Tetrahydroisoquinoline derivatives as highly selective and potent rho kinase inhibitors

Article abstract of DOI:10.1021/jm100579r

Rho kinase (ROCK) is a promising drug target for the treatment of many diseases including hypertension, multiple sclerosis, cancer, and glaucoma. The structure-activity relationships (SAR) around a series of tetrahydroisoquinolines were evaluated utilizing biochemical and cell-based assays to measure ROCK inhibition. These novel ROCK inhibitors possess high potency, high selectivity, and appropriate pharmacokinetic properties for glaucoma applications. The lead compound, 35, had subnanomolar potency in enzyme ROCK-II assays as well as excellent cell-based potency (IC₅₀ = 51 nM). In a kinase panel profiling, 35 had an off-target hit rate of only 1.6% against 442 kinases. Pharmacology studies showed that compound 35 was efficacious in reducing intraocular pressure (IOP) in rats with reasonably long duration of action. These results suggest that compound 35 may serve as a promising agent for further development in the treatment of glaucoma. 2010 American Chemical Society. r2010 American Chemical Society.

Full text of DOI:10.1021/jm100579r

J. Med. Chem. 2010, 53, 5727–5737 5727
DOI: 10.1021/jm100579r
Tetrahydroisoquinoline Derivatives As Highly Selective and Potent Rho Kinase Inhibitors
Xingang Fang,† Yan Yin,† Yen Ting Chen,† Lei Yao,† Bo Wang,† Michael D. Cameron,† Li Lin,† Susan Khan,† Claudia Ruiz,†
†
†
†
†
‡
,†
€
€
Thomas Schroter, Wayne Grant, Amiee Weiser, Jennifer Pocas, Alok Pachori, Stephan Schurer, Philip LoGrasso,* and
Yangbo Feng*,†
†Translational Research Institute and Department of Molecular Therapeutics, The Scripps Research Institute, Florida, 130 Scripps Way, 2A1,
Jupiter, Florida 33458, and ‡Department of Pharmacology and Center for Computational Science, University of Miami, Miami, Florida 33136
Received May 11, 2010
Rho kinase (ROCK) is a promising drug target for the treatment of many diseases including hyper-
tension, multiple sclerosis, cancer, and glaucoma. The structure-activity relationships (SAR) around a
series of tetrahydroisoquinolines were evaluated utilizing biochemical and cell-based assays to measure
ROCK inhibition. These novel ROCK inhibitors possess high potency, high selectivity, and appropriate
pharmacokinetic properties for glaucoma applications. The lead compound, 35, had subnanomolar
potency in enzyme ROCK-II assays as well as excellent cell-based potency (IC50 = 51 nM). In a kinase
panel profiling, 35 had an off-target hit rate of only 1.6% against 442 kinases. Pharmacology studies
showed that compound 35 was efficacious in reducing intraocular pressure (IOP) in rats with reasonably
long duration of action. These results suggest that compound 35 may serve as a promising agent for
further development in the treatment of glaucoma.
Introduction
ROCKa is a protein serine/threonine kinase of ∼160 kDa
wherethe kinase domain islocated atthe N-terminusfollowed
by the coiled-coil domain, the Rho-binding domain, and the
pleckstrin homology (PH) domain with an internal cysteine-
rich region/domain (CRD) at the C-terminus.1 ROCK be-
Figure 1. General structures of R-amino anilides.
