Synthesis and evaluation of 4-(1-aminoalkyl)-N-(4-pyridyl) cyclohexanecarboxamides as Rho kinase inhibitors and neurite outgrowth promoters

Article abstract of DOI:10.1016/j.bmcl.2004.07.025

The influence of stereochemistry and alkyl side chain length on the bioactivity of the Rho kinase inhibitor Y-27632 [(+)-1, R = Me] was examined by the synthesis of (+)- and (-)-1, and two alkyl chain analogs (+)- and (-)-2 (R = n-propyl) and (-)-3 (R = n

Full text of DOI:10.1016/j.bmcl.2004.07.025

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4931–4934
Synthesis and evaluation of 4-(1-aminoalkyl)-N-(4-pyridyl)-
cyclohexanecarboxamides as Rho kinase inhibitors and
neurite outgrowth promoters
a
Karine Gingras, Hovsep Avedissian, Eryk Thouin, Veronique Boulanger,
b
a
a
´
Charles Essagian,a Lisa McKerrachera,c and William D. Lubellb,
*
a
´
´
´
BioAxone Therapeutique Inc, 3575 Ave. Du Parc., suite 5322, Montreal, Quebec, Canada H2X 3P9
b
´
Departement de Chimie, Universite de Montreal, C. P. 6128, Succ. Centre Ville, Montreal, Quebec, Canada H3C 3J7
Departement de Pathologie et de Biologie Cellulaire, Universite de Montreal, C. P. 6128, Succ. Centre Ville, Montreal,
´
´
´
´
c
´
´
´
´
´
Quebec, Canada H3C 3J7
Received 25 May 2004; revised 12 July 2004; accepted 13 July 2004
Available online 4 August 2004
Abstract—The influence of stereochemistry and alkyl side chain length on the bioactivity of the Rho kinase inhibitor Y-27632 [(+)-1,
R = Me] was examined by the synthesis of (+)- and (ꢀ)-1, and two alkyl chain analogs (+)- and (ꢀ)-2 (R = n-propyl) and (ꢀ)-3
(R = n-octyl) as well as their evaluation in enzymatic and neurite outgrowth assays.
Ó 2004 Elsevier Ltd. All rights reserved.
Protein kinase inhibitors have become important targets
for the development of novel therapeutics.1 For exam-
ple, Fasudil (1-(5-isoquinolinesulfonyl)-homopiper-
azine, Fig. 1), a Rho kinase inhibitor, has been
approved for the treatment of cerebral vasospasm and
is presently in clinical trials for the treatment of angina
pectoris.2 A related potent Rho kinase inhibitor, com-
pound Y-27632 ((+)-R-trans-4-(1-aminoethyl)-N-(4-pyr-
idyl)cyclohexanecarboxamide, (+)-1, Fig. 1) has been
commonly employed as a research tool.3 Evaluation of
its biological activity has shown that (+)-1 can inhibit
smooth muscle contractility, tumor cell invasion neutro-
phil chemotaxis, and hypertension.4
Figure 1. Representative Rho kinase inhibitors.
Rho kinase is an effector of Rho, a GTPase linked to the
membrane in its active state. Recognizing the impor-
tance of Rho kinase in the Rho signaling pathway in-
volved in axonal growth,5 we synthesized (+)-1
(Scheme 1) and evaluated its potential to serve as a neu-
roregenerative agent.6 Inactivation of Rho kinase with
(+)-1 allowed neurons to extend axons on growth inhib-
itory substrates when plated on myelin in vitro.6 More-
over, treatment of mice, after injury of their CNS, with
(+)-1 stimulated axon regeneration and functional
recovery when applied in vivo to the injured spinal
cord.6
e-Amino amide (+)-1 is composed of a rigidifying cyclo-
hexane body possessing a pyridyl amide head and amino
ethyl tail. Numerous modifications of the carboxamide
head group had been made; a few leading to improved
activity.7 For example, the replacement of the 4-pyridyl
group by a 1H-pyrrolo[2,3,b]pyridine-4-yl heterocycle
gave a 10-fold increase in inhibitor potency against
Rho kinase.3 On the other hand, there has been no
Keywords: Kinase; Enzyme inhibitor; Rho; Synthesis.
