Synthesis of enantiopure angiotensin II type 2 receptor [AT2R] antagonist EMA401

Article abstract of DOI:10.1016/j.tet.2015.07.018

We report a facile synthesis of the angiotensin II type 2 receptor antagonist EMA401, which recently passed phase II clinical trials, in high overall yield. The synthesis of the key phenylalanine intermediate involved the formation of an α-nitro cinnamic ester and its reduction followed by a Pictet-Spengler cyclization, which furnished the tetrahydroisoquinoline core structure. Next, EMA401 was separated from its enantiomer EMA402 by four recrystalizations of a diastereomeric salt in 98% ee. All steps were performed on gram scale with emphasis on avoiding column purification and using readily available low cost starting materials and reagents.

Full text of DOI:10.1016/j.tet.2015.07.018

Tetrahedron xxx (2015) 1e7
Contents lists available at ScienceDirect
Tetrahedron
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Synthesis of enantiopure angiotensin II type 2 receptor [AT2R]
antagonist EMA401
,
Prasad B. Wakchaure a, Ulf Bremberg b, Johan Wannberg b, Mats Larhed b
*
a Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University, BMC, Box 574, SE-751 23 Uppsala, Sweden
b Department of Medicinal Chemistry, Science for Life Laboratory, Uppsala University, BMC, Box 574, SE-751 23 Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
We report a facile synthesis of the angiotensin II type 2 receptor antagonist EMA401, which recently
passed phase II clinical trials, in high overall yield. The synthesis of the key phenylalanine intermediate
Received 13 April 2015
Received in revised form 22 June 2015
Accepted 6 July 2015
involved the formation of an
a-nitro cinnamic ester and its reduction followed by a PicteteSpengler
cyclization, which furnished the tetrahydroisoquinoline core structure. Next, EMA401 was separated
from its enantiomer EMA402 by four recrystalizations of a diastereomeric salt in 98% ee. All steps were
performed on gram scale with emphasis on avoiding column purification and using readily available low
cost starting materials and reagents.
Available online xxx
Keywords:
EMA401
Angiotensin II type 2 receptor
Synthesis
Ó 2015 Elsevier Ltd. All rights reserved.
Enantiopure
1. Introduction
used various peptide derivatives,3 or preferably metabolically sta-
ble compounds such as the selective agonist M24/C21, the non-
Some 12e15 years ago a drug discovery program aimed at
identifying drug-like angiotensin II type 2 receptor [AT2R] agonists
was initiated at our department with Prof. Anders Hallberg as the
project leader.1 We applied two different approaches; a) starting
from non-peptide, non-selective AT1/AT2 receptor ligands,2 and b)
transforming the native parent peptide angiotensin II, via the AT2R
selective agonistic derivative (p-amino-Phe6)Ang II (Fig. 1),3 into
selective drug like ligands exhibiting agonism at the AT2R. In the
research program, a new class of selective AT2R antagonists was
also discovered, with compound M132/C38 as the most promising
example.4 In fact, the AT2R antagonist M132/C38 was found to be
considerably more effective as an antagonist in the neurite out-
growth model than PD-123,319,5 which was previously used in
most laboratories as the standard selective AT2R antagonist
(Fig. 1).6
While angiotensin II type 1 receptor [AT1R] antagonists such as
Losartan (Fig.1) have been used in the clinic for almost two decades
to treat hypertension,7 the clinical use of AT2R modulators has been
more elusive. The recent successful phase II study of the AT2R an-
tagonist EMA401 (Fig. 1) for treatment of postherpetic neuralgia
has put the spotlight on the therapeutic potential of also the AT2R.8
Previous efforts to investigate the biological function of AT2R have
selective antagonist PD123,319,5 or the selective antagonist
M132/C38.4 After the clinical results with EMA401, this compound
should also become a new important pharmacological tool.
We were curious to study and benchmark the properties
exhibited by EMA401 as part of our ongoing research projects to
further develop the M132/C38 class of AT2R antagonists. Synthesis
of racemic EMA400 (EMA401/EMA402 1:1) has been reported by
several authors9 and a patent application describing the prepara-
tion of EMA400 as well as the partial resolution to the active en-
antiomer EMA401 have been published.10 However, these
preparative protocols have been difficult to reproduce in our hands,
and the problem of preparing the pure enantiomer EMA401 is still
unresolved in the scientific literature. The method to produce the
pharmaceutical material used in clinic may never be published, and
at the time of writing there are no commercial sources.11 We hereby
report a synthetic protocol for preparing EMA401, including a res-
olution of the enantiomers EMA401 and EMA402 by selective
crystallization of diastereomeric salts.
2. Result and discussion
We started the investigation by scouting the first synthetic
routes already published for EMA401 by the Hodges team (Scheme
1).9 The literature strategy involved synthesis of the appropriately
substituted phenylalanine derivative from hydantoin 3, starting
from commercially available 2-hydroxy-3-methoxybenzaldehyde.
