A practical synthesis of Rho-Kinase inhibitor Y-27632 and fluoro derivatives and their evaluation in human pluripotent stem cells

Article abstract of DOI:10.1039/c1ob05332a

A practical synthesis of the Rho-Kinase inhibitor Y-27632 and two new fluoro derivatives was achieved in seven steps and with a good overall yield of 45% starting from commercially available (R)-1-phenylethylamine. Compared to Y-27632 the new fluoro deriv

Full text of DOI:10.1039/c1ob05332a

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PAPER
A practical synthesis of Rho-Kinase inhibitor Y-27632 and fluoro derivatives
and their evaluation in human pluripotent stem cells†
Jirˇ´ı Palecˇek,a Robert Zweigerdt,b Ruth Olmer,b Ulrich Martin,b Andreas Kirschning*a and Gerald Dra¨ger*a
Received 2nd March 2011, Accepted 26th April 2011
DOI: 10.1039/c1ob05332a
A practical synthesis of the Rho-Kinase inhibitor Y-27632 and two new fluoro derivatives was achieved
in seven steps and with a good overall yield of 45% starting from commercially available
(R)-1-phenylethylamine. Compared to Y-27632 the new fluoro derivatives showed reduced or no effect
on hPSC vitality and expansion after dissociation in human pluripotent stem cells.
The synthesis of 1 and its analogues was first published by
Introduction
Yoshitomi Pharmaceutical Industries.3 The a-alkylbenzylamines
were used as chiral starting material. However, these reports3 do
not provide sufficient analytical data. In fact, only the melting
point and the optical rotation [a]D +4.6 (c 1, MeOH) was
reported.3b
Later two syntheses of ROCK-inhibitor 1 were published
that started from 1,4-cyclohexyldimethanol4a and from trans-1,4-
cyclohexanedicarboxylic acid.4b Again, these publications lacked
sufficient description of analytical data.
As part of our investigations in stem cell research2 we first
required sufficient amounts of 1 as well as of fluoro analogues.
During our efforts to follow the patent descriptions3 we noted
major difficulties as well peculiarities when comparing analytical
data with those reported.
(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)-cyclohexane carboxam-
ide dihydrochloride (1) (Y-27632, Fig. 1) is highly potent, cell-
permeable and selective ROCK (Rho-associated coiled coil form-
ing protein serine/threonine kinase) inhibitor. This compound has
played a major role for better understanding the physiological roles
of this Rho-kinase. Furthermore, Y-27632 (1) is widely utilised
and tested in the treatment of various diseases. These include
different cellular functions, cell adhesion, cell motility, vascular
and smooth muscle contraction, cytokinesis,1a,b cardiovascular
diseases, such as hypertension and arteriosclerosis,1c bronchial
asthma,1d cancer,1e Alzheimer’s disease,1f coronary artery spasm1c,g
and vasospastic angina.1c,h Recently, we and others disclosed the
first applications of 1 in human pluripotent embryonic stem (hPS)
cell research, particularly in scalable mass expansion in suspension
culture which is of high relevance to clinical applications.2 Due to
its broad importance this ROCK inhibitor 1 or derivatives should
become pharmaceutical candidates for several future clinical
applications.
Results and discussion
The synthesis of Y-27632 (1) commenced from the commercially
available and inexpensive (R)-1-phenylethylamine (2) (Scheme 1)
which was transformed according to the published procedure3c
to the benzoic acid 3 in the three-step sequences that included
N-acylation (95%), Friedel–Crafts acylation (90%) and haloform
reaction (91%).
Hydrogenation (H2, 5% Ru/C, 26% aqueous ammonia) of the
aromatic moiety was the key step of the synthesis. It was carried
out in an autoclave under different conditions with respect to
pressure and temperature. We repeatedly observed that only one
isomer of two possible cyclohexane products was formed, the
N-acetylated acid 4 or/and the deacetylated acid 5, respectively
(Scheme 1). Lower initial hydrogen pressure, lower temperature
Fig. 1 Rho-kinase inhibitor Y-27632 (1).
aInstitut fu¨r Organische Chemie and Biomolekulares Wirkstoffzentrum
(BMWZ), Leibniz Universita¨t Hannover, Schneiderberg 1b, 30167, Han-
nover, Germany. E-mail: andreas.kirschning@oci.uni-hannover.de; Fax: +49
(0)511-7623011; Tel: +49 (0)511-7624614
◦
◦
and shorter reaction time (50 bar, 90 C, 6 h, then 120 C, 6 h)
led to the acetamidoethyl acid 4. Still the starting benzoic acid 3
(ca 10–20%) was always detectable in the NMR spectra. When the
hydrogenation was carried out at higher pressure and temperature
bHannover Medical School (MHH), Department of Cardiac, Thoracic,
Transplantation and Vascular surgery (HTTG), Leibniz research labora-
tories for biotechnology and artificial organs (LEBAO), Carl-Neuberg-Str.
1, 30625, Hannover, Germany
◦
(70 bar, 150 C) for 1–3 days, formation of a mixture of the two
trans-isomers 4a and 5a in varying ratios were observed. In the
following, the resulting mixture of 4a and 5a was heated in an
† Electronic supplementary information (ESI) available: Copies of spectra
and analytical results. See DOI: 10.1039/c1ob05332a
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Scheme 1 Synthesis of (R)-4-(1-aminoethyl) cyclohexanecarboxylic acid
5a (1,4-trans) and isomerisation attempts.
Fig. 2 1H-NMR spectra of trans-(R)-4-(1-aminoethyl)cyclohexane car-
boxylic acid hydrochloride (7a).
autoclave with 26% aqueous ammonia at 160 ◦C for three days
which resulted in full conversion to the aminoethyl acid 5a.
Longer reaction times (>3 days) under identical hydrogenation
conditions afforded the acid 5a in one step in 70% yield. In all
these hydrogenation experiments the carboxylic acids 4a or 5a
were isolated as pure 1,4-trans-isomers while formation of the
1,4-cis isomer 4b or 5b, respectively, was not detected. However,
comparison of the melting point of our 1,4-trans-carboxylic acid
5a (255 ◦C) revealed a discrepancy with the reported one (1,4-
TBTU/DIPEA10 (85%) were probed. Finally, removal of the Boc
group using 1 N HCl in diethyl ether afforded the target ROCK-
inhibitor 1 (Scheme 2).