*
Corresponding author. Tel.: +1-514-3437339; fax: +1-514-
3437586; e-mail: lubell@chimie.umontreal.ca
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.025

4932
K. Gingras et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4931–4934
one route, aldehyde 6 was condensed quantitatively with
S- and R-a-methylbenzylamine in the presence of
MgSO4 in CH2Cl2 to furnish the corresponding imine,
(S)- and (R)-7, respectively.9 a-Methylbenzyl aldimine
7 may be reacted with a variety of nucleophilic organo-
metallic reagents to diastereoselectively furnish different
alkyl-branched amines.10–12 For example, treatment of 7
with methyl magnesium bromide in the presence of CuI
and BF3ÆEt2O in THF furnished the secondary amine 8
in >70% yield.
The newly formed center was assumed to possess the
(S)-stereochemistry because these conditions had previ-
ously been shown to convert the (S)-a-methylbenzyl-
imine of cyclohexane carboxaldehyde into the
corresponding (S,S)-amine.11 Similarly, the reaction of
7 with allyl magnesium chloride and CuI in THF gave
the corresponding secondary amine in P75% yield.12
Hydrogenation of 8 using Pd/C and ammonium formate
in MeOH at reflux13 removed the chiral auxiliary and
liberated the primary amine, which was protected as
tert-butyl carbamate 9. N-Boc-e-amino acid 10 was then
prepared from 9 by fluoride mediated silyl ether depro-
tection and oxidation of the resulting alcohol with
TEMPO, NaClO2, and NaOCl.14 Amino acid 10 was
coupled to 4-aminopyridine using TBTU15 and DIEA
in DMF to provide selectively the trans-diatereoisomer,
which on deprotection of the Boc group with HCl bub-
bles in CH2Cl2 afforded amino amide 1 as the bis-hydro-
chloride salt. The enantiomeric purity of (+)- and (ꢀ)-1
was evaluated by coupling the amine to N-(p-toluene-
sulfonyl)-L-prolyl chloride16 and examination of the
Scheme 1. Reagents and conditions: (a) TBDMS-Cl, Et3N, DMAP,
61%; (b) PCC, Celitee, DCM, 80%; (c) (R)-(+) or (S)-(ꢀ)-phenyleth-
ylamine, MgSO4, DCM; 91–96%; (d) MeMgBr, CuI, THF, ꢀ30 to
ꢀ65°C, BF3ÆEt2O, 71–81%; (e) HCO2NH4, Pd/C, MeOH, reflux, 90–
93%; (f) (Boc)2O, NaHCO3, Na2CO3, DME–H2O (1:1), 85–94%; (g)
TBAF, THF, 0°C, 89–90%; (h) TEMPO, phosphate buffer, NaClO2,
NaOCl, CH3CN, 35°C, 68–74%; (i) 4-aminopyridine, TBTU, DIEA,
DMF, rt, 79–80%; (j) HCl gas, DCM, 0°C–rt, 73–75%.
report, to the best of our knowledge, of the influence of
modification of the stereochemistry and the length of the
amino alkyl chain of (+)-1 on its kinase inhibitor activity
and biology. To explore the potential of such analogs,
two alternative methods have been developed for syn-
thesizing derivatives of (+)-1 modified at the alkyl
amine.
1
resulting amides after aqueous work-up by H NMR
spectroscopy. Measurement of the diastereotopic signals
at 3.87 and 3.97ppm showed a 9:1 isomeric ratio and
indicated that the corresponding hydrochloride salts 1
possessed an enantiomeric excess of 80%.