longs to the AGC kinase family and is most homologous to
myotonic dystrophy kinase (DMPK), DMPK-related cell divi-
sion control protein 42 (Cdc42)-binding kinases (MRCK),
and citronkinase(CRIK), and is also closelyrelatedtoprotein
kinase A (PKA) and protein kinase B (Akt). ROCK is auto-
inhibited in its free form and is activated by association with
the GTP-binding form of RhoA. There are two isoforms of
ROCK: ROCK-I (ROKβ or p160ROCK) and ROCK-II
(ROKR), which share 65% identity overall and 92% of
identity in the kinase domain. Both ROCK-II and ROCK-I
are widely expressed in several tissues but ROCK-II is more
abundant in brain tissue.1
ROCK plays a pivotal role in stress fiber formation,2 focal
adhesion,2 smooth muscle contraction,3 and regeneration of
nerve fibers.4 Therefore, ROCK inhibitors have potential for
the treatment of myocardial ischemia,5 hypertension,3,6,7 erectile
dysfunction,8 glaucoma,9 cancer migration,10,11 spinal-cord
injury,12 multiple sclerosis,13 and inflammatory disorders.14
Fasudil,15 the only approved ROCK inhibitor, has been mar-
keted as a treatment for cerebral vasospasm in Japan since
1995. Encouraged by the success of Fasudil, various classes of
ROCK inhibitors have been reported including isoquino-
lines,16,17 pyridines (the Y series),3 aminofurazans,18 dihydro-
pyrimidines,19 indazoles,20 and azaindoles.21 For a recent re-
view, see LoGrasso and Feng 2009.14 Our group has recently
reported potent and selective ROCK inhibitors based on ben-
zodioxane amides,22 chromane amides,23 benzimidazoles/
benzoxazoles,24 benzothiazoles,25 and aniline-ureas.26
Amino acids are common building blocks in medicinal
chemistry. Because of the ready availability of chiral R-amino
acids, optically pure compounds can quickly be accessed. In
this study, the identification of a new class of potent and
selective R-amino anilide-based ROCK inhibitors with general
structures 1 and 2 (Figure 1) are reported, and the optimiza-
tion for structure 2 is described. This class of compounds
consisted of a heterocycle that bound to the hinge site in the
ROCK-II ATP binding pocket as determined by molecular
modeling, an aromatic ring in the middle as a spacer, and an
amino acid based amide moiety that is expected to bind to the
hydrophobic region under the P-loop in the enzyme. The
major findings from this class of compounds include: potent
enzyme inhibition of ROCK (IC50 < 1 nM), potent cell-based
activity (IC50s = 10-50 nM), low oral bioavailability to pre-
vent systemic exposure after topical application, and IOP-
lowering ability with long duration of action.
*To whom correspondence should be addressed. For Y.F.: phone,
561-228-2201; Fax, 561-228-3089; E-mail, yfeng@scripps.edu. For P.L.:
phone, 561-228-2230; E-mail, lograsso@scripps.edu.
a Abbreviations: AUC, pharmacokinetic area under curve; Cl, phar-
macokinetic clearance; Cmax, pharmacokinetic maximum concentration;
Vd, volume of distribution; F, oral bioavailability; HLMS, human liver
microsomal stability; HTS, high-throughput screening; ppMLC, bis-
phospho myosin light chain; IOP, intraocular pressure; ROCK, Rho
kinase; aqd, once daily.
Chemistry
For compounds based on commercially available amino
acids (phenylalanines, phenylglycines, and unsubstituted
r
2010 American Chemical Society
Published on Web 07/20/2010
pubs.acs.org/jmc

5728 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Scheme 1. General Synthesis of R-Amino Anilides 1a
Fang et al.
Scheme 3. Synthesis of N-Boc-6-chloro-1,2,3,4-tetrahydroiso-
quinoline-3-carboxylic acid 17a
a Reaction and conditions: (a) HATU, DIPEA, DMF, rt (or 40 °C),
2 h; (b) R4 boronic acid or its pinacol ester, Pd(PPh3)4, dioxane/water,
140 °C, 30 min, microwave; (c) 1:2 TFA/CH2Cl2, triisopropylsilane.
a Reaction conditions: (a) (i) SOCl2, MeOH, (ii) EtOCOCl, Et3N;
(b) 3:1 AcOH/H2SO4, 4 equiv paraformaldehyde, 10 h; (c) 6 M HCl,
reflux, 16 h; (d) (Boc)2O, 1 M NaOH, 1 h.
Scheme 2. General Synthesis of 1,2,3,4-Tetrahydroisoquino-
line-Based Compounds 2a
S enantiomers) of either 9 or 10 was confirmed by NMR
analysis of the corresponding Mosher amides.