◦
trans-isomer 5a: mp >290 C),5 while the melting point for the
1,4-cis-isomer 5b was reported to be 243 ◦C. In order to clarify
the situation, we more closely analysed the 1,4-configurations in
the cyclohexane ring in 4a and 5a, respectively. As shown below,
1
two isomerisation experiments on 5a and 6a, H-NMR analysis
of an analytically pure sample of 7a and final NMR comparison
of the synthesised ROCK inhibitor Y-27632 (1) with an authentic
sample (see ESI†) confirmed the correct stereochemistry.
Scheme 2 Synthesis of ROCK-inhibitor Y-27632 (1).
Firstly, we attempted two isomerisation experiments3c,5,6
(Scheme 1) on carboxylic acid 5a and its methyl ester 6a which was
first straightforwardly prepared by esterification7 using methanolic
HCl (1.25 N solution). Thus, both cyclohexane derivatives 5a
and 6a were heated under refluxing conditions in an ethanolic
solution of tBuOK for 3 days. NMR-analysis revealed no changes
except that ester 6a was hydrolysed to the carboxylic acid
5a. From these results it can be concluded that the 1,4-trans
isomer is the thermodynamically more stable isomer which is
also formed under hydrogenation conditions. Recrystallisation
of cyclohexanecarboxylic acid hydrochloride 7a from ethanol-
acetonitrile gave an analytically pure sample of 7a which allowed
to assign all coupling constants for the axially oriented protons
at H-1 and H-4 (3Jaa = 12 Hz) that further confirmed the desired
1,4-trans-configuration (Fig. 2).
In addition, we found that the optical rotations reported in the
literature substantially differed from the one that we measured.
We measured a specific optical rotation value of ([a]D -11.5 (c
0.5, MeOH) compared to [a]D +4.6 (c 1, MeOH).3b Nevertheless,
all physical and analytical data were in full agreement with those
measured for an authentic sample of 1 (see ESI†).11 Correctness of
our material was proven in biological tests with human embryonic
stem (hES) cells. Both the synthetic as well as an authentic sample
of Y-27632 showed identical activity.2
In order to study the influence of electronic effects in the pyridine
ring on ROCK inhibition without introducing large substituents
we prepared two new fluoro derivatives 10 and 11 by following the
sequence described in Scheme 2 (Scheme 3).
Human pluripotent stem cells (hPSC)—including human em-
bryonic stem cells (hESC) and human induced pluripotent stem
cells (hiPSC)—unlike mouse ones, are vulnerable to apoptosis
upon dissociation.2 Based on our fundamental work showing that
1 markedly supports expansion of hPS cells as floating aggregates
in a serum-free suspension culture,2d we further evaluated the
Finally, the synthesis of Y-27632 (1) was finalised after N-
Boc protection (89%)8 and amidation of the carboxylic acid
8 with 4-aminopyridine to yield 9. For this step two different
methods, the Mukaiyama reaction9 (97%) and the reagent system
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bly, cell motility and contraction. The ROCK protein consists
of several domains, an N-terminal region, a kinase catalytic
domain, a coiled-coil domain containing a RhoA binding site,
and a pleckstrin homology domain. The C-terminal region of
ROCK binds to and inhibits the kinase catalytic domains, and
this inhibition is reversed by binding RhoA, a small GTPase.
Jacobs and coworkers12 determined crystal structures of four
ROCK protein–ligand complexes containing the inhibitors Y-
27632, fasudil (HA-1077), hydroxyfasudil (HA-1100), and a
dimethylated analog of fasudil (H-1152P). Y-27632 is a pyridine
compound that is chemically distinct from the other three
isoquinoline-based ligands. Each of these compounds binds with
reduced affinity to cAMP-dependent kinase (PKA), a highly
homologous kinase. The X-ray crystal structure of ROCK bound
to ATP-competitive inhibitors revealed two kinase domains linked
by an N-terminal dimerisation domain comprised of five a-helices
from each monomer. In this arrangement, the active sites of the
two kinases share a common face, possibly facilitating interactions
with dimeric substrates or inhibitory domains. Each kinase
domain appears to have a catalytically competent conformation in
the absence of phosphorylation. Comparison of the four ROCK-
ligand structures - both among themselves and against their
corresponding counterparts in PKA - suggested that a very specific
active site contact is important for selective ROCK inhibition.12
In the present study we did not define protein–ligand complex
structures of ROCK with our novel Y-27632 derivatives 10 and 11.
However, our functional evaluation in two hPS cell lines suggest
that 3-fluoro substitution in the pyridine ring in 11 interferes
with the active site contact of ROCK, resulting in a diminished
biological activity i.e. >50% compared to Y-27632 (1). This
effect is even more pronounced in derivative 10, when fluorine
is substituted in the 2-position complete loss of biological activity
was provoked.
Scheme
inhibitor 1.
3
Synthesis of fluoro derivatives 10 and 11 of ROCK-
activity of the novel fluoro derivatives. The data presented in
Fig. 3 suggest that fluoro derivative 11 at least partially rescued
hPSC. This was in the range of 25–40% compared to the
activity of 1 regarding the efficiency of supporting cell expansion
(Fig. 3 A). In contrast, the activity of fluoro derivative 10 was
equivalent to non-supplemented medium controls in the same
assay, meaning that survival, re-aggregation and expansion of
single-cell dissociated hPSC was not supported (Fig. 3 A-B).
Conclusions
In summary, the present work discloses a practical and scalable
synthesis of the ROCK inhibitor Y-27632 (1) with complete
characterisation by 1H-, 13C-NMR- and IR-spectroscopy, MS-
and HRMS-spectrometry and m.p. as well as specific optical
rotation. The title compound was obtained in 45% yield over
seven steps. The key hydrogenation step afforded the trans-(R)-4-
(1-aminoethyl) cyclohexane carboxylic acid 5a as a single isomer.
The optimised synthesis also served to access two new fluoro
derivatives. Biological evaluation of new derivatives 10 and 11 in
human pluripotent stem cells revealed that fluorine substitution in
the pyridine ring partially or completely diminished the supporting
effect of the original molecule Y-27632 on hPSC vitality and
expansion after dissociation.