Previous syntheses of (+)-1 and its analogs have relied
on the use of a-alkylbenzylamines as chiral educts,
which were subsequently acylated under Friedel–Crafts
conditions at the para-position and reduced at the aro-
matic ring to provide the 1,4-disubstituted cyclohexane
system.7,8 One drawback to this approach has been the
limited number of enantiomerically enriched a-alk-
ylbenzylamines that are available commercially. Two
alternative approaches to synthesize the 1,4-substituted
cyclohexane system were developed as stereoselective
means for generating analogs possessing a wider diver-
sity of alkyl-branched amine moieties. In particular,
the methyl group of 1 was replaced by longer alkyl
chains to explore the influence of the hydrocarbon moi-
ety on Rho kinase inhibition and neurite outgrowth pro-
motion. Furthermore, we have evaluated the importance
of the amine-bearing carbon stereochemistry on biolog-
ical activity.
In an alternative route, aldehyde 6 was reacted with
either (S,S)- or (R,R)-diisopropyl-2-allyl-1,3,2-dioxa-
borolane-4,5-dicarboxylate 11 in toluene, which provided
respectively (R)- or (S)-homoallylic alcohol 12 (Scheme
2).17 (R)-Azide 14 was subsequently prepared by activa-
tion of (S)-alcohol 12 as a methanesulfonate and
displacement with inversion using sodium azide.
Reduction of azide 14 with triphenylphosphine and
water in THF18 followed by Boc-protection of the
resulting amine provided carbamate 15. Olefin metathe-
sis19 was next used to extend the alkyl chain of carba-
mate 15. Treatment of a solution of carbamate 15 and
6-dodecene in dichloromethane with ruthenium catalyst
16 provided a 75:25 trans:cis mixture of octenyl carba-
mate isomers 17 in 77% yield.20 Employing a similar
sequence as described for the conversion of silyl ether
9 to its corresponding amide, N-(Boc)amino butenyl
and nonenyl analogs 15 and 17 were transformed into
their respective carboxamides 19 and 20.
1,4-Cyclohexyldimethanol 4 was employed as an inex-
pensive starting material (Scheme 1). Selective protec-
tion of one of the hydroxyl groups as silylether 5
followed by oxidation provided aldehyde 6 in 49% over-
all yield (as a 70:30 mixture of trans:cis isomers by inte-
gration of the isomeric protons at 3.37 and 3.42ppm). In
Amino butyl and nonanyl analogs 2 and 3 were finally
obtained by hydrogenation of the double bond with
H2 and Pd/C in MeOH and Boc group deprotection
with HCl gas in MeOH to afford their bis-hydrochloride
salts.21,22 The S-enantiomer of 2 was prepared using the

K. Gingras et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4931–4934
4933
Table 1. Effect of 1–3 on Rho kinase activity and neurite outgrowth
Compound
R
Rho kinase
activitya with 10lM
(% control SD)
Neurite
outgrowthb
at 35lM
(% SD)
DMSOc
(+)-1
––
CH3
100
6
12
6
3
68 15
70 10
(ꢀ)-1
CH3
12
15
35
16
1
2
2
3
(+)-2
(ꢀ)-2
n-C3H7
n-C3H7
n-C8H17
––
48
23
0
7
1
(ꢀ)-3
(R)-Y 27632e
13 2d
82
5
a Rho kinase assays were performed using Kinase profilerTM service
from Upstate Ltd, Dundee, UK. ATP was present at 100lM in all
assays.
b Neurite outgrowth assay on cell culture-treated plastic. NG108-15
cells were incubated for 4h with test sample and fixed with 4% para-
formaldehyde and 1% glutaraldehyde. Cells were stained with 0.05%
cresyl violet and counted by microscopy. % Neurite outgrowth is
stated as cells with neurites longer than cell body/total cells, where
total cells is 125 20 cells. Results represent mean% neurite out-
growth standard deviation for each experiment. This assay is semi-
quantitative.
c DMSO 0.35% used as vehicle solution.
d Results taken from Davies et al. (2000).4
e Y27632 purchased from Calbiochem, USA.