Because of insufficient reactivity of the unprotected 3-chloro-
phenylalanine, an electron withdrawing group N-ethoxycarbonyl
was applied to provide extra reactivity in the Pictet-Spengler
condensation28 in the synthesis of 6-chloro-1,2,3,4-tetrahy-
droisoquinoline-3-carboxylic acid (Scheme 3). Thus, amino
acid 13 was protected as an ester and an ethyl carbamide in
two steps to provide intermediate 14. Compound 14 under-
went Pictet-Spengler reaction with paraformaldehyde in a
mixture of acetic acid and sulfuric acid (3:1 by volume) to
provide the protected 6-chloro-1,2,3,4-tetrahydroisoquino-
line 15. Both the ester and the carbamide groups of 15 were
cleaved by refluxing in 6 M HCl. The Boc protection of the
amino group furnished intermediate 17.
Results and Discussion
a Reaction conditions: (a) 37% formaldehyde in water, 3-6 M HCl
solution, 12 h; (b) (Boc)2O, 1N NaOH, 12 h; (c) HATU, DIPEA,
4-bromoaniline 4, DMF, rt (or 40 °C), 2 h; (d) TFA/CH2Cl2, triisopro-
pylsilane, 1 h; (e) R2 aldehyde, NaBH(OAc)3, CH2Cl2, rt, 14 h, then
NaBH4; (f) R4 boronic acid pinacol ester, Pd(PPh3)4, dioxane/water,
140 °C, 30 min, microwave.
Our initial literature-based structure design and in-house
high-throughput screening29 led to the identification of amide
18 (Figure 2), an R-amino acid amide phenyl-pyrazole structure,
as an early lead. Compared to the well-studied (R)-4-(1-amin-
oethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide (Y-27632,
one of the best Y series compounds) and Fasudil, this com-
pound (Table 1) had higher affinity for ROCK-II (IC50 = 12
nM) as well as good human microsomal stability (T1/2 = 67
min), although it had moderate potency in cell-based myosin
1,2,3,4-tetrahydroisoquinoline-3-carboxlic acids), the typical
synthesis of the R-amino anilides with general structure 1 is
outlined in Scheme 1. The Boc protected R-amino acid 3
(commercial or obtained in one step from R-amino acids) was
condensed with various 4-bromoaniline 4 using HATU as the
coupling reagent to provide intermediate 5, which was then
coupled with 1H-4-pyrazoleboronic acid pinacol ester under
Suzuki conditions to provide amide 6. Deprotection of Boc
group using TFA/CH2Cl2 in the presence of 5% triisopropyl-
silane (TIS) asthe scavenger gave thefinalcompound1, which
was purified by preparative HPLC.
The preparation of structure 2 is summarized in Scheme 2.
The Boc protected 1,2,3,4-tetrahydroisoquinoline-3-carboxlic
acid 9 was synthesized from phenylalanine 7 by a Pictet-
Spengler step with aqueous formaldehyde in an acidic med-
ium followed by the Boc protection of the secondary amino
group. The resulting Boc protected tetrahydroisoquinoline 9
was coupled with 4-bromoaniline 4 to form the amide 10.
Suzuki coupling of 10 with the corresponding boronic acid
pinacol ester followed by deprotection of the Boc group
provided compound 2 containing a free N-H group on
tetrahydroisoquinoline moiety. The N-alkylated analogue 2
was synthesized by deprotection of the Boc on intermediate
10, reductive amination27 of 11 with an aldehyde, and Suzuki
coupling with the corresponding boronic acid pinacol ester to
furnish the final compound 2. The optical purity (both R and
light chain bis-phosphorylation assays (ppMLC)30 (IC50
=
713 nM) and low selectivity against PKA (chosen as routine
kinase counter screen because it is closely related to ROCK)
(IC50 = 41 nM, 3.4-fold). Our optimization started by repla-
cing the 3-fluorophenylalanine moiety in 18 with a couple of
other phenylalanine and phenylglycine-based structures. As
shown in Table 1, the 4-chlorophenylalanine-based anilide 19
had higher ROCK-II potency (IC50 = 2 nM) and much
improved cell potency (IC50 = 18 nM), but the PKA selec-
tivity still remained low (IC50 = 7 nM, 3-fold). Replacement
of the 4-chlorophenylalanine moiety with 4-chlorophenylgly-
cinegavethe potent amide20(IC50 = 1 nM) with excellent cell
activities (IC50 = 25 nM) as well as improved PKA selectivity
(IC50 = 23 nM, 23-fold). These results were encouraging but
the PKA selectivity for these ROCK inhibitors still needed
improvement.
