × 150 mm (YMC), elution with
water/CH3CN (1/4), over 30 min, 1.0 mL/min, at 40 °C, with UV detection at 254 nm; 8: 10.9 min;
9, 2.9 min
HPLC Method B - Column: Unison UK-Phenyl, 5 μm, 4.6 mm × 250 mm (Imtakt), elution with 50
mmol/L HClO4 buffer (adjusted to pH 2.0)/CH3CN (27/13), over 30 min, 1.0 mL/min., at 50 °C,
with UV detection at 254 nm; 9: 11.9 min; 11, 23.1 min
HPLC Method C - Column: Unison UK-Phenyl, 5 μm, 4.6 mm × 250 mm (Imtakt), elution with 50
mmol/L HClO4 buffer (adjusted to pH 3.0)/CH3CN (1/1), over 30 min, 1.0 mL/min, at 50 °C, with
UV detection at 254 nm; 11: 8.4 min; 12, 23.3 min
HPLC Method D - Column: COSMOSIL® Cholester, 5 μm, 4.6 mm × 250 mm (Nacalai Tesque),
elution with 50 mmol/L HClO4 buffer (adjusted to pH 2.0)/CH3CN (1/3), over 30 min, 1.0 mL/min,
at 45 °C, with UV detection at 254 nm; 12: 16.7 min; cis-12: 13.3 min; 15, 9.4 min
HPLC Method E - Column: Unison UK-Phenyl, 5 μm, 4.6 mm× 250 mm (Imtakt), elution with 50
mmol/L HClO4 buffer (adjusted to pH 2.0)/CH3CN (23/17), over 60 min, 1.0 mL/min, at 45 °C,
with UV detection at 254 nm; 15: 13.8 min; 1, 19.1 min, cis-1: 22.1 min
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tert-butyl 4-cyclohexyl-4-hydroxypiperidine-1-carboxylate (4)
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A suspension of cerium (III) chloride anhydrous (37.1 kg, 151 mol) and THF (326 kg) was agitated
at 20 °C for 16 h. Cyclohexyl magnesium chloride (20% [w/w] in toluene-THF, 107.6 kg, 151 mol)
was added at 20 °C, and the mixture was aged for 1 h and then cooled to 7 °C. A solution of
tert-butyl 4-oxopiperidine-1-carboxylate 3 in THF (20.0 kg, 100.4 mol/THF 78.1 kg) was then
added at 6 to 8 °C, and the mixture was aged for 3 h at the same temperature; GC analysis
subsequently indicated that < 1% starting material 3 remained (GC method A). After the
completion of the reaction, the batch was poured into an aqueous solution of AcOH (AcOH: 37.0
kg / water: 333 kg) at around 10 °C and EtOAc (100 kg) was added to this mixture. After the phase
separation, the resulting aqueous layer was extracted with EtOAc (100 kg). Organic layers were
combined and washed with aqueous solution of NaCl (NaCl: 22.4 kg/ water: 89.6 kg). The resulting
organic layer was then washed with aqueous solution of NaHCO3 (NaHCO3: 9.0 kg/ water 103 kg)
and then with aqueous solution of NaCl (NaCl: 22.4 kg/ water: 89.6 kg). The organic later was
concentrated in vacuo using a vacuum pump to 60 L. To the residue was added THF (88.7 kg). The
mixture was then concentrated in vacuo using a vacuum pump to 100 L. To the residue was added
THF (88.7 kg), and the resulting solution was concentrated in vacuo using a vacuum pump to 100 L.
The resulting solution was added THF (88.7 kg) and concentrated in vacuo using a vacuum pump
to 100 L. The resulting solution was used in the next step without purification and isolation (28.4
kg from GC assay, quantitative yield). An analytical sample of 4 was prepared by concentration in
labs (oily material).