Scheme 2. Reagents and conditions: (a) inverse addition of 6 on 11,
˚
4A molecular sieves, toluene, ꢀ78°C, 70%; (b) MsCl, Et3N, DCM,
0°C–rt, 74%; (c) NaN3, DMF, 75°C, 79%; (d) (i) PPh3, H2O, THF,
45°C; (ii) (Boc)2O, NaHCO3, Na2CO3, DME–H2O (1:1), 53%; (e) 6-
dodecene, 16, DCM, rt, 77%; (f) TBAF, THF, 0°C, 77–84%; (g)
TEMPO, phosphate buffer, NaClO2, NaOCl, CH3CN, 35°C, 88–92%;
(h) 4-aminopyridine, TBTU, DIEA, DMF, rt, 63–72% (i) H2, Pd/C,
MeOH, rt, 80–90%; (j) HCl/MeOH, 0°C–rt, 91–95%.
same strategy starting from (R)-homoallylic alcohol 12.
The extent of stereoinduction during the allyl borona-
tion of aldehyde 6 was determined by the synthesis of
diastereomeric L- and D-(toluenesulfonyl)prolylamides
on removal of the Boc group of 19 with HCl gas fol-
lowed by coupling to the prolyl chlorides and examina-
tion of the resulting amides after aqueous work-up by
1H NMR spectroscopy. Measurement of the diastereo-
topic allylic signals at 5.05 and 5.15 and 5.72 and
5.82ppm showed a ratio of 9:1 and indicated that the
corresponding hydrochloride salts 2 and 3 both pos-
sessed an 80% enantiomeric excess.
trans-4-(1-Aminoalkyl)-N-(4-pyridyl)cyclohexanecarbox-
amides 1–3 were examined as inhibitors of Rho kinase
and for their ability to stimulate neurite outgrowth on
tissue culture treated surface in a semi-quantitative
cell-based assay (Table 1). Enantiomerically enriched
(80% ee) compounds 1–3 were tested directly without
further resolution. All compounds inhibited Rho kinase
activity at 10lM by P 65% relative to vehicle (DMSO)
in the presence of 100lM ATP. Compounds 1 and 2
demonstrated similar results for Rho kinase assay; (+)-
1 and (+)-2 are more potent than (ꢀ)-1 and (ꢀ)-2. The
ability of compounds (+)-1, (ꢀ)-1, and (+)-2 to promote
neurite outgrowth was demonstrated relative to vehicle
(5.7, 5.8, and 4-fold vehicle, respectively). To a lesser
extent (1.9-fold vehicle control), compound (ꢀ)-2 pro-
Figure 2. Neurite outgrowth assay on MAG (upper panel) and myelin
(lower panel) substrates. NG108-15 cells were plated in chamber slides
coated with 100lg/mL poly-L-lysine (PLL) or with 100lL of 80lg/mL
MAG or myelin solution, with and without compound (+)-1 and (ꢀ)-1
at concentrations of 0.35 and 35lM. Cells were fixed after 24h. Cells
on MAG were stained with 0.05% cresyl violet and cells with neurites
longer than 1 cell body diameter were counted. Cells on myelin were
counted as described in Dergham et al. 2002.6

4934
K. Gingras et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4931–4934
1991, 32, 1367. Spectral characterization of the product
from addition of allyl magnesium chloride to (S)-7: 1H
NMR (400MHz, CDCl3) signals for the major isomer are
as follows: d 0.04 (s, 6H), 0.74–1.55 (m, 19H), 1.63 (m,
1H), 1.80 (m, 2H), 1.93 (m, 1H), 2.18–2.40 (m, 3H), 3.38
(d, 2H, J = 6.4Hz), 3.92 (m, 1H), 5.10 (m, 2H), 5.80 (m,
1H), 7.20–7.40 (m, 5H). Distinct signals for the minor
isomer include: d 0.06 (s, 6H), 3.42 (d, 2H, J = 7.1Hz),
5.62 (m, 1H); 13C NMR (100MHz, CDCl3) d 146.2, 135.9,
128.1, 126.7, 126.5, 116.6, 68.7, 65.6, 58.5, 55.2, 40.8, 40.5,
34.5, 29.5, 28.7, 28.2, 25.8, 24.9, 18.2, ꢀ5.5; MS (FAB) m/z
402.4 ([MH+]).
moted neurite outgrowth. Although compound (ꢀ)-3
demonstrated a significant effect on Rho kinase activity,
it failed to promote neurite outgrowth and caused cell
rounding instead.