The introduction of a 2-(N,N-dimethylamino)ethoxy side
chain onto the central phenyl ring (compound 21, IC50 = 22
nM for ROCK-II) was discovered to be an efficient way to
improve the inhibitor’s selectivity against PKA (IC50 =2524nM,
>100-fold). This change in PKA selectivity was likely due to a
much reduced PKA activity. The 2-(N,N-dimethylamino)-
ethoxy side chain bound to residue Asp176 in the ROCK-II
binding pocket (based on our docking pose). The space around

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5729
Figure 2. SAR evolution and identification of novel tetrahydroisoquinoline-based ROCK inhibitors.
Table 1. Assay Data for Compounds 18-23
IC50a (nM)
selectivity
(ROCK-II
ppMLC
cell assay T1/2 (min)b
compd ROCK-II PKA
over PKA) IC50a (nM)
HLM
18
19
20
21
22
23
12
2
41
7
3.4
3.5
713
18
67
67
84
14
14
98
1
23
23
25
310
22
371
7
2524
>20000
2514
115
>54
359
>2700
32
a Average of two or more experiments and deviation less than 50%.
b 2 mg/mL human liver microsome (HLM) was employed.
this area of the ATP binding pocket is larger in ROCK than in
PKA (the corresponding residue is Glu127 in PKA),31,32 and
therefore, a large side chain was more tolerated in ROCK. To
further improve the PKA selectivity, the bicyclic phenylgly-
cine-based 1,2,3,4-tetrahydroisoquinoline-1-carboxamide 22
was synthesized and evaluated. As shown in Table 1, com-
pound 22 exhibited good selectivity against PKA (>54-fold)
but its ROCK potency was much reduced (IC50 = 371 nM).
On the other hand, the bicyclic analogue of the phenylalanine-
based compound, 1,2,3,4-tetrahydroisoquinoline-3-carboxa-
Figure 3. Compound 23 docked into the ROCK-II ATP-binding
pocket. H-bonds are shown by dashed lines.
characterized by residues Phe103, Ala102, Leu123, and Phe136.
As suggested in our previous studies, this hydrophobic inter-
action is a major contributor to the high potency of ROCK
inhibitors.22
mide 23 exhibited excellent selectivity against PKA (IC50
=
2510 nM, 360-fold) as well as high ROCK potency in both
enzyme and cell-based assays (IC50 = 7 and 32 nM, respe-
ctively), and high microsomal stability (T1/2 = 98 min). As a
result, all subsequent optimizations were focused on analo-
gues containing the 1,2,3,4-tetrahydroisoquinoline-3-carbox-
amide moiety.
On the basis of this model, substitutions with reasonable
size are expected to be tolerated on the 5- and 6-position of the
tetrahydroisoquinoline phenyl ring (Figure 3). Thus, substitu-
tion with both electron-donating (methoxy and ethoxy) and
electron-withdrawing (F, Cl) groups at the 6-position of the
tetrahydroisoquinoline ring was investigated. The 3-position
chirality effect of the tetrahydroisoquinoline moiety was first
studied. As demonstrated in Table 2, among the four pairs of
enantiomers, the unsubstituted (23, 24) and the 6-methoxy
substitution (29, 30) favored the R configuration while the
6-fluoro substitution (25, 26) favored the S configuration. On
the other hand, no clear chirality effect was observed for the
6-chloro-substituted ROCK inhibitors (27, 28). The relative
configurations of R and S compounds were confirmed by
comparing the 1H NMR chemical shifts and split patterns of
certain hydrogen nuclei on their corresponding Mosher
amides,33 and the results suggested the agreement of the
assignment on all 6-OMe, 6-Cl, and 6-F compounds.