* Corresponding author. Tel.: þ46 18 471 4667; fax: þ46 18 471 4474; e-mail
address: Mats.Larhed@orgfarm.uu.se (M. Larhed).
http://dx.doi.org/10.1016/j.tet.2015.07.018
0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.
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P.B. Wakchaure et al. / Tetrahedron xxx (2015) 1e7
Fig. 1. Angiotensin II type 1 and 2 receptor ligands.
Scheme 1. First attempt to prepare EMA400.
Thus, 2-hydroxy-3-methoxybenzaldehyde was O-benzylated to
furnish 2-benzyloxy-3-methoxybenzaldehyde (1). In the next step,
the obtained O-protected 1 was condensed with hydantoin in
possibilities to rapidly access the appropriately substituted phe-
nylalanine derivative (intermediate B). Since 2-hydroxy-3-
methoxybenzaldehyde is commercially available and cheap, we
decided to again explore the possibilities to use this benzyl pro-
tected derivative as the starting material. The selected route was
based on a report by Dauzonne et al.12 and involved the synthesis of
presence of b-alanine in acetic acid to furnish 2 in 60% isolated yield
(Scheme 1). When compound 2 was subjected to reduction using
reported conditions9 using zinc dust and conc. HCl in methanol
reflux, we observed formation of compound 4 in 61% yield instead
of forming hydantoin 3.
Under controlled conditions, e.g., lowering the temperature and
reducing the equivalents of zinc dust, resulted into incomplete
reaction and gave only either over reduced product 4 or unreacted
starting 2. Probably, the reduction of hydantoin 3 to compound 4 is
faster than the reduction of compound 2 to 3, as LCMS was not able
to detect any considerable peak for compound 3.
a-nitro cinnamate ester 5 from 1 by condensation with commer-
cially available ethyl nitroacetate and its subsequent reduction to
intermediate B (Scheme 2).
Condensation of aldehyde 1 with ethyl nitroacetate in presence
of diethyl amine hydrochloride in anhydrous toluene with the re-
moval of formed water by a DeaneStark apparatus smoothly fur-
nished intermediate A. We did not isolate this intermediate and
after aqueous work up it was directly subjected for reduction with
sodium borohydride in isopropanolechloroform. The obtained re-
duced nitro ester 5 was purified by column chromatography to
We therefore decided to switch to an alternative strategy
(Scheme 2). A literature survey revealed that there are multiple
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P.B. Wakchaure et al. / Tetrahedron xxx (2015) 1e7
3
Scheme 2. Synthetic route to EMA400.
afford a 67% isolated yield over two steps. The next transformation
was chemoselective reduction of the nitro functionality to the
corresponding aminoester intermediate B. With H2/PdeC as the
reducing system we observed faster O-debenzylation than re-
duction of nitro group. However, Zn dust in acetic acid provided the
desired reduction of the nitro functionality without affecting the
benzyl protection group. The reaction was exothermic and the
temperature was maintained by controlling the addition of the Zn
dust. After aqueous work up, the obtained intermediate B was
smoothly cyclized with Pictet-Spengler conditions.13 To our delight
the highly pure hydrochloride salt of the isoquinoline (6a) pre-
cipitated out from the aqueous phase during the reaction (76%
isolated yield over two steps). Next, salt 6a was reacted with
diphenylacetyl chloride to furnish ester 7 in high isolated yield
(85%). It was found advantageous to have the ester protected iso-
quinoline as the precursor for this reaction (Scheme 2).9 Thus, after
synthesizing ester 7 in less than five steps, it was hydrolyzed using
aqueous NaOH to furnish EMA400 in 84% yield. Although there are
no published spectroscopic data available for EMA400 or EMA401,
the spectroscopic data concurred well with the obtained product.
After accessing the EMA400 in 0.87 g scale, the next task was to
separate enantiomers EMA401 and EMA402 since the former is
more active and of higher interest as an AT2R antagonist.
Strategy 1B did not yield sufficient separation of diastereomers
with attempted chromatographic and fractional crystallization
methods. Strategy 2A gave good separation of the EMA400 menthyl
ester diastereomers using preparative HPLC (see Supplementary
data, S5 for details). The preparative value of strategies 1B and 2A
was, however, limited since diastereomerically enriched carba-
mates and esters under the examined cleavage conditions un-
derwent racemization (basic) or O-debenzylation (acidic). Also, it
was observed that free base 6b was not fully stable as it was turning
yellow after 1e2 days storage under air. All the above observations
led us to focus on Strategy 2B.
First we decided to investigate Strategy 2B, in an attempt to
improve the partial resolution methodology as reported by
Klutchko et al.9 This methodology involved the resolution of
EMA400 using S-methyl benzylamine as a chiral resolving agent.
Unfortunately, we could only obtain poor resolution despite in-
vestigating crystallizations in different solvents (ethyl aceta-
teehexane, ethanol). It has been reported that (1S,2S)-
(þ)-thiomicamine is useful for partly resolving EMA401.10 Screen-
ing various crystallization solvents for the resolution using (1S,2S)-
(þ)-thiomicamine (Table 1, See Supplementary data, S6), we found
that the use of ethanol, ethyl acetate or toluene were not successful,
providing only low resolution of EMA401 after single crystalliza-
tions. However, when the salt obtained from crystallizations in
ethanol was subjected to three additional crystallizations in etha-
nol; it was observed to generate the EMA401 salt in 93% ee
according to LC.