The ability of compounds (+)- and (ꢀ)-1 to promote
neurite outgrowth was examined on two inhibitory sub-
strates, myelin-associated glycoprotein (MAG) and
myelin (Fig. 2). After 24h on MAG or myelin, few
NG108-15 cells showed neurites. However, following
treatment with either (+)- or (ꢀ)-1, the percentage of
NG108-15 cells with neurites significantly increased as
compared to untreated cells plated on MAG or myelin.
Both (+)- and (ꢀ)-1 helped overcome the inhibitory
effect of MAG and myelin on neurite outgrowth promo-
tion.
13. Bringmann, G.; Geisler, J.-P.; Geuder, T.; Kunkel, G.;
¨
Kinzinger, L. Liebigs Ann. Chem. 1990, 795.
14. (a) Zhao, M.; Li, J.; Mano, E.; Song, Z.; Tschaen, D. M.;
Grabowski, E. J. J.; Reider, P. J. J. Org. Chem. 1999, 64,
2564; (b) Dettwiler, J. E.; Lubell, W. D. J. Org. Chem.
2003, 68, 177.
15. (a) Avenoza, A.; Busto, J. H.; Peregrina, J. M.; Rodriguez,
F. J. Org. Chem. 2002, 67, 4241; (b) Poulain, R. F.; Tartar,
Stereochemistry at the amine-bearing center had a lim-
ited influence on the biological activity of compounds
1 and 2; both enantiomers possessed the ability to inhi-
bit Rho kinase activity and promote neurite outgrowth.
In contrast, the octyl analog (ꢀ)-3 did not stimulate
neurite outgrowth and induced cell rounding, suggesting
cell toxicity. Although no clear relationship between
Rho kinase inhibition and neurite outgrowth promotion
was established, several Rho kinase inhibitors did pro-
mote neurite outgrowth. Based on the influences of
modifications of the stereochemistry and alkyl chain
length of compounds 1–3, opportunity may exist to
modify these features to create cell permeable com-
pounds with improved activity.
´
A. L.; Deprez, B. P. Tetrahedron Lett. 2001, 42,
1495.
16. Cluzeau, J.; Lubell, W. D. J. Org. Chem. 2004, 69,
1504.
17. Stereochemical assignment for the alcohol 12 was made
based on the product from the addition of (R,R)-
diisopropyl-2-allyl-1,3,2-dioxaborolane-4,5-dicarboxylate
to cyclohexane carboxaldehyde, which produced predom-
inantly the (S)-alcohol (dr 93:7): (a) Roush, W. R.;
Hoong, L. K.; Palmer, M. A. J.; Park, J. C. J. Org.
Chem. 1990, 55, 4109; (b) Roush, W. R.; Walts, A. E.;
Hoong, L. K. J. Am. Chem. Soc. 1985, 107, 8186; (c)
Roush, W. R.; Palkowitz, A. D.; Ando, K. J. Am. Chem.
Soc. 1990, 112, 6248.
18. Nicolaou, K. C.; Caulfield, T.; Kataoka, H.; Kumazawa,
T. J. Am. Chem. Soc. 1988, 110, 7910.
References and notes
19. (a) Blackwell, H. E.; OꢀLeary, D. J.; Chatterjee, A. K.;
Washenfelder, R. A.; Bussmann, D. A.; Grubbs, R. H. J.
Am. Chem. Soc. 2000, 122, 58; (b) Trnka, T. M.; Grubbs,
R. H. Acc. Chem. Res. 2001, 34, 18; (c) Randl, S.; Blechert,
S. J. Org. Chem. 2003, 68, 8879.
20. The isomeric ratio 75:25 trans:cis was assigned by
integration of isomeric signals at 5.26 and 5.35 and 5.47
and 5.57ppm. The major isomer was assigned the trans
isomer based on the 15.3Hz coupling constant for the
olefin protons.
1. Noble, M. E. M.; Endicott, J. A.; Johnson, L. N. Science
2004, 303, 1800.