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1H NMR (400 MHz, DMSO-d6) δ 3.65-3.76 (2H, m), 2.81-3.10 (2H, m), 1.68-1.78 (4H, m),
1.55-1.62 (2H, m), 1.41 (1H, br), 1.38 (9H, s), 1.31-1.37 (5H, m), 1.09-1.18 (2H, m), 0.90-1.01 (2H,
m)
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13C NMR (100 MHz, DMSO-d6) δ 154.4, 78.8, 70.0, 48.7, 33.7, 30.9 (2 carbons), 28.6 (3 carbons),
26.9 (2 carbons), 26.7 (2 carbons), 26.6 (2 carbons)
MS (FAB, positive mode) m/z 284.1; MS (FAB, negative mode) m/z 282.1
tert-butyl 4-cyclohexyl-4-methoxypiperidine-1-carboxylate (5)
THF (141 kg) was added to a solution of compound 4 in THF (from previous step), with sodium
hydride (60%, 11.9 kg, 301 mol) and more THF (10.1 kg) added subsequently at 20 °C. This
mixture was then heated to 33 °C, and methyl iodide (28.5 kg, 201 mol) was added at 30 to 40 °C;
GC analysis subsequently indicated that < 1% compound 4 remained (GC method A). After the
completion of the reaction, the batch was poured into the mixture of ice and water (134 kg). To the
batch was added EtOAc (100 kg). After the phase separation, the resulting aqueous layer was
extracted with EtOAc (100 kg). The combined organic layer was washed with aqueous solution of
NaCl (NaCl: 27.0 kg / water 104 kg). The resulting organic layer was concentrated in vacuo using a
vacuum pump to 60 L. The residue was treated three times each with EtOAc (56.4 kg), evaporating
each time in vacuo using a vacuum pump to 60L. The resulting solution was used in the next step
without purification and isolation. (28.4 kg from GC assay, 95% yield). An analytical sample of 5
was prepared by concentration in labs (oily material).
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1H NMR (400 MHz, DMSO-d6) δ 3.66-3.80 (2H, m), 3.32 (3H, s), 2.75-2.90 (2H, m), 1.70-1.78
(2H, d, J = 12.8 Hz), 1.48-1.64 (6H, m), 1.38 (9H, s), 1.00-1.25 (5H, m), 0.90-1.01 (2H, m)
13C NMR (100 MHz, DMSO-d6) δ 154.5, 78.8, 75.2, 47.6, 42.0, 29.5 (3 carbons), 28.6, 27.0 (2
carbons), 26.9 (2 carbons), 26.7 (2 carbons), 26.5 (2 carbons)
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MS (FAB, positive mode) m/z 298.1; MS (FAB, negative mode) m/z 296.1
4-cyclohexyl-4-methoxypiperidine monohydrochloride (6)
EtOAc (5.4 kg) and 14.6% (w/w) HCl in EtOAc (72.3 kg, 289 mol) were added to a solution of
compound 5 in EtOAc (from previous step) at 10 °C, and the mixture was aged for 1 h at 20 °C;
GC analysis subsequently indicated that < 0.5% compound 5 remained (GC method B). After
reaction completion, n-heptane (71.5 kg) was added to the cooled reaction mixture at 20 °C and
aged for 3 h at 20 °C, and the mixture was then filtered and washed with n-heptane (71.5 kg). Loss
to the filtrate was < 7%. The wet cake was dried in vacuo at 45 °C to afford the desired 6 at 98.0%
purity via GC method B (20.4 kg, 87.0% yield from 3 in three steps).