When repeating the multiple crystallization strategy in in-
creased scale, we observed high insolubility of the diastereomeric
salt after 2e3rd crystallization. This resulted in the need for large
amounts of ethanol to dissolve the EMA401/EMA402 salt at 80 ꢀC.
The consequence of this was that the crystallization became un-
predictable. Thus, it became necessary to identify an alternative
solvent in which the salt had higher solubility at an elevated tem-
perature. An acetone solution of EMA400 and (1S,2S)-(þ)-thio-
micamine at room temperature was observed to form a precipitate
upon addition of water. By the use of an acetoneewater (1:1)
mixture, we were able to achieve 20% ee of EMA401 in the first
crystallization (Table 1, Entry 8, See Supplementary data, S6).
This partly resolved material was subjected for repeated crys-
tallizations, providing improved enantiomeric purity of EMA401.
The acetoneewater solvent system was therefore selected for
multiple crystallizations for the generation of enantiopure EMA401.
EMA400 was next prepared with a modified procedure in multi
gram scale, allowing facile purification without column chroma-
tography (Scheme 4). The synthetic sequence involved conversion
of the precipitated hydrochloride salt 6a to amide 7 followed by its
hydrolysis to furnish crude EMA400 (Scheme 4, Step 1e2).
Identified EMA400 resolution strategies are depicted in Scheme
3. Resolution by chiral acid (Strategy 1A) and chiral base (Strategy
2B) appeared to have the best scaling potentials. Since it is ad-
vantageous to have the resolution in an early step of the synthesis
to improve the atom economy it was decided to first evaluate
Strategy 1A. We screened various chiral acid (R-mandelic acid, R-
camphor sulfonic acid, D-tartaric acid, D-dibenzoyl tartarate) and
solvent (acetone, toluene, ethanol, ethyl acetate, ethanolewater,
ethyl acetate-isohexane, acetonitrile etc.) combinations for the
resolution of 6b but none of the strategy 1A crystallization condi-
tions investigated were able to separate the diastereomeric salts.
Scheme 3. Strategies for enantiomeric separation.
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4
P.B. Wakchaure et al. / Tetrahedron xxx (2015) 1e7
Scheme 4. Multi gram scale procedure for synthesis of racemic EMA400. Resolution of EMA401 using (1S,2S)-(þ)-thiomicamine.
Precipitation of unresolved EMA400 was performed with (1S,2S)-
(þ)-thiomicamine in ethanol and ethyl acetate according to Entry 3
and 4, Table 1 (See Supplementary data) providing the racemic
material in 4 g scale and in 86% isolated yield over three steps.
Minor colored impurities formed in Step 1 and 2 were removed
during the filtration of the formed salt.
of the synthesis was improved by racemization of EMA402 to
EMA400. We hope that this scalable synthetic procedure, with full
purification, analysis and characterization data for EMA401 will be
helpful to academic and industrial researchers in need of this im-
portant AT2R antagonist.
By employing the resolution condition developed in acetonee-
water, we were able to obtain enantiopure EMA401 in 4 crystalli-
zations with 10% yield starting from salt 8 (Step 4, Scheme 4). The
pure enantiomer EMA401 (>98% ee) was separated from (1S,2S)-
(þ)-thiomicamine by washing it with aqueous 2N HCl.
4. Experimental section
4.1. General information
All purchased chemicals were used as received without further
purification. Analytical thin layer chromatography was performed
on silica gel 60 F-254 plates and visualized with UV light. Purifi-
cations were performed with flash column chromatography by
Traditional diastereomeric resolution strategies for the separa-
tion of enantiomers are extensively used in the pharmaceutical
industry today. It is quite often less expensive compared to asym-
metric synthesis methods due to the cost of catalyst, ligands etc.
The major demerit of classical resolution strategies is the maximum
yield of 50%, adding extra cost to the overall synthesis and in-
creasing the chemical waste. Thus, it is highly desirable to develop
procedure in which unwanted chiral waste is isolated, racemized
and recycled.
ꢀ
silica gel (60 A pore size, 230e400 mesh). Analytical HPLC-MS was
performed with UV detection (214, 254 and 280 nm) and ion trap
MS (ESIꢁ or ESIþ), using a C18 column (50ꢂ3.0 mm, 2.6
mm particle
ꢀ
size, 100 A pore size) and a flow rate of 1.5 mL/min. A gradient of
CH3CN in 0.05% aqueous HCOOH solution was used. 1H and 13C
NMR spectra were recorded at 400 and 100 MHz, respectively,
using DMSO-d6, acetone-d6 or chloroform-d as a solvent. Chemical
shifts for 1H and 13C are referenced via the residual solvent signal.
The HRMS experiment was performed on a 7-T hybrid ion trap
(LTQ) FT mass spectrometer modified with a nanospray ion source.