2. Shimokawa, H.; Iinuma, H.; Kishida, H.; Nakashima, M.;
Kato, K. Circulation 2001, 104, 2843.
3. Ishizaki, T.; Uehata, M.; Tamechika, I.; Keel, J.; Nono-
mura, K.; Maekawa, M.; Narumiya, S. Mol. Pharmacol.
2000, 57, 976.
4. Davies, S. P.; Reddy, H.; Caivano, M.; Cohen, P.
Biochem. J. 2000, 351, 95.
5. McKerracher, L.; Winton, M. J. Neuron 2002, 36, 345.
6. Dergham, P.; Ellezam, B.; Essagian, C.; Avedissian, H.;
Lubell, W. D.; McKerracher, L. J. Neurosci. 2002, 22,
6570.
7. Arita, M.; Saito, T.; Okuda, H.; Sato, H.; Uehata, M. U.S.
Patent 5,478,838, 1995.
8. Muro, T.; Seki, T.; Abe, M.; Inui, J.; Sato, H. U.S. Patent
4,997,834, 1991.
9. Cristau, H.-J.; Monbrun, J.; Tillard, M.; Pirat, J.-L.
Tetrahedron Lett. 2003, 44, 3183.
10. Bloch, R. Chem. Rev. 1998, 98, 1407.
11. The 9:1 diastereomeric ratio observed after coupling 1 to
N-(toluenesulfonyl)prolyl chloride was in close agreement
with the 93:7 dr obtained in the addition of MeMgBr to a-
methylbenzyl cyclohexylaldimines: (a) Alvaro, G.; Savoia,
D.; Valentinetti, M. R. Tetrahedron 1996, 52, 571; (b)
Boga, C.; Savoia, D.; Umani-Ronchi, A. Tetrahedron:
Asymmetry 1990, 1, 291.
12. Alvaro, G.; Boga, C.; Savoia, D.; Umani-Ronchi, A. J.
Chem. Soc., Perkin Trans. 1 1996, 875; (b) Bocoum, A.;
Boga, C.; Savoia, D.; Umani-Ronchi, A. Tetrahedron Lett.
21. In the case of 2 the Boc group was removed before
hydrogenation; with 3 the hydrogenation preceded Boc
deprotection.
22. The physical properties for 1 were the same as observed in
Ref. 5: 13C NMR (100MHz, CD3OD) d 178.2, 155.5,
143.1, 115.8, 53.1, 46.6, 41.7, 29.5 (2C), 28.9, 27.6, 15.9.
Specific rotation concentrations are reported in g/100mL:
(+)-2: 1H NMR (400MHz, CD3OD) d 1.01 (t, 3H,
J = 7.1Hz), 1.25–1.65 (m, 10H), 1.88 (m, 2H), 2.10 (d,
2H, J = 12Hz), 2.54 (m, 1H), 3.08 (m, 1H), 8.21 (d, 2H,
J = 7.31Hz), 8.60 (d, 2H, J = 7.3Hz); 13C NMR
(100MHz, CD3OD) d 178.5, 155.9, 142.1, 114.8, 56.2,
20
45.7, 41.4, 39.4, 31.9, 28.7, 27.6, 27.5, 18.6, 13.2; ½aꢁD 2:7
(c 0.58, MeOH); HRMS calcd for C16H26NO3 ([MH+]):
276.2075. Found: 276.2079.
20
Compound 2: ½aꢁD ꢀ 2:7 (c 0.4, MeOH); HRMS calcd for
C16H26NO3 ([MH+]): 276.2075. Found: 276.2081.
Compound 3: 1H NMR (400MHz, CD3OD) d 0.93 (t, 3H,
J = 7Hz), 1.40 (m, 15H), 1.65 (m, 5H), 1.92 (m, 2H), 2.08
(d, 2H, J = 14Hz), 2.49 (m, 1H), 3.08 (m, 1H), 8.15 (d, 2H,
20
J = 6.5Hz), 8.58 (d, 2H, J = 6.8Hz); ½aꢁD ꢀ21:6
(c 0.125, MeOH); MS (TIC) m/z 346.2 ([MH+]).