Fig. 3 In vitro assessment of 1, 10 and 11 regarding their efficiency to
support survival, re-aggregation and expansion of single cell-inoculated
suspension cultures of human ES and human iPS cells. (A) Fold expansion
of hESC or hiPSC 4 days after inoculation relative to the seeding density.
Controls were cultured in mTeSR medium only. In test cultures mTeSR
was supplemented with the respective compound at a 10 mM concentration
each. Every bar represents n = 9. (B) Representative pictures of aggregates
formed in suspension culture 4 days after single cell inoculation in controls
or in the presence of the respective compound; scale bars represents
200 mm.
Experimental
General remarks
All solvents were dried by conventional methods. Starting ma-
terials and reagents were purchased from commercial suppli-
ers and used without further purification. Preparative column
chromatography was performed using silica gel 60, particle size
0.040–0.063 mm (230–240 mesh, flash). Analytical TLC was
The serine/threonine kinase ROCK is an effector of Rho-
dependent signalling and is involved in actin-cytoskeleton assem-
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carried out employing silica gel 60 F254 plates from Merck,
Darmstadt. Visualisation of the developed chromatograms was
performed with detection by UV (254 nm), by colouration with
a phosphomolybdic acid solution in EtOH or ninhydrin solution
in EtOH. NMR spectra were recorded on a Bruker ARX-400
(1H, 400 MHz; 13C, 100 MHz) spectrometer. All spectra were
measured using standard Bruker pulse sequences. Mass spectra
were obtained from a Micromass LCT via loop-mode injection
from a Waters (Alliance 2695) HPLC system. HRMS data were
recorded on a Micromass Q-TOF in combination with a Waters
Aquity Ultraperformance LC system. Ionisation was achieved by
ESI or APCI. Melting points were measured on a SRS OptiMelt
apparatus and are uncorrected. Optical rotation was measured
with a Perkin Elmer 341 polarimeter. IR spectra were recorded
with a Bruker Vektor 22 FT-IR spectrophotometer (GoldenGate
ATR unit).
CH3CO), 5.14 (quint, 1H,3JHH = 7.0 Hz, CH), 5.90 (bs, 1H, NH),
7.41 (d, 2H,3JHH = 8.3 Hz, 2 ¥ Ar-H), 7.92 (d, 2H,3JHH = 8.3 Hz, 2 ¥
Ar-H); 13C-NMR (CDCl3, 100 MHz): d 21.8 (CH3), 23.3 (CH3),
26.6 (CH3), 48.6 (CH), 126.3 (Ar-CH), 128.5 (Ar-CH), 136.1 (Ar-
C), 148.7 (Ar-C), 169.2 (C O), 197.7 (C O); MS (+ESI): m/z
(%) = 228.0920 (75) [M+Na]+, 206.0948 (100) [M+H]+; HR-MS:
206.1176 (206.1186 calc. for C12H16N1O2).
The spectroscopic data of (R)-N-(1-acetylphenylethyl)-
acetamide were in full agreement with those reported in the
literature.3c,13d
(R)-4-(1-Acetamidoethyl)benzoic acid (3)
Sodium hypochlorite (12% solution, 130 mL) was slowly added
to a mixture of (R)-N-(1-(4-acetylphenyl)ethyl)acetamide (10 g,
48.7 mmol, 1 equiv.), and sodium hydroxide (2.2 g, 52.9 mmol,
1.1 equiv.) in methanol (100 mL) at r.t. The mixture was heated to
60 ◦C for 2 h. After completion of the reaction (checked by TLC,
ethyl acetate/methanol 9 : 1), the solvent was removed and the
residue obtained was poured onto ice water (200 mL). Then, conc.
hydrochloride acid was added which resulted in a yellow crystalline
precipitate. This was filtered off and the acidic aqueous filtrate
was extracted with ethyl acetate (3 ¥ 100 mL). The organic layers
were combined and evaporated, dried overnight under vacuum
which gave another crop of yellow crystals which was combined
with the yellow crystalline material obtained before to yield the
title compound 3 (9.18 g, 44.3 mmol; 91%). mp decomposition at
(R)-N-(1-Phenylethyl)acetamide
Acetic anhydride (33.7 g, 330 mmol, 2 equiv.) was added dropwise
to a solution of (R)-1-phenylethylamine (2) (20 g, 165 mmol,
1 equiv) in chloroform (150 mL) at 0 ◦C. After completion of
the reaction (checked by TLC, ethyl acetate/methanol, 9 : 1), ice
water (150 mL) was added and the mixture was extracted with
chloroform (3 ¥ 150 mL). The extracts were washed with 1 N
aqueous solution of NaOH (100 mL), dried over MgSO4, filtered,
concentrated under reduced pressure and dried overnight under
vacuum to afford the title compound (25.6 g, 156.8 mmol; 95%)
as colourless crystals.
◦
20
about 175 C; [a]D +116.7 (c 1, MeOH) [ref. 3c [a]D +136.8 (c
1, MeOH). 1H-NMR (MeOH-d4, 400 MHz): d 1.49 (d, 3H,3JHH
=
7.1 Hz, CH3), 2.01 (s, 3H, CH3CONH), 5.06 (q, 1H,3JHH = 7.1 Hz,
CH), 7.49 (d, 2H,3JHH = 8.2 Hz, 2 ¥ Ar-H), 8.01 (d, 2H,3JHH
mp 100–101 ◦C (ref. 13a mp 99–100 ◦C, ref. 13b mp 97–100 ◦C);
=
20
[a]D +140.4 (c 1, CHCl3) [ref. 3c [a]D +143.5 (c 1, EtOH), ref.
8.2 Hz, 2 ¥ Ar-H). The signals for NH and COOH could not be
detected in the spectrum; 13C-NMR (MeOH-d4, 100 MHz): d 23.1
(CH3), 23.4 (CH3), 50.9 (CH), 127.9 (Ar-CH), 131.4 (Ar-C), 131.5
(Ar-CH), 151.5 (Ar-C), 170.5 (C O), 173.3 (C O); MS (+ESI):
m/z (%) = 230.0850 (30) [M+Na]+, 208.0799 (100) [M+H]+; HR-
MS: 208.0949 (208.0974 calc. for C11H14N1O3).
13a [a]D +138.8 (c 2.35, EtOH), ref. 13b [a]D +141.0 (c 1, CHCl3)].