During our studies of resolution strategies 1B and 2A (Scheme
3), we observed racemization during hydrolysis of the separated
carbamates and esters, indicating the value of the filtrate contain-
ing enriched EMA402 (66% ee) from Step 4 in Scheme 4. Next,
EMA402 was esterified by employing SOCl2 in dry ethanol (Scheme
5). When attempting to racemize ethyl ester 7 using aqueous 2N
NaOH, we observed incomplete racemization. Hence compound 7
was treated with KOtBu in ethanol at 80 ꢀC and was thereafter
hydrolyzed in the same reaction flask with 2N NaOH. Removal of
ethanol and acidification precipitated fully racemized EMA400 in
high purity. This combined approach on gram scale furnished ra-
cemic EMA400 in 84% overall yield from EMA402.
4.2. 2-(Benzyloxy)-3-methoxybenzaldehyde 1
To a stirred solution of 2-hydroxy-3-methoxybenzaldehyde
(15.2 g, 100.0 mmol) in anhydrous EtOH (150 mL) was added dry
K2CO3 (34 g, 250.0 mmol) and benzyl bromide (11.9 mL,
100.0 mmol) at room temperature. The reaction mixture was
heated at 100 ꢀC under nitrogen atmosphere. After 2 h of heating
the reaction mixture was cooled to room temperature, insoluble
salts were removed by filtration and was washed by EtOH (50 mL).
The combined filtrate was evaporated to dryness. The obtained
crude oil was dissolved in diethyl ether (200 mL), washed by 2N aq
NaOH (3ꢂ40 mL), brine (3ꢂ40 mL) and dried over MgSO4. The
organic layer was filtered and evaporated to dryness to provide low
melting colorless solid (1, 22.2 g, 92% yield). Mp 42e44 ꢀC. The
obtained product was used in next step without any purification. 1H
NMR (400 MHz, CDCl3)
7.23e7.04 (m, 2H), 5.18 (s, 2H), 3.95 (s, 3H); 13C NMR (101 MHz,
CDCl3) 190.4, 153.2, 151.2, 136.5, 130.5, 128.8, 128.7, 128.7, 124.4,
d
10.24 (d, J¼1 Hz, 1H), 7.53e7.29 (m, 6H),
d
Scheme 5. Racemization of less active EMA402 to EMA400.
119.2, 118.1, 76.5, 56.2.
3. Conclusion
4.3. 5-(2-(Benzyloxy)-3-methoxybenzylidene)imidazolidine-
2,4-dione 2
In summary, we have achieved the gram-scale preparation of
enantiopure EMA401 in 8 steps starting from readily available 2-
hydroxy-3-methoxybenzaldehyde and by resolving the racemic
EMA400 to EMA401 with (1S,2S)-(þ)-thiomicamine. The efficiency
2-(Benzyloxy)-3-methoxybenzaldehyde (1, 5.0 g, 20.0 mmol),
hydantoin (2.34 g, 23.0 mmol) and
b-alanine (0.38 g, 4.0 mmol)
were refluxed in AcOH (50 mL) for 6 h. The reaction mixture was
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P.B. Wakchaure et al. / Tetrahedron xxx (2015) 1e7
5
then cooled to room temperature and poured onto water (200 mL)
under stirring. The precipitated product was collected by filtration
and washed with water and MeOH. The obtained product was dried
under vacuum to provide colorless solidꢁ1(2, 3.80 g, 60%). Mp
1H), 5.08 (d, J¼11.1 Hz, 1H), 4.22 (q, J¼7.1 Hz, 2H), 3.91 (s, 3H),
3.47e3.33 (m, 2H), 1.20 (t, J¼7.1 Hz, 3H); 13C NMR (101 MHz, ace-
tone-d6)
d 165.2, 153.7, 147.0, 138.8, 129.2, 129.2, 129.1, 128.9, 125.0,
123.2, 113.4, 88.8, 75.1, 63.5, 56.2, 32.4, 14.1; HRMS (EI) m/z calcd.
190e192 ꢀC. IR (Neat) 1753, 1707, 1650 cm
;
1H NMR (400 MHz,
For C19H22NO6 [MþH]þ: 360.1447, found: 360.1444.
DMSO-d6)
J¼7.4,1.8 Hz,1H), 7.14e7.05 (m, 2H), 6.62 (s,1H), 4.95 (s, 2H), 3.86 (s,
3H); 13C NMR (101 MHz, DMSO-d6)
165.4, 155.6, 152.6,145.7, 137.1,
d 11.19 (s, 1H), 10.35 (s, 1H), 7.44e7.28 (m, 5H), 7.23 (dd,
4.6. Ethyl 5-(benzyloxy)-6-methoxy-1,2,3,4-
tetrahydroisoquinoline-3-carboxylate hydrochloride 6a
d
128.7, 128.3, 128.3, 128.1, 127.3, 124.5, 120.8, 113.3, 102.8, 74.6, 55.9;
HRMS (EI) m/z calcd. For C18H17N2O4 [MþH]þ: 325.1188, found:
325.1193.