1H NMR (400 MHz, DMSO-d6) δ 9.08 (1H, br), 3.04 (3H, s), 3.00-3.10 (2H, m), 2.85 (2H, dd, J =
12.4, 11.6 Hz), 1.84 (2H, dd, J = 13.8 Hz), 1.74 (2H, J = 12.8 Hz), 1.50-1.71 (6H, m), 1.14-1.28
(2H, m), 1.02-1.13 (1H, m), 0.88-1.01 (2H, m)
13C NMR (100 MHz, DMSO-d6) δ 73.9, 47.8, 41.7, 39.1, 26.9 (2 carbons), 26.7 (2 carbons), 26.6
(2 carbons), 26.3 (2 carbons)
MS (ESI, positive mode) m/z; 198.0, MS (ESI, negative mode) m/z; 196.0
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Anal. Calcd for C12H23NO.HCl: C (61.65%), H (10.35%), Cl (15.17%), N (5.99%). Found: C
(61.75%), H (10.43%), Cl (15.11%), N (5.88%); mp: 218 °C (by DSC)
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ethyl 4-(4-cyclohexyl-4-methoxypiperidin-1-yl)benzoate (8)
Compound 6 (20.4 kg, 87.3 mol), ethyl 4-fluorobenzoate 7 (15.4 kg, 91.6 mol), and potassium
carbonate (24.1 kg, 174.5 mol) were added to stirred DMSO (112 kg), and the mixture was heated
to 110 °C and aged for 24 h; GC analysis subsequently indicated that < 1.5% compound 6 remained
(GC method B). After reaction completion, the batch was cooled to 65 °C, and water (143 kg) was
added. The resulting slurry was then cooled to 25 °C, aged for 2 h, filtered, and washed with water
(143 kg). Loss to the filtrate was less than < 0.01%. The wet cake was dried in vacuo at 40 °C to
afford the desired 8 at 94.0% purity via HPLC method A (27.3 kg, 90.7% yield).
1H NMR (400 MHz, DMSO-d6) δ 7.76 (2H, d, J = 9.2 Hz), 6.97 (2H, d, J = 9.2 Hz), 4,23 (2H, dd,
J = 7.0, 6.8 Hz), 3.67 (2H, d, J = 12.8 Hz), 3.08 (3H, s), 2.29-3.02 (2H, m), 1.73 (2H, d, J = 12.8
Hz), 1.52-1.66 (8H, m), 1.28 (3H, t, J = 7.0 Hz), 0.88-1.20 (5H, m)
13C NMR (100 MHz, DMSO-d6) δ 166.2, 154.3, 131.2 (2 carbons), 118.1 (2 carbons), 113.7 (2
carbons), 75.1, 60.3, 47.7, 43.2, 41.9, 29.0 (2 carbons), 27.1 (2 carbons), 26.9 (2 carbons), 26.7 (2
carbons)
MS (ESI, positive mode) m/z; 346.0, MS (ESI, negative mode) m/z; 344.0
Anal. Calcd for C21H31NO3: C (73.01%), H (9.04%), N (4.05%). Found: C (73.31%), H (9.00%), N
(3.98%); mp: 250 °C (by DSC)
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4-(4-cyclohexyl-4-methoxypiperidin-1-yl)benzohydrazide (9)
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Compound 8 (27.3 kg, 79.0 mol) and hydrazine monohydrate (79.1 kg, 1580 mol) were added to
stirred EtOH (108 kg), and the mixture was heated to 80 °C and aged for 20 h; HPLC analysis
subsequently indicated that < 0.5% compound 8 remained (HPLC method A). After reaction
completion, water (191 kg) was added at 50 °C, and the mixture was cooled to 25 °C. The resulting
slurry was then aged for 2 h, filtered, and washed with water (137 kg). Loss to the filtrate was 1.0%.
The wet cake was dried in vacuo at 40 °C to afford the desired 9 at 100.0% purity via HPLC
method A (25.1 kg, 95.9% yield).
1H NMR (400 MHz, DMSO-d6) δ 9.45 (1H, s), 7.69 (2H, d, J = 8.8 Hz), 6.91 (2H, d, J = 9.2 Hz),
4.35 (2H, br), 3.59 (2H, d, J = 12.0 Hz), 3.07 (3H, s), 2.80-2.95 (2H, m), 1.73 (2H, d, J = 12.4 Hz),
1.52-1.68 (8H, m), 1.20-1.26 (3H, m), 0.88-1.02 (2H, m)
13C NMR (100 MHz, DMSO-d6) δ 166.5, 153.2, 128.7 (2 carbons), 122.3, 114.1 (2 carbons), 75.0,
47.6, 43.7, 41.9, 29.1 (2 carbons), 27.1 (2 carbons), 26.9 (2 carbons), 26.8 (2 carbons)
MS (ESI, positive mode) m/z; 332.30, MS (ESI, negative mode) m/z; 330.30
Anal. Calcd for C19H29N3O2: C (68.85%), H (8.82%), N (12.68%). Found: C (68.80%), H (8.88%),
N(12.77%); mp: 210 °C (by DSC)
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methyl
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trans-4-{2-[4-(4-cyclohexyl-4-methoxypiperidin-1-yl)benzoyl]hydrazine-1-carbonyl}cyclohexane-
1-carboxylate (11)
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Compound 9 (24.0 kg, 72.4 mol) and trans-4-(methoxycarbonyl)cyclohexanecarboxylic acid 10
(14.8 kg, 79.7 mol) were added to DMF (182 kg), and then HOBt (2.94 kg, 21.7 mol),
triethylamine (11.0 kg, 109 mol), EDC∙HCl (20.8 kg, 109 mol), and DMF (22.8 kg) were added.