1H-NMR (CDCl3, 400 MHz): d 1.50 (d, 3H,3JHH = 6.9 Hz, CH3),
2.01 (s, 3H, CH3CONH), 5.21 (quint, 1H,3JHH = 6.9 Hz, CH), 5.83
(bs, 1H, NH), 7.31–7.39 (m, 5H, 5 ¥ Ar-H);
13C-NMR (CDCl3, 100 MHz): d 21.7 (CH3), 23.5 (CH3), 48.8
(CH), 126.2 (Ar-CH), 127.4 (Ar-CH), 128.7 (Ar-CH), 143.1 (Ar-
C), 169.0 (C O); MS (+ESI): m/z (%) = 186.0822 (90) [M+Na]+,
164.0918 (100) [M+H]+; HR-MS: 164.1068 (164.1075 calc. for
C10H14N1O1).
The spectroscopic data of (R)-4-(1-acetamidoethyl)benzoic acid
(3) were in full agreement with those reported in the literature.3c
Catalytic hydrogenation – general procedure
The spectroscopic data of (R)-N-(1-phenylethyl)acetamide were
in full agreement with those reported in the literature.3c,13
A solution of benzoic acid 3 and 5% ruthenium on carbon in 26%
aqueous ammonia was stirred in an autoclave. Then, the catalyst
was filtered off through a short pad of CeliteTM. The filter pad
was washed with water and the filtrate was concentrated and dried
overnight under vacuum to afford 1,4-trans 4a and ca. 10–20%
of starting material 3 (method A), a mixture of 1,4-trans 4a and
1,4-trans 5a in varying ratios (method B) or only 1,4-trans 5a
(method C).
(R)-N-(1-(4-Acetylphenyl)ethyl)acetamide
Aluminium chloride (44 g, 331.3 mmol, 2 equiv.) was added
portionwise to a solution of (R)-N-(1-phenylethyl)acetamide (27 g,
165.6 mmol, 1 equiv.) in 1,2-dichloroethane (150 mL) at r.t.
Then, acetyl chloride (15.6 g, 198.7 mmol, 1.2 equiv.) was added
dropwise. The mixture was stirred at r.t. for 1 h and subsequently
at 60 ◦C for 3 h. After completion of the reaction (checked by TLC,
ethyl acetate), the mixture was poured onto ice water and extracted
with dichloromethane (3 ¥ 150 mL). The extracts were washed with
water (100 mL) dried over MgSO4, filtered, concentrated and the
residue obtained was crystallised (isopropyl ether/ethanol 9 : 1) to
afford the title compound (30.6 g, 149.1 mmol; 90%) as orange
crystals. mp 125–126 ◦C; [a]D20 +173.8 (c 0.5, CHCl3) [ref. 3c [a]D
Method A: trans-(R)-4-(1-Acetamidoethyl)cyclohexane carboxylic
acid (4a)
Benzoic acid 3 (1 g, 4.83 mmol) and 5% ruthenium on carbon (0.7
g) in 26% aqueous ammonia (5 mL) were reacted for 6 h at 90 ◦C
and for 6 h at 120 ◦C according to the general procedure. The
initial hydrogen pressure was set at 50 bar. Trans-4a (0.80 g, 78%)
was obtained which was contaminated with the starting material
3 (ca 10%, judged by 1H NMR spectroscopy).
1
+162.0 (c 1, MeOH). H-NMR (CDCl3, 400 MHz): d 1.49 (d,
3H,3JHH = 7.0 Hz, CH3), 1.99 (s, 3H, CH3CONH), 2.56 (s, 3H,
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Method B: mixture of trans-4a and trans-5a
mixture of ethanol/acetonitrile 1 : 1 to afford an analytically pure
sample of the title compound 7a (0.28 g, 1.35 mmol; 76.9%). mp
Benzoic acid 3 (5 g, 24.1 mmol) and 5% ruthenium on carbon (5 g)
in 26% aqueous ammonia (25 mL) were reacted for 3 h at 90 ◦C and
then for 1–3 d at 150 ◦C according to the general procedure. The
initial hydrogen pressure was set at 70 bar. A mixture of trans-4a
and trans-5a (3–3.9 g, calc. ca 69–80%) was obtained.
254 ◦C [ref. 5 mp >290 ◦C]; [a]D +7.5 (c 0.5, MeOH). 1H-NMR
20
3
2
3
(D2O, 400 MHz): d 1.16 (dq, 2H, JHH = 3.1 Hz, JHH = JHH
=
3
13 Hz, H-3a and H-5a), 1.26 (d, 3H, JHH = 6.8 Hz, CH3), 1.42
(dq, 1H, 3JHH = 3.1 Hz, 2JHH = JHH = 13 Hz, H-2a or H-6a), 1.43
3
3
(dq, 1H, 3JHH = 3.1 Hz, 2JHH = JHH = 13 Hz, H-6a or H-2a), 1.59
(dtt, 1H, 3JHH = 3.1 Hz, 3JHH = 6.5 Hz, 3JHH = 12.6 Hz, H-4), 1.83
(m, 2H, H-3e and H-5e), 2.05 (m, 2H, H-2e and H-6e), 2.35 (tt, 1H,
3JHH = 3.4 Hz, 3JHH = 12.3 Hz, H-1), 3.22 (quint, 1H, 3JHH = 6.5 Hz,
CHCH3). The signals for NH2 and COOH were not detected in
the spectrum; 13C-NMR (D2O, 100 MHz): d 14.9 (CH3), 26.1
(CH2), 27.2 (CH2), 27.8 (CH2), 27.9 (CH2), 39.9 (CH-4), 42.5
(CH-1), 51.9 (CHCH3), 181.0 (C O); MS (+ESI): m/z (%) =
172.1407 (100) [M+H]+; HR-MS: 172.1334 (172.1338 calc. for
C9H18N1O2).
Method C: trans-(R)-4-(1-Aminoethyl)cyclohexanecarboxylic acid
(trans-5a)
Benzoic acid 3 (5 g, 24.1 mmol) and 5% ruthenium on carbon (5
g) in 26% aqueous ammonia (25 mL) were reacted at 90 ◦C for
3 h and then at 150 ◦C for 3 d to the according to the general
procedure. The initial hydrogen pressure was set at 70 bar. 1,4-
Trans 5a (2.89 g, 16.9 mmol; 70%) was obtained.