Step I (intermediate B): To a stirred solution of compound 5
(4.5 g, 13.0 mmol) in AcOH (45 mL) at room temperature was added
zinc dust (6.55 g, 100.0 mmol). The exotherm was maintained be-
low 60e65 ꢀC by controlled addition of Zn dust. After the com-
pletion of addition, the reaction was continued further at 60 ꢀC. LC-
MS and TLC after 2 h of stirring confirmed the completion of re-
action. The reaction mixture was then cooled to room temperature
and filtered. The insoluble inorganic solid was washed by AcOH
(20 mL). The combined colorless filtrate was evaporated to dryness.
The crude product obtained was dissolved in DCM (100 mL),
washed with water (3ꢂ50 mL), satd aq NaHCO3 (3ꢂ50 mL) and
brine, dried over MgSO4, filtered and evaporated to dryness to
provide aminoester intermediate B (3.90 g) as colorless oil, which
was used in next step without any purification.
4.4. 4-[2-(Benzyloxy)-3-methoxybenzyl]-1,3-dihydro-2H-imi-
dazol-2-one 4
To the stirred suspension of compound 2 (3.00 g, 9.0 mmol) and
Zn dust (2.40 g, 37.0 mmol) in MeOH (60 mL), conc. HCl (3 mL) was
added dropwise under stirring. The reaction mixture was heated at
70 ꢀC for 30 min. During this time the starting material started
dissolving slowly. A second portion of conc. HCl (3 mL) was added
slowly, dissolving all starting material. After stirring for 30 min the
reaction mixture was then cooled to room temperature and the
excess unreacted Zn dust was removed by filtration. The filtrate was
then poured to water (200 mL) under stirring. The precipitated
product was separated by filtration and washed with water. The
obtained product was recrystallized from MeOH to provide color-
less solid (4, 1.74 g, 61%). Mp 155e157 ꢀC. IR (Neat) 1685,
Step II: The solution of above obtained aminoester intermediate
B in 2N HCl (aq) (39 mL) was purged with nitrogen gas for 30 min
and then 37% aq formaldehyde solution (4 mL, 39.9 mmol, 3 equiv)
was added under stirring. The reaction was continued further for
24 h at room temperature under nitrogen atmosphere. The pre-
cipitated hydrochloride salt of product was filtered, washed with
water (40 mL) and suction dried. The obtained moist product was
dried under vacuum to furnish pure compound 6a (3.6 g, 76% over
1600 cmꢁ1; 1H NMR (400 MHz, DMSO-d6)
d 9.71 (br s, 1H), 9.43 (br
s, 1H), 7.49e7.42 (m, 2H), 7.42e7.31 (m, 3H), 7.05e6.93 (m, 2H),
6.75 (dd, J¼7.5, 1.7 Hz, 1H), 5.83e5.68 (m, 1H), 4.92 (s, 2H), 3.83 (s,
3H), 3.52 (s, 2H); 13C NMR (101 MHz, DMSO-d6)
d 154.9, 152.4,
145.2,137.8, 132.2,128.3,128.1,127.9,123.9,121.4,120.5,111.3, 104.9,
73.8, 55.8, 25.4; HRMS (EI) m/z calcd. For C18H19N2O3 [MþH]þ:
311.1396, found: 311.1407.
two steps). Mp 165e167 ꢀC. IR (Neat) 1748, 1452 cmꢁ1
;
1H NMR
(400 MHz, DMSO-d6) 10.09 (s, 2H), 7.55e7.29 (m, 5H), 7.07 (d,
d
J¼8.6 Hz, 1H), 7.01 (d, J¼8.6 Hz, 1H), 4.98 (d, J¼1.2 Hz, 2H), 4.38 (dd,
J¼11.0, 5.2 Hz, 1H), 4.32e4.15 (m, 4H), 3.85 (s, 3H), 3.21 (dd, J¼17.3,
4.5. Ethyl 3-(2-(benzyloxy)-3-methoxyphenyl)-2-
nitropropanoate 5
5.2 Hz, 1H), 2.90 (dd, J¼17.3, 11.0 Hz, 1H), 1.24 (t, J¼7.1 Hz, 3H); 13
C
NMR (101 MHz, DMSO-d6)
d 168.2, 151.2, 144.4, 137.5, 128.4, 128.0,
124.8, 122.2, 121.0, 112.1, 73.7, 62.0, 56.0, 52.8, 43.2, 23.6, 13.9;
HRMS (ESI) m/z calcd. For C20H24NO4 [MþH]þ: 342.1705, found:
342.1705.
Step
I (intermediate A): A mixture of 2-(benzyloxy)-3-
methoxybenzaldehyde (1, 5.00 g, 21.0 mmol), ethyl nitroacetate
(3.29 g, 25.0 mmol) and Et2NH$HCl (3.39 g, 31.0 mmol) in anhy-
drous toluene (100 mL) was heated under nitrogen atmosphere at
130 ꢀC. The formed water was removed using a DeaneStark as-
sembly. Heating was stopped after 12 h and reaction mixture was
cooled to room temperature. The toluene was evaporated to dry-
ness and the obtained residue was dissolved in DCM (50 mL). The
organic layer was washed with water (3ꢂ50 mL), brine and dried
over MgSO4, filtered and evaporated to dryness. The yellow oily
compound obtained (8.10 g) was directly subjected for next syn-
thetic step without any purification.