The reaction mixture was aged for 3 h at 25 °C; HPLC analysis subsequently indicated that < 0.1%
compound 9 remained (HPLC method B). After reaction completion, water (120 kg) was added to
the mixture, and the resulting slurry was aged for 18 h at 25 °C, then filtered and washed with
water (240 kg). Loss to the filtrate was < 0.01%. The resulting wet cake was then added to water
(240 kg), and the mixture was agitated for 0.5 h and filtered followed by washing with water (120
kg). Loss to the filtrate was < 0.01%. The wet cake was dried in vacuo at 55 °C to afford desired 11
at 100.0% purity via HPLC method B (34.1 kg, 94.3% yield).
1H NMR (400 MHz, DMSO-d6) δ 9.95 (1H, s), 9.68 (1H, s), 7.73 (2H, d, J = 8.8 Hz), 6.94 (2H, d,
J = 9.2 Hz), 3.64 (2H, d, J = 12.0 Hz), 3.60 (3H, s), 3.08 (3H, s), 2.88-3.00 (2H, m), 2.10-2.34 (2H,
m), 1.92-1.98 (2H, m), 1.80-1.88 (2H, m), 1.70-1.78 (2H, m), 1.56-1.67 (6H, m), 1.30-1.50 (5H, m),
1.11-1.22 (4H, m), 0.90-1.02 (2H, m)
13C NMR (100 MHz, DMSO-d6) δ 175.8, 174.8, 165.7, 153.5, 129.3 (2 carbons), 121.2, 113.9 (2
carbons), 75.1, 51.9, 47.6, 43.6, 42.1, 41.9, 41.8, 29.0 (2 carbons), 28.6 (2 carbons), 28.3 (2
carbons), 27.1 (2 carbons), 26.9 (2 carbons), 26.8 (2 carbons)
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MS (ESI, positive mode) m/z; 500.0, MS (ESI, negative mode) m/z; 498.0
Anal. Calcd for C28H41N3O5: C (67.31%), H (8.27%), N (8.41%). Found: C (67.45%), H (8.38%),
N (8.22%); mp: 207 °C (by DSC)
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methyl
trans-4-{5-[4-(4-cyclohexyl-4-methoxypiperidin-1-yl)phenyl]-1,3,4-thiadiazol-2-yl}cyclohexane-1
-carboxylate (12)
Compound 11 (34.1 kg, 68.3 mol) was added to THF (424 kg), followed by phosphorus
pentasulfide (P2S5, 19.7 kg, 88.8 mol) at 30 °C, and the mixture was aged for 2 h at 30 °C; HPLC
analysis subsequently indicated that < 0.7% compound 11 remained (HPLC method C and method
D). After reaction completion, an aqueous solution of Na2CO3 (Na2CO3: 21.7 kg, 205 mol/water:
512 kg) was added to the reaction mixture at 7 °C, and the resulting slurry was agitated for 17 h,
filtered, and then washed with water (68.0 kg). Loss to the filtrate was 0.7%. The resulting wet cake
was then added to a solution of water (205 kg) and THF (121 kg), and the mixture was then
agitated for 1 h at 10 °C, filtered, and washed with water (68.0 kg). Loss to the filtrate was < 0.03%.