Transformation of the mixture of 1,4-trans 4a and 1,4-trans 5a
trans-(R)-4-(1-Aminoethyl)cyclohexanecarboxylic acid methyl
ester (6a)
A solution of a mixture of 4a and 5a (4 g) obtained from insufficient
hydrogenation in 26% aqueous ammonia (40 mL) was stirred
under a hydrogen atmosphere (70 bar) in an autoclave at 160 ◦C
for 3 d. Then, the solvent was evaporated and the product was
dried overnight under vacuum to exclusively afford the carboxylic
acid 5a (3.7 g).
Methanolic HCl (1.25 N solution, 9.3 mL, 11.7 mmol, 2 equiv.) was
added to cyclohexanecarboxylic acid 5a (1 g, 5.84 mmol, 1 equiv.)
in dry methanol (20 mL) under an argon atmosphere. The mixture
was heated under refluxing conditions for 2 h. Then, the solvent
was removed under vacuum and chloroform (30 mL) and water
(30 mL) were added. After addition of solid potassium carbonate
(1 g) the mixture was extracted with chloroform (3 ¥ 30 mL).
The combined organic layers were dried over MgSO4, filtered and
concentrated under reduced pressure to afford the title compound
6a (0.86 g, 4.64 mmol; 80%) as an oil which was used without
further purification in the isomerisation experiment.
4a: In all instances 4a contained traces of benzoic acid 3 as
judged from NMR-analysis.
1H-NMR (MeOH-d4/DMSO-d6, 400 MHz): d 1.05 (d, 3H,
3JHH = 6.8 Hz, CH3), 1.17–1.68 (m, 7H), 1.92 (s, 3H, CH3CO),
2.05 (m, 2H), 2.52 (m, 1H, H-1), 3.81 (quint, 1H, 3JHH = 6.8 Hz,
CHCH3). The signals for NH and COOH were not detected in the
spectrum.
1H-NMR (CDCl3, 400 MHz): d 1.00 (m, 2H, H-3a and H-5a),
13C-NMR (MeOH-d4/DMSO-d6, 100 MHz): d 16.9 (CH3),
21.4 (CH3CO), 25.5 (CH2), 25.6 (CH2), 26.1 (CH2), 26.3 (CH2),
39.5 (CH-4), 41.7 (CH-1), 47.8 (CHCH3), 170.8 (CH3CO), 177.2
(C O); MS (-ESI): m/z (%) = 212.1570 (100) [M-H]-; MS (+ESI):
m/z (%) = 277.1996 (80) [M+ CH3CN + Na]+, 213.1982 (100)
[M]+; mixture of acetonitrile/H2O was used as a solvent; HR-
MS: 277.1529 (277.1528 calc. for C11H19N1O3 + CH3CN + Na+);
mixture of acetonitrile/H2O was used as a solvent.
3
1.01 (d, 3H, JHH = 6.5 Hz, CH3), 1.12 (m, 1H, H-4), 1.39 (dq,
3
2
3
1H, JHH = 3.1 Hz, JHH = JHH = 13 Hz, H-2a or H-6a), 1.40
3
2
3
(dq, 1H, JHH = 3.1 Hz, JHH = JHH = 13 Hz, H-6a or H-2a),
1.50 (bs, 2H, NH2), 1.81 (m, 2H, H-3e and H-5e), 1.99 (m, 2H,
H-2e and H-6e), 2.19 (tt, 1H, 3JHH = 3.4 Hz, 3JHH = 12.3 Hz, H-1),
2.65 (quint, 1H, 3JHH = 6.5 Hz, CHCH3), 3.62 (s, 3H, COOCH3);
13C-NMR (CDCl3, 100 MHz): d 20.9 (CH3), 27.8 (CH2), 27.9
(CH2), 28.8 (CH2), 27.9 (CH2), 43.2 (CH-4), 44.4 (CH-1), 51.2
(COOCH3), 51.4 (CHCH3), 176.4 (C O); MS (+ESI): m/z (%) =
186.1375 (100) [M+H]+; HR-MS: 186.1488 (186.1494 calc. for
C10H20N1O2).
5a: mp 255 ◦C [ref. 5 mp >290 ◦C]; [a]D +0.4 (c 0.5, H2O).
20
1H-NMR (D2O, 400 MHz): d 1.16 (m, H-3a and H-5a), 1.18 (d,
3H, 3JHH = 6.7 Hz, CH3), 1.37 (m, 1H, H-4), 1.45 (dq, 2H, 3JHH
=
=
3
3.4 Hz, 2JHH = JHH = 13 Hz, H-2a or H-6a), 1.46 (dq, 2H, 3JHH
3
3.4 Hz, 2JHH = JHH = 13 Hz, H-6a or H-2a), 2.01 (m, 2H, H-3e and
Isomerisation attempts with the acids 5a and 6a
H-5e), 2.05 (m, 2H, H-2e and H-6e), 2.18 (tt, 1H, 3JHH = 3.5 Hz,
3
3JHH = 12.3 Hz, H-1), 2.88 (quint, 1H, JHH = 6.4 Hz, CHCH3).
Potassium tert-butoxide (0.1 g, 0.88 mmol, 3 equiv.) was added to
a solution of cyclohexanecarboxylic acid 5a (0.05 g, 0.29 mmol,
1 equiv.) in ethanol (2 mL) and the mixture was heated under
refluxing conditions for 2 d. Then, the mixture was concentrated
and the residue was dried overnight under vacuum. The starting
The signals for NH2 and COOH were not detected in the spectrum;
13C-NMR (D2O, 100 MHz): d 18.9 (CH3), 28.1 (CH2), 28.3 (CH2),
30.2 (2 ¥ CH2), 43.7 (CH-4), 47.6 (CH-1), 51.4 (CHCH3), 186.8
(C O); MS (+ESI): m/z (%) = 172.1392 (100) [M+H]+.
material 5a was completely recovered as judged by H- and 13C-
1
NMR spectroscopy.
trans-(R)-4-(1-Aminoethyl)cyclohexanecarboxylic acid
Likewise, potassium tert-butoxide (0.09 g, 0.81 mmol, 3 equiv.)
was added to a solution of methyl ester 6a (0.05 g, 0.27 mmol, 1
equiv.) in ethanol (2 mL) and the mixture was heated under reflux-
ing conditions for 2 d. Then, the mixture was concentrated and
the residue dried overnight under vacuum to quantitatively afford
the acid 5a as was judged by 1H- and 13C-NMR spectroscopy.