4.7. Ethyl 5-(benzyloxy)-2-(2,2-diphenylacetyl)-6-methoxy-
1,2,3,4-tetrahydroisoquinoline-3-carboxylate 7
To a stirred solution of compound 6a (1.0 g, 2.64 mmol) in DCM
(20 mL) was added diphenylacetyl chloride (0.85 g, 3.70 mmol)
under nitrogen atmosphere. After adding catalytic DMAP (16 mg,
0.132 mmol), Et3N (1.1 mL, 7.92 mmol) was added drop wise at
room temperature and reaction was continued. After 30 min when
TLC and LC-MS analysis showed complete consumption of the
starting material, the reaction mixture was diluted with DCM
(20 mL). The organic layer was washed by 2N HCl (3ꢂ30 mL), satd
aq NaHCO3 (3ꢂ30 mL), brine and dried over MgSO4. The organic
layer was filtered, evaporated to dryness and further purified by
flash column chromatography with 15% EtOAc in petroleum ether
to provide a colorless solid (7, 1.20 g, 85% yield). Mp 96e98 ꢀC. IR
(Neat) 1732, 1639, 1294 cmꢁ1; NMR shows presence of amide
Step II: The above obtained intermediate A was dissolved in
CHCl3 (150 mL) and IPA (50 mL). The reaction mixture was cooled to
0
ꢀC and then silica gel (50 g, 230e400 mesh) was added under
stirring. Subsequently NaBH4 (3.8 g, 103.0 mmol) was added over
5e7 min. The reaction mixture was stirred at room temperature for
2 h and then quenched by adding 5 mL AcOH. The reaction mixture
was filtered to remove silica gel, the silica gel was washed with
CHCl3 (100 mL) and the combined filtrate evaporated to dryness.
The crude residue obtained was dissolved in DCM (100 mL) and
washed by water (3ꢂ100 mL), brine and dried over MgSO4. The
organic layer was filtered, evaporated to dryness and further pu-
rified by flash column chromatography with 10% EtOAc in petro-
leum ether to provide thick oil (5, 4.95 g, 67% over two steps). IR
rotamers. 1H NMR (400 MHz, acetone-d6)
d 7.53e7.43 (m, 2H),
7.43e7.16 (m, 13H), 6.94 (d, J¼2.4 Hz, 0.7H), 6.88 (d, J¼8.4 Hz, 0.7H),
6.72 (dt, J¼8.4, 0.8 Hz, 0.7H), 5.66 (s, 0.7H), 5.57 (s, 0.3H), 5.26 (dd,
J¼6.2, 4.0 Hz, 0.7H), 5.23 (dd, J¼5.9, 2.6 Hz, 0.3H), 5.05 (d, J¼11.0 Hz,
0.7H), 5.01 (d, J¼11.0 Hz, 0.3H), 4.97e4.83 (m, 2H), 4.55 (d,
J¼15.1 Hz, 0.7H), 4.41 (d, J¼17.1 Hz, 0.3H), 4.09e3.88 (m, 2H), 3.86
(s, 1H), 3.85 (s, 2H), 3.48 (dd, J¼16.4, 2.5 Hz, 0.3H), 3.43 (dd, J¼16.1,
4.0 Hz, 0.7H), 2.87 (dd, J¼16.2, 6.2 Hz, 0.7H), 2.56 (dd, J¼16.4,
5.9 Hz, 0.3H), 1.08 (t, J¼7.1 Hz, 2H), 1.01 (t, J¼7.1 Hz, 1H); 13C NMR
(Neat) 1748, 1559, 1269 cmꢁ1
7.51e7.46 (m, 2H), 7.41e7.31 (m, 3H), 7.06e6.98 (m, 2H),
6.78e6.75 (m, 1H), 5.59 (dd, J¼9.4, 6.0 Hz, 1H), 5.16 (d, J¼11.1 Hz,
;
1H NMR (400 MHz, acetone-d6)
d
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6
P.B. Wakchaure et al. / Tetrahedron xxx (2015) 1e7
(101 MHz, acetone-d6)
d
172.0, 171.6, 171.1, 152.4, 152.0, 146.0, 145.8,
The above obtained colorless salt (0.41 g, 0.57 mmol) was sus-
pended in DCM (20 mL) and 2N HCl (20 mL) was slowly added. The
suspension was next stirred for 10e20 min to dissolve the solid
material. The organic layer was separated, washed by 2N HCl
(2ꢂ20 mL), brine and dried over MgSO4. The organic layer was
filtered and evaporated to dryness. Thereafter the obtained pure
EMA401 was dissolved in acetonitrile and subjected for freeze
drying to provide a colorless solid (0.28 g, 98% yield, 98% ee). NMR
141.2, 141.0, 140.8, 140.7, 139.0, 138.9, 130.3, 130.1, 129.9, 129.3,
129.2, 129.1, 129.1, 129.1, 129.0, 129.0, 128.8, 128.7, 128.7, 127.9, 127.7,
127.5, 127.5, 127.4, 127.0, 127.0, 126.7, 122.8, 122.3, 112.5, 112.1, 75.2,
75.0, 61.8, 61.4, 56.2, 56.2, 55.4, 55.3, 55.2, 52.9, 46.0, 43.7, 26.9,
26.0, 14.4, 14.3; HRMS (ESI) m/z calcd. For C34H34NO5 [MþH]þ:
536.2437, found: 536.2432.