To gain a better understanding of how our inhibitors bind
in the kinase domain of ROCK-II, and more importantly, to
direct our optimization strategy, compound 23 was docked
into a human ROCK-II model using the method previously
described.22 The binding pose illustrated in Figure 3 demon-
strated that several key interactions are responsible for the
high potency and selectivity of compound 23. The pyrazole
forms a typical 2-hydrogen bond interaction pattern with the
kinase hinge residues. The amide carbonyl group of 23 formed
a third hydrogen bond with the side chain NH on residue
Lys121 in the phosphate binding region. The last important
hydrogen bonding element was found between the tertiary
amine on the dimethylaminoethoxy side chain of 23 with the
carboxylate side chain of Asp176, which explains the high
selectivity of 23 compared to those compounds without a side
chain on the central phenyl ring. In addition, the pose of
compound 23 indicated a hydrophobic interaction in which
the aromatic ring of the tetrahydroisoquinoline moiety is
immersed into a hydrophobic region under the flexible P-loop
Our data (Table 2) indicated that the R configured tetra-
hydroisoquinolines intrinsically bind better to Rho kinase as
is the case in isomers 23 vs 24, and 29 vs 30. The reversal of the
activity trend of the R and S enantiomers (Table 2) in 25 vs 26
suggests that the 6-F substitution resulted in modulation of

5730 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Table 2. SAR Studies on the 1,2,3,4-Tetrahydroisoquinoline Moiety
Fang et al.
IC50a (nM)
ROCK-II
selectivity
(ROCK-II
over PKA)
ppMLC
cell assay
IC50a (nM)
T1/2 (min)b
HLM
compd
R
chirality c
PKA
2514
23
24
25
26
27
28
29
30
31
32
33
H
H
R
7
59
359
68
32
98
S
4040
6860
868
ndd
52
ndd
ndd
121
17
6-F
6-F
R
290
7
24
564
S
3950
4435
6-Cl
6-Cl
R
23
51
193
226
85
S
11540
ndd
41
ndd
101
ndd
ndd
ndd
ndd
6-OMe
6-OMe
6-OEt
7-OMe
8-OMe
R
10
>20000
>20000
4300
>2000
>128
5
S
156
850
261
334
ndd
ndd
ndd
ndd
Racemate
R
Racemate
>20000
>20000
>77
>60
a Average of two or more experiments, and deviation less than 50%. b 2 mg/mL human liver microsome was employed. c Chirality came from starting
material and was confirmed by NMR analysis of the corresponding Mosher amide. d Not determined.
the interactions of the inhibitor distinct from the 6-OMe or
unsubstituted tetrahydroisoquinoline derivatives. Different
types of interactions between fluorine atoms with protein
functional groups have been reported, and in many cases
compounds with fluorine substitution behave differently in
protein environments compared to similar compounds with
different or even other halogen substituents.34 Such fluorine
interactions include F-H hydrogen bonds, π-interactions
with aromatic or guanidine groups, multipolar interactions,
and/or F-nitrogen interactions (halogen bonding).34 These
special F-interactions (or the general halogen-interactions)
could result in a change of the orientation of the tetrahydroi-
soquinoline ring under the P-loop. Indeed, this kind of halo-
gen bonding has been shown to be important for 1-aryl-3,4-
dihydroisoquinoline inhibitors of JNK3, suggesting this ob-
servation does impart potency enhancing elements to kinase
inhibitors.35 Because of the direct F-bonding or indirect
F-substitution effects, the S configuration is preferred for
the 6-F tetrahydroisoquinoline-based compounds. In the case
of 6-Cl compounds, no clear preference was observed between
the R and S configurations(IC50 is 23 nM vs 51 nM), probably
because the halogen-bonding effect of Cl35 is not as significant
as that of the F atom.