hydrochloride (7a)
Concentrated HCl (0.5 mL) was slowly added to a solution of
1,4-trans 5a (0.3 g, 1.44 mmol) in water (2 mL). The mixture was
stirred overnight, the solvent was evaporated and the resulting
solid dried overnight under vacuum and then recrystallised from a
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trans-(R)-4-(1-(tert-Butoxycarbonylamino)ethyl)
182 ◦C; [a]D +4.5 (c 0.7, MeOH). 1H-NMR (CDCl3, 400 MHz):
d 1.07 (m, 2H, H-3a and H-5a), 1.08 (d, 3H, 3JHH = 6.7 Hz, CH3),
1.42 (s, 9H, C(CH3)3), 1.43 (m, 3H, H-2a, H-4, H-6a), 1.85 (m,
2H, H-3e and H-5e), 2.02 (m, 2H, H-2e and H-6e), 2.33 (tt, 1H,
3JHH = 3.4 Hz, 3JHH = 12 Hz, H-1), 3.48 (quint, 1H, 3JHH = 6.7 Hz,
CHCH3), 4.48 (bs, 1H, NH), 7.59 (d, 2H, 3JHH = 7.2 Hz, 2 ¥ Py-
20
cyclohexanecarboxylic acid (8)
Cyclohexanecarboxylic acid 5a (5 g, 29.2 mmol, 1 equiv.) was
dissolved in a mixture of water (150 mL) and THF (150 mL)
at 0 ◦C. Then, sodium hydroxide (2.35 g, 58.5 mmol, 2 equiv.)
was added and after stirring for 30 min, di-tert-butyl dicarbonate
(7.65 g, 35.1 mmol, 1.2 equiv.) was added. The mixture was
warmed to r.t and stirred for 2 d (progress was checked by
TLC, ethyl acetate/petroleum ether 1 : 1). THF was removed
under reduced pressure, the aqueous layer was acidified with
HCl (1 N) to pH 3-4 and extracted with ethyl acetate (3 ¥
150 mL). The combined organic layers were dried over MgSO4,
filtered and concentrated under reduced pressure. The crude
product 8 was purified by flash column chromatography (SiO2,
ethyl acetate/petroleum ether 1 : 2; Rf = 0.42) to afford the Boc-
protected acid 8 (7 g, 25.8 mmol; 89%) as colourless crystals. mp
106–108 ◦C; [a]D20 +0.8 (c 0.6, MeOH). 1H-NMR (MeOH-d4, 400
MHz): d 1.08 (m, 2H, H-3a and H-5a), 1.10 (d, 3H,3JHH = 6.8 Hz,
CH3), 1.32 (m, 3H, H-2a, H-4 and H-6a), 1.47 (s, 9H, C(CH3)3),
1.88 (m, 2H, H-3e and H-5e), 2.05 (m, 2H, H-2e and H-6e), 2.22
3
H), 8.44 (d, 2H, JHH = 7.2 Hz, 2 ¥ Py-H); 13C-NMR (CDCl3,
100 MHz): d 18.6 (CH3), 28.4 ((CH3)3), 28.4 (CH2), 29.0 (CH2),
29.1 (CH2), 31.8 (CH2), 38.6 (CH), 42.7 (CH), 46.2 (CH), 79.1
(C-(CH3)3), 113.6 (Py-CH), 145.6 (Py-C), 150.4 (Py-CH), 155.6
(NHC O), 175.3 (C O); MS (+ESI): m/z (%) = 348.2146 (100)
[M+H]+; HR-MS: 348.2288 (348.2287 calc. for C19H30N3O3).
Rho-Kinase inhibitor Y-27632 (1)
A hydrogen chloride solution (1.0 N in diethyl ether, 202 mL,
202 mmol, 10 equiv.) was added dropwise at 0 ◦C to a stirred
mixture of amide 9 (7 g, 20.2 mmol, 1 equiv.) in dichloromethane
(200 mL) under an argon atmosphere. The reaction was warmed
up to r.t. and stirred for 2 d (progress was checked by TLC,
dichloromethane/methanol 1 : 1). After completion of the reac-
tion, the solvents were evaporated, dried overnight under vacuum
and recrystallised from a mixture of diethyl ether/methanol 9 : 1
to afford the title compound 1 (6.1 g, 19.1 mmol; 95%) as yellowish
crystals. mp 258 ◦C [ref. 3b mp 286–287 ◦C for bis-hydrochloride
(tt, 1H, 3JHH = 3.4 Hz, 3JHH = 12 Hz, H-1), 3.40 (quint, 1H,3JHH
=
6.8 Hz, CHCH3). The signals of NH and COOH were not detected
in the spectrum. 13C-NMR (CDCl3, 100 MHz): d 18.3 (CH3), 28.4
((CH3)3), 27.7 (CH2), 28.0 (CH2), 28.5 (2 ¥ CH2), 42.7 (CH-4), 43.0
(CH-1), 50.4 (CHCH3), 79.1 (C-(CH3)3), 155.5 (NHC O), 181.6
(C O); MS (-ESI): m/z (%) = 270.1447 (100) [M-H]-; HR-MS:
270.1705 (270.1705 calc. for C14H24N1O4).