shows presence of amide rotamers. ½a D20
ꢃ
þ24ꢀ (0.9% in MeOH). Mp
4.8. 5-(Benzyloxy)-2-(2,2-diphenylacetyl)-6-methoxy-1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid EMA400
82e84 ꢀC. IR (Neat) 3128e2837 (br s), 1696, 1620 cmꢁ1 1H NMR
;
(400 MHz, CDCl3)
d 7.43e7.26 (m, 11H), 7.25e7.12 (m, 4H), 6.87 (d,
J¼8.5 Hz, 0.2H), 6.81 (d, J¼8.5 Hz, 0.2H), 6.72 (d, J¼8.4 Hz, 0.8H),
6.56 (d, J¼8.4 Hz, 0.8H), 5.32 (s, 0.8H), 5.17e5.12 (m, 1H), 5.04 (d,
J¼10.9 Hz, 0.8H), 5.00e4.89 (m, 1.2H), 4.86e4.81 (m, 0.4H), 4.60 (d,
J¼14.9 Hz, 0.8H), 4.52 (d, J¼17.2 Hz, 0.2H), 4.42 (d, J¼14.9 Hz, 0.8H),
3.85 (s, 2.4H), 3.84 (s, 0.6H), 3.41 (dd, J¼16.3, 2.6 Hz, 0.2H), 3.34 (dd,
J¼16.2, 5.0 Hz, 0.8H), 2.87 (dd, J¼16.2, 6.3 Hz, 0.8H), 2.40 (dd,
To a stirred solution of compound 7 (1.1 g, 2.05 mmol) in THF
(11 mL) was added 2 N NaOH (11 mL) and biphasic reaction mixture
was stirred vigorously at 60 ꢀC for 12 h. Reaction mixture was cooled
to room temperature and THF was evaporated to dryness. To the ob-
tained basic aqueous residue, 2N HCl (30 mL) was added slowly. The
gummy precipitate obtained was extracted to EtOAc (3ꢂ20 mL). The
organic layer was washed with brine and dried over MgSO4. The or-
ganic layer was filtered and evaporated to dryness. The crude product
obtained was purified by flash column with 60% EtOAc in petroleum
ether to provide a colorless solid (EMA400, 0.87 g, 84% yield).
J¼16.4, 6.0 Hz, 0.2H); 13C NMR (101 MHz, CDCl3)
d 175.8, 175.6,
172.6, 172.0, 151.8, 151.1, 145.0, 145.0, 139.2, 139.0, 138.8, 138.3, 137.7,
137.5, 129.5, 129.3, 129.2, 129.1, 128.9, 128.8, 128.71, 128.67 (2 Car-
bon), 128.6, 128.5, 128.4, 128.3, 128.1, 127.6, 127.3, 127.3, 127.2, 127.1,
125.7, 125.5, 125.3, 122.4, 121.5, 111.8, 111.2, 75.0, 74.9, 56.0 (2 Car-
bon), 55.8, 55.6, 54.4, 52.4, 45.6, 43.4, 25.9, 25.0; HRMS (ESI) m/z
calcd. For C32H30NO5 [MþH]þ: 508.2124, found: 508.2102.
4.9. Improved procedure for the synthesis of racemic EMA400
salt 8, Scheme 4
4.11. Racemization of EMA402 to EMA400
Step I: To a stirred solution of compound 6a (2.5 g, 6.61 mmol) in
DCM (50 mL) was added diphenylacetyl chloride (2.14 g, 9.28 mmol)
under nitrogen atmosphere. After adding catalytic DMAP (40 mg,
0.33 mmol), Et3N (2.8 mL, 19.8 mmol) was added drop wise at room
temperature and reaction was continued further. After 30 min of
stirring, the reaction mixture was diluted with DCM (50 mL). The
organic layer was washed by 2N HCl (3ꢂ50 mL), satd aq NaHCO3
(3ꢂ50 mL), brine and dried over MgSO4. The organic layer was fil-
tered, evaporated to dryness to provide the crude product 7 (3.50 g),
which was used without further purification in the next step.
Step II: To a stirred THF (34 mL) solution of compound 7 ob-
tained in step I was carefully added 2N NaOH (34 mL) and the
resulting biphasic reaction mixture was stirred vigorously at 60 ꢀC.
After 12 h of stirring, the reaction mixture was cooled to room
temperature and the THF was evaporated to dryness. To the ob-
tained aqueous basic residue, 2N HCl (100 mL) was added slowly.