The wet cake was dried in vacuo at 60 °C to afford the desired 12 at 99.4% purity via HPLC
method C (31.9 kg, 93.8% yield). cis-12 (0.5%) was observed on HPLC method D.
1H NMR (400 MHz, CDCl3) δ 7.76 (2H, d, J = 8.8 Hz), 6.91 (2H, d, J = 8.8 Hz), 3.67 (3H, s), 3.60
(2H, d, J = 12.4 Hz), 3.14 (3H, s), 3.04-3.14 (2H, m), 2.35-2.42 (2H, m), 2.24-3.32 (2H, m),
2.08-2.17 (2H, m), 1.50-1.84 (14H, m), 1.14-1.26 (3H, m), 0.92-1.04 (2H, m)
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13C NMR (100 MHz, CDCl3) δ 175.9, 172.9, 168.3, 153.1, 129.1 (2 carbons), 119.9, 114.9 (2
carbons), 75.3, 68.1, 51.8, 47.7, 43.9 (2 carbons), 42.5, 42.3, 39.2, 32.8 (2 carbons), 29.2, 28.6 (2
carbons), 27.1 (2 carbons), 27.0 (2 carbons), 26.7
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MS (ESI, positive mode) m/z; 498.0, MS (ESI, negative mode) m/z; 496.0
Anal. Calcd for C28H39N3O3S: C (67.57%), H (7.90%), N (8.44%), S (6.44%). Found: C (67.45%),
H (7.99%), N (8.22%), S (6.55%); mp: 173 °C (by DSC)
(trans-4-{5-[4-(4-cyclohexyl-4-methoxypiperidin-1-yl)phenyl]-1,3,4-thiadiazol-2-yl}cyclohexyl)m
ethanol (15)
Compound 12 (31.8 kg, 63.9 mol) was added to THF (536 kg), and then the mixture was cooled to
6 °C. Red-Al (70% in toluene, 27.7 kg, 95.8 mol) was then added, followed by washing with THF
(28.2 kg) and aging for 1 h at 15 °C; HPLC analysis subsequently indicated that < 0.1% compound
12 remained (HPLC method D). After reaction completion, the batch was cooled to 3 °C, and
MeOH (12.6 kg) and aqueous hydrochloric acid (conc. HCl: 93.8 kg/water 398 kg) were added.
The batch was then warmed to 25 °C, and dichloromethane (527 kg) was added. After the phase
separation, the resulting aqueous layer was first extracted with dichloromethane (316 kg) and then
re-extracted with dichloromethane (211 kg) again. After combining these resulting organic layers
and washing with water (159 kg), anhydrous MgSO4 (32.0 kg) and activated charcoal (1.59 kg)
were then added, and the batch was filtered through Celite® 545 (6.4 kg). After the resulting filtrate
was concentrated in vacuo using a vacuum pump to 320 L, MeOH (252 kg) was added. After this
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solution was concentrated in vacuo using a vacuum pump to 320 L, more MeOH (252 kg) was
added, and the solution was concentrated again in vacuo using a vacuum pump to 320 L. The
residue was then warmed to 47 °C, and water (95.0 kg) was added to the batch. The resulting slurry
was cooled to 2 °C, agitated for 2 h, and filtered followed by washed with MeOH (63.0 kg)/water
(79.5 kg). Loss to the filtrate was 0.7%. The wet cake was dried in vacuo at 60 °C to afford the
desired 15 at 100.0% purity via HPLC method D (27.9 kg, 93.1% yield).