monohydrate and 276 ◦C for bis-hydrochloride hydrate]; [a]D
1
2
20
-11.5 (c 0.5, MeOH) [ref. 3b [a]D +4.6 (c 1, MeOH); ref. 4b [a]D
-1
˜
+4.3 (c 1, MeOH)]. IR (film): u (cm ) = 2858, 1713, 1639, 1609,
1573, 1502, 1480, 1386, 1312, 1292, 1246, 1176, 1139, 1039, 929,
82; 1H-NMR (MeOH-d4, 400 MHz): d 1.27 (dq, 1H, 3JHH = 3.4 Hz,
trans-tert-Butyl (R)-1-[(1-(1R,4R)-4-(pyridine-4-
ylcarbamoyl)cyclohexyl]ethylcarbamate (9)
3
2JHH = JHH = 13 Hz, H-3a or H-5a), 1.28 (dq, 1H, 3JHH = 3.4 Hz,
3
3
2JHH = JHH = 13 Hz, H-5a or H-3a), 1.33 (d, 3H, JHH = 6.8 Hz,
CH3), 1.62 (m, 3H, H-2a, H-4, CH-6a), 1.95 (m, 2H, H-3e and
H-5e), 2.13 (m, 2H, H-2e and H-6e), 2.57 (tt, 1H, 3JHH = 3.4 Hz,
Procedure according to Mukaiyama. 1-Methyl-2-chloro-
pyridinium iodide (5.65 g, 22.1 mmol, 1.2 equiv.) was added to
a stirred mixture of 8 (5 g, 18.5 mmol, 1 equiv.), 4-aminopyridine
(1.8 g, 19.4 mmol, 1.05 equiv.) and triethylamine (6.3 mL,
44.2 mmol, 2.4 equiv.) in dichloromethane (250 mL) under
an argon atmosphere at r.t. The reaction was stirred for 2 d
(checked by TLC, dichloromethane/methanol 95 : 5 and ethyl
acetate/petroleum ether 1 : 1). After completion of the reaction,
water (250 mL) was added and the mixture was extracted with
dichloromethane (3 ¥ 200 mL). The combined organic layers were
dried over MgSO4, filtered and concentrated under reduced pres-
sure. The crude product was purified by column chromatography
(SiO2; dichloromethane/methanol 95 : 5; Rf = 0.15) to afford the
title compound 9 (6.2 g, 17.8 mmol; 97%) as yellow crystals.
3JHH = 11.9 Hz, H-1), 3.20 (quint, 1H, JHH = 6.8 Hz, CHCH3),
3
8.25 (d, 2H, 3JHH = 7.2 Hz, 2 ¥ Py-H), 8.64 (d, 2H, 3JHH = 7.2 Hz,
2 ¥ Py-H). The signals for NH and NH2 were not detected in
the spectrum; 13C-NMR (MeOH-d4, 100 MHz): d 16.8 (CH3),
28.5 (CH2), 29.8 (CH2), 30.3 (CH2), 30.4 (CH2), 42.7 (CH-4), 47.5
(CH-1), 54.0 (CHCH3), 116.7 (Py-CH), 144.0 (Py-CH), 156.4 (Py-
C), 178.9 (C O); MS (+ESI): m/z (%) = 248.1512 (100) [M+H]+;
MS (-ESI): m/z (%) = 282.1715 (100) [M+Cl]-; HR-MS: 248.1763
(248.1763 calc. for C14H22N3O1).
The spectroscopic data of the Rho-Kinase inhibitor Y-27632
(1) were in full agreement with those recorded for an authentic
sample.11
TBTU-method. DIPEA (0.1 mL, 0.55 mmol, 1.5 equiv.) and
TBTU (0.14 g, 0.44 mmol, 1.2 equiv.) were successively added to a
stirred mixture of 8 (0.1 g, 0.37 mmol, 1 equiv.), 4-aminopyridine
(0.04 g, 0.41 mmol, 1.1 equiv.) in dichloromethane (5 mL) under
an argon atmosphere at 0 ◦C. The reaction mixture was warmed
to r.t. and checked by TLC (dichloromethane/methanol 95 : 5 and
ethyl acetate/petroleum ether 1 : 1). After stirring overnight, water
(5 mL) was added and the mixture extracted with dichloromethane
(3 ¥ 4 mL). The combined organic layers were dried over
MgSO4, filtered and concentrated under reduced pressure. The
crude product was purified by column chromatography (SiO2;
dichloromethane/methanol 95 : 5; Rf = 0.15) to afford the title
compound 9 (0.11 g, 0.32 mmol; 85%) as yellow crystals. mp
(R)-trans-4-(1-aminoethyl)-N-(2-fluoro-4-pyridyl)-
cyclohexanecarboxamide dihydrochloride (10)
1-Methyl-2-chloropyridinium iodide (0.09 g, 0.34 mmol, 1.2
equiv.) was added at r.t to a stirred mixture of 8 (0.075 g,
0.28 mmol, 1 equiv.), 2-fluoro-4-aminopyridine (0.03 g, 0.29 mmol,
1.05 equiv.) and triethylamine (0.09 mL, 0.67 mmol, 2.4 equiv.) in
dichloromethane (5 mL) under an argon atmosphere. The reaction
was stirred for 2 d (checked by TLC, ethyl acetate/petroleum
ether 1 : 1). After completion of the reaction, water (5 mL)
was added and the mixture was extracted with dichloromethane
(3 ¥ 5 mL). The combined organic layers were dried over
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MgSO4, filtered and concentrated under reduced pressure. The
crude product was purified by column chromatography (SiO2;
ethyl acetate/petroleum ether 1 : 1; Rf = 0.33) to afford the Boc-
protected amide (0.04 g, 0.11 mmol; 39%) as a viscous oil.
1H-NMR (MeOH-d4, 400 MHz): d 1.32 (m, 5H, H-3a, H-5a and
CH3), 1.65 (m, 3H, H-2a, H-4, CH-6a), 1.94 (m, 2H, H-3e and H-
5e), 2.10 (m, 2H, H-2e and H-6e), 2.72 (m, 1H, H-1), 3.19 (m, 1H,
CHCH3), 8.59 (m, 1H, Py-H), 8.98 (m, 2H, 2 ¥ Py-H). The signals
for NH and NH2 were not detected in the spectrum. 13C-NMR
(MeOH-d4, 100 MHz): d 16.9 (CH3), 28.5 (CH2), 29.8 (CH2), 30.6
(CH2), 30.7 (CH2), 42.6 (CH-4), 47.0 (CH-1), 54.1 (CHCH3), 118.2
(Py-CH), 132.8 (d, Py-CH, 2JCF = 30.5 Hz), 142.4 (Py-CH), 145.1
(Py-C), 150.4 (Py-C, 1JCF = 249.0 Hz), 178.9 (C O); MS (-ESI):
m/z (%) = 264.1559 (100) [M-H]-; HR-MS: 264.1516 (264.1512
calc. for C14H19N3O1F1); MS (+ESI): m/z (%) = 266.1399 (100)
[M+H]+; HR-MS: 266.1661 (266.1669 calc. for C14H21N3O1F1).