The gummy precipitate obtained was extracted to EtOAc
(3ꢂ30 mL). The organic layer was washed with brine and dried over
MgSO4. The organic layer was filtered and evaporated to dryness to
provide crude EMA400 (3.20 g), which was used in next step
without further purification.
The filtrates obtained from the first and second crystallizations
in the resolution of EMA401 were evaporated to dryness (1.91 g).
The obtained colorless salt was suspended in DCM (20 mL) and 2N
HCl (20 mL) was added to the stirred suspension to dissolve the salt.
The layers were separated and the organic layer was washed by 2N
HCl (2ꢂ20 mL) and brine, dried over MgSO4, filtered and evapo-
rated to dryness to provide EMA402 (66% ee, 1.31 g, 2.58 mmol).
EMA402 was dissolved in anhydrous EtOH (20 mL) and SOCl2
(2 mL) was added under nitrogen atmosphere. After stirring the
reaction mixture at room temperature for 24 h, it was evaporated to
dryness and the obtained crude product was dissolved in EtOAc
(20 mL). The organic layer was washed by satd aq NaHCO3
(3ꢂ20 mL), brine and dried over MgSO4. Next, the organic layer was
filtered and evaporated to dryness. The obtained ester 7 (1.32 g)
was dissolved in anhydrous EtOH (20 mL) and KOtBu (0.29 g,
2.58 mmol) was added under nitrogen atmosphere and reaction
mixture was stirred at 80 ꢀC. After 1 h, 2N NaOH (10 mL) was
carefully added and reaction was further stirred at the same tem-
perature for 1 h. The reaction mixture was then concentrated under
reduced pressure to remove the ethanol. The obtained aqueous
residue was slowly acidified by 2N HCl (40 mL) to precipitate the
EMA400 as white solid (1.1 g, 84% over 2 steps). Chiral purity
analysis showed 1:1 enantiomeric ratio.
Step III: To the stirred solution of above obtained crude EMA400
in EtOH (40 mL) was added (1S,2S)-(þ)-thiomicamine (1.4 g,
6.61 mmol). After heating the reaction mixture to reflux for
10e20 min, it was cooled to room temperature and stirred for an-
other 3e4 h. The precipitated racemic salt was then filtered, suction
dried and washed by EtOH (20 mL). The obtained colorless product
was further evaporated to dryness to provide EMA400 salt 8 (4.12 g,
86% over 3 steps).
4.12. Analysis of the enantiomeric excess (ee) of EMA401
The diastereomeric salt (8, 2e5 mg)wassuspendedinDCM (2 mL)
and washed by 2N HCl (2 mL) in a sample vial. The DCM layer was
dried over MgSO4 and filtered. A drop of (S)-methyl benzylamine and
HBTU (20e30 mg) was added to the filtrate and the reaction vial was
shaken. After 5e10 min the formed diastereomers were analyzed for
chiral purity by HPLC (Note: The use of EDCI as dehydrating agent
shown racemization of the separated enantiomer. Hence HBTU was
used instead for the formation of diastereomer.)
4.10. Resolution of EMA401
The above obtained salt (8, 4.0 g, 5.55 mmol) was dissolved in
minimum amount of acetone (40 mL) at 75 ꢀC under stirring. After
complete dissolution of salt, water (50 mL) was added dropwise.
The solution was then cooled gradually and when the temperature
reached 25e30 ꢀC, the crystallized product was filtered, suction
dried and further evaporated to dryness to provide the product
(2.6 g). Repetition of additional 3 crystallizations in the similar way
provided 0.42 g EMA401 salt (10.5% yield).
4.13. HPLC analysis condition
Reverse phase HPLC (UV-triggered, 214 nm) was performed
with C18 column (50ꢂ3 mm, 2.6
mm particle size) and a H2O/
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P.B. Wakchaure et al. / Tetrahedron xxx (2015) 1e7
7
€
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Acknowledgements
We thank Uppsala University and Science for Life Laboratory for
support.
7. (Renin and Angiotensin; Jackson E. K., 789e821)Goodman
& Gilman’s The
Pharmacological Basis of Therapeutics, 11th ed.; Brunton, L. L., Lazo, J. S., Parker,
K. L., Eds.; New York McGraw Hill: 2006.
Supplementary data
8. (a) Rice, A. S. C.; Dworkin, R. H.; McCarthy, T. D.; Anand, P.; Bountra, C.;
McCloud, P. I.; Hill, J.; Cutter, G.; Kitson, G.; Desem, N.; Raf, M. Lancet 2014, 383,
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(b) Blankley, C. J.; Hodges, J. C.; Sylvester, K.; US Patent Application, Publication
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2015.07.018.
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Pharmaceuticals Ltd., Australia. Repeated enquires to Glycosyn has not resulted
in any quotes.
12. Dauzonne, D.; Royer, R. Synthesis 1987, 399.
13. Zhang, X.; Wang, W.; Cheng, S.; Zhao, M.; Zheng, M.; Chang, H. W.; Wu, J.; Peng,
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