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1H NMR (400 MHz, CDCl3) δ 7.76 (2H, d, J = 9.2 Hz), 6.91 (2H, d, J = 8.4 Hz), 3.56 (2H, d, J =
12.0 Hz), 3.49 (2H, d, J = 6.0 Hz), 3.14 (3H, s), 3.01-3.12 (3H, m), 2.18-2.28 (2H, m), 1.90-1.99
(2H, m), 1.86 (1H, br), 1.50-1.82 (13H, m), 1.08-1.26 (4H, m), 0.90-1.13 (3H, m)
13C NMR (100 MHz, CDCl3) δ 173.8, 168.2, 153.0, 129.0 (2 carbons), 119.9, 114.9 (2 carbons),
75.3, 68.2, 47.7, 43.9 (2 carbons), 42.3, 40.1, 39.8, 33.4 (2 carbons), 29.5 (2 carbons), 29.3, 29.1,
27.1 (2 carbons), 26.9 (2 carbons), 26.7
MS (ESI, positive mode) m/z; 470.0, MS (ESI, negative mode) m/z; 468.0
Anal. Calcd for C27H39N3O2S: C (69.04%), H(8.37%), N(8.95%), S(6.83%). Found: C (69.15%), H
(8.39%), N(8.79%), S(6.85%); mp: 195 °C (by DSC)
trans-4-{5-[4-(4-cyclohexyl-4-methoxypiperidin-1-yl)phenyl]-1,3,4-thiadiazol-2-yl}cyclohexane-1
-carbaldehyde (1)
Compound 15 (24.0 kg, 51.1 mol), dichloromethane (572 kg), and triethylamine (31.0 kg, 307 mol)
were added to DMSO (106 kg), and then dichloromethane (31.8 kg) was added to the batch. Sulfur
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trioxide pyridine complex (24.4 kg, 153 mol) was then added to the mixture, followed by washing
with dichloromethane (31.8 kg) at 25 °C and aging for 3 h at 25 °C; HPLC analysis subsequently
indicated that 0.4% compound 15 remained (HPLC method E). After reaction completion, an
aqueous solution of ammonium chloride (48.0 kg/water 480 kg) was added to the mixture, followed
by dichloromethane (318 kg). After the phase separation, the resulting aqueous layer was then
extracted with dichloromethane (159 kg). The combined organic layer was concentrated in vacuo
using a vacuum pump to 240 L. CH3CN (188 kg) was added to the residue at 30 °C, and the
material was concentrated in vacuo using a vacuum pump to 240 L. More CH3CN (94.0 kg) and
water (120 kg) were then added at 30 °C, and the resulting slurry was agitated for 3 h at 25 °C, and
then filtered and washed with CH3CN (28.2 kg)/water (36.0 kg). Loss to the filtrate was 1.1%. The
resulting wet cake was then added to CH3CN (188 kg) and agitated for 1 h at 50 °C, cooled to
25 °C, and then filtered and washed with CH3CN (37.5 kg). Loss to the filtrate was 0.2%. The wet
cake was dried in vacuo at 40 °C to afford the desired 1 at 99.9% purity via HPLC method E (21.7
kg, 90.9% yield). cis-1 (0.02%) was observed on HPLC method E.
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1H NMR (400 MHz, CDCl3) δ 9.65 (1H, d, J = 0.4 Hz), 7.76 (2H, d, J = 8.8 Hz), 6.91 (2H, d, J =
8.8 Hz), 3.57 (2H, d, J = 12.4 Hz), 3.14 (3H, s), 3.02-3.13 (3H, m), 2.28-2.38 (3H, m), 2.10-2.20
(2H, m), 1.72-1.82 (3H, m), 1.62-1.71 (8H, m), 1.38-1.61 (3H, m), 1.05-1.26 (3H, m), 0.92-1.04
(2H, m)
13C NMR (100 MHz, CDCl3) δ 203.8, 172.7, 168.3, 153.1, 129.1 (2 carbons), 119.8, 114.9 (2
carbons), 75.3, 49.4, 47.7, 43.9 (2 carbons), 42.3, 39.4, 32.4 (2 carbons), 29.3 (2 carbons), 27.1 (2
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carbons), 27.0 (2 carbons), 26.7 (2 carbons), 25.6
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MS (ESI, positive mode) m/z; 468.0, MS (ESI, negative mode) m/z; 466.0
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Anal. Calcd for C27H37N3O2S: C (69.34%), H(7.97%), N(8.99%), S(6.86%). Found: C (69.25%), H
(7.88%), N(8.93%), S(6.82%). Water: 0.04% (KF); mp: 211 °C (by DSC)
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