A hydrogen chloride solution (1.0 N in diethyl ether, 1 mL,
1.0 mmol, 10 equiv.) was added dropwise at 0 ◦C to a stirred
mixture of Boc-protected amide (0.035 g, 0.1 mmol, 1 equiv.) in
dichloromethane (2 mL) under an argon atmosphere. The reaction
was warmed up to r.t. and stirred for 2 d (progress was checked
by TLC, dichloromethane/methanol 1 : 1). After completion of
the reaction, the solvents were evaporated, the crude product
was dried overnight under vacuum and recrystallised from a
mixture of diethyl ether/methanol 99 : 1 to afford amide 10 (0.03 g,
0.09 mmol; 94%) as colourless crystals. mp decomposition at 88–
In vitro assessment of 1, 10 and 11
118 ◦C; [a]D -4.8 (c 0.25, MeOH). IR (film): u (cm ) = 2933,
20
-1
˜
The cell lines hES3 (ES Cell International, Singapore2c) and
hCBiPS2, (LEBAO, MHH Hannover2a) were applied for in vitro
functional testing, respectively. For conventional 2D cultures,
cells were grown on g-irradiated human foreskin fibroblasts or
mouse embryonic fibroblasts in KO medium (KnockOut DMEM,
20% KnockOut Serum Replacement, 50 ng/ml bFGF, 2mM L-
glutamine, and 1% MEM Non Essential Amino Acids; Invitrogen)
and passaged every 4–7 days.
Feeder-based hESC or hiPSC cultures were dissociated into
single cells by collagenase B (Roche) treatment and inoculated
in mTeSR medium (STEMCELL Technologies Inc.) at 100.000
cells per 3 ml in low attachment 6 well dishes to generate
suspension cultures (culture set up was described in detail by
Zweigerdt et al.2d ). Cells were cultured for 4 days (one passage)
without medium change either in mTeSR alone (controls) or
supplemented with 10 mM of 1, 10 or 11, respectively. On day
four, aggregate formation was monitored by light microscopy. For
subsequent passaging, aggregates were dissociated into single cells
by collagenase B treatment and re-inoculated at the initial density
of 100.000 cells per 3 ml medium per well. Cell yields were analysed
by Trypan blue staining for cell vitality and cell counting was
performed using a Haemocytometer; 3 parallel wells were counted
for each condition; the cell yield was analysed for 3 independent
passages applying supplementation with the respective compound.
1680, 1596, 1503, 1413, 1328, 1254, 1171, 1121, 1096, 1002, 970,
1
929, 903, 856, 740; H-NMR (MeOH-d4, 400 MHz): d 1.25 (m,
2H, H-3a and H-5a), 1.31 (d, 3H, 3JHH = 6.8 Hz, CH3), 1.60 (m, 3H,
H-2a, H-4, CH-6a), 1.92 (m, 2H, H-3e and H-5e), 2.06 (m, 2H, H-
2e and H-6e), 2.44 (tt, 1H, 3JHH = 3.4 Hz, 3JHH = 12.0 Hz, H-1), 3.18
(quint, 1H, 3JHH = 6.5 Hz, CHCH3), 7.42 (dt, 1H, 3JHH = 5.8 Hz,
4
4JHH = 1.4 Hz, Py-H), 7.54 (d, 1H, JHH = 1.4 Hz, Py-H), 8.08
(d, 1H, 3JHH = 5.8 Hz, Py-H). The signals for NH and NH2 were
not detected in the spectrum; 13C-NMR (MeOH-d4, 100 MHz): d
15.9 (CH3), 27.7 (CH2), 29.1 (CH2), 29.6 (CH2), 29.7 (CH2), 41.9
(CH-4), 46.5 (CH-1), 53.2 (CHCH3), 99.4 (d, Py-CH, 2JCF = 41.5
Hz), 113.0 (d, Py-CH, 4JCF = 3.6 Hz), 148.1 (d, Py-CH, 3JCF = 15.4
Hz), 152.5 (d, Py-C, 3JCF = 12.2 Hz), 165.8 (d, Py-C, 1JCF = 234.9
Hz), 177.7 (C O); MS (-ESI): m/z (%) = 264.1606 (100) [M-H]-;
HR-MS: 264.1517 (264.1512 calc. for C14H19N3O1F1).
(R)-trans-4-(1-aminoethyl)-N-(3-fluoro-4-pyridyl)-
cyclohexanecarboxamide dihydrochloride (11)
1-Methyl-2-chloropyridinium iodide (0.14 g, 0.55 mmol, 1.2
equiv.) was added at r.t to a stirred mixture of 8 (0.125 g,
0.46 mmol, 1 equiv.), 3-fluoro-4-aminopyridine (0.05 g, 0.48 mmol,
1.05 equiv.) and triethylamine (0.15 mL, 1.10 mmol, 2.4 equiv.) in
dichloromethane (6 mL) under an argon atmosphere. The reaction
was stirred for 2 d (checked by TLC, ethyl acetate/petroleum
ether 1 : 1). After completion of the reaction, water (6 mL) was
added and the mixture was extracted with dichloromethane (3 ¥
6 mL). The combined organic layers were dried over MgSO4,
filtered and concentrated under reduced pressure. The crude
product was purified by column chromatography (SiO2; ethyl
acetate/petroleum ether 1 : 1; Rf = 0.51) to afford the Boc-
protected amide (0.085 g, 0.23 mmol 50%) as yellowish crystals.
A hydrogen chloride solution (1.0 N in diethyl ether, 2.2 mL,
2.2 mmol, 10 equiv.) was added dropwise at 0 ◦C to a stirred
mixture of Boc-protected amide (0.08 g, 0.22 mmol, 1 equiv.) in
dichloromethane (4 mL) under an argon atmosphere. The reaction
was warmed up to r.t. and stirred for 2 d (progress was checked
by TLC, dichloromethane/methanol 1 : 1). After completion of
the reaction, the solvents were evaporated, the crude product was
dried overnight under vacuum and recrystallised from a mixture
of diethyl ether/methanol 9 : 1 to afford the amide 11 (0.07 g,
0.21 mmol; 95%) as yellow crystals. mp decomposition at 180–
Acknowledgements
This work was supported by the DFG (EXC REBIRTH - From
Regenerative Biology to Reconstructive Therapy) An authentic
sample of Y-27632 was kindly provided by Prof. Dr Ulrich
Martin, Leibniz Research Laboratories for Biotechnology and
Artificial Organs (LEBAO), Department of Cardiac, Thoracic,
Transplantation and Vascular Surgery.
Notes and references
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˜
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©

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