Results in Table 2 demonstrated that, when the preferred
enantiomer was applied (i.e., “R” in 6-OMe-tetrahydroiso-
quinoline and “S” in 6-F-tetrahydroisoquinoline; compounds
29 and 26, respectively), small substitutions did not change the
inhibitor’s ROCK-II affinity much as compared to the non-
substituted compound 23. On the other hand, a more sig-
nificant change in ROCK-II activity was observed when the
less preferred enantiomer was used (“S” in 6-OMe-tetrahy-
droisoquinoline and “R” in 6-F-tetrahydroisoquinoline; com-
pounds 30 and 25, respectively). Moreover, a larger substi-
tution such as an ethoxy group (compound 31) significantly
reduced the ROCK-II potency. In terms of selectivity against
PKA, when the preferred enantiomer was applied, both fluoro
(26, 564-fold) and methoxy (29, > 2000-fold) substitutions
were favored as compared to the nonsubstituted 23 (359-fold).
However, the large ethoxy substitution (31) notably decreased
this PKA selectivity (5-fold) apparently due to reduced
ROCK-II potency. This conclusion was also true even when
comparing the less preferred enantiomers of tetrahydroiso-
quinolines whose derivatives contain smaller group substitu-
ents (compounds 24, 25, and 30; PKA selectivity was 68, 24,
and >128-fold, respectively). These results suggested that
bulky groups at the 6-position of the tetrahydroisoquinoline
ring were not as well tolerated in the ROCK-II binding pocket
as they were in the corresponding region of the PKA binding
pocket. Thus, smaller substitution such as H, F, and OMe
were favored for both potency and PKA selectivity when the
preferred chirality of the tetrahydroisoquinoline ring was
applied. The methoxy group was then applied to different
positions of the phenyl ring in the tetrahydroisoquinoline
moiety. Results in Table 2 showed that substitutions at the
6-position (29) were well tolerated, while substitutions at the
7- and 8-position (32 and 33) reduced the ROCK potency.
These SAR results are consistent with our docking pose
shown in Figure 3, which demonstrated that there was not
much room around the 7- and 8-positions of the tetrahydroi-
soquinoline ring to incorporate a methoxy group.
As described above, the side chain on the central phenyl
ring is critical to the inhibitor’s selectivityagainst PKA (and to
some extent, to the high potency of our ROCK inhibitors). To
gain a better understanding on how different side chains
would affect these properties, various side chains from several
different classes on the central phenyl ring ortho- to the amide
group were introduced to the 6-methoxy-1,2,3,4-tetrahydroi-
soquinoline pyrazole compounds. Large meta-substitutions
were not investigated in this study because they are not well-
tolerated as illustrated in our previous study with a different
chemotype.14 Assummarizedin Table3, both the potency and
PKA selectivity for many of those compounds with a sub-
stitution on the central phenyl ring ortho- to the aniline amide
group were much improved compared to the unsubstituted
compound 34. We believe that the extra binding of the side
chain with residue Asp176 in the ROCK-II enzyme is the basis
of this effect.
Compounds 35, 36, and 37, which had a tertiary amine
containing alkoxy side chain, were potent in both enzymatic
and cell-based assays, selective against PKA, and stable in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5731
Table 3. Substitution Effect on the Central Phenyl Ring
Table 4. Effects of Fluorination on the Central Ring and Alkylation of
the 1,2,3,4-Tetrahydroisoquinoline
a Average of two or more experiments, and deviation less than 50%.
b 2 mg/mL human liver microsomes were employed. c Not determined.
a Average of two or more experiments, and deviation less than 50%.
b 2 mg/mL human liver microsomes were employed. c Not determined.
d With 3-methylpyrazol-4-yl as the hinge binding moiety.
Thus, N-substitution on this group was investigated (Table 4)
because our modeling pose shown in Figure 3 suggested that a
substitution on the NH might be tolerated. Results in Table 4
demonstrated that N-ethyl (compounds 48 and 51) and N-
cyclopropylmethyl (compound 49) substitutions led to im-
proved selectivity against PKA, while the methyl group (47)
slightly reduced the potencyand PKA selectivity. On the other
hand, N-acylation of NH produced a compound (50) with low
ROCK-II activity (IC50 = 775 nM). This is probably because
the amide carbonyl group is a strong H-bond acceptor,
resulting in negative modulation of the enzyme-ligand inter-
actions. It is also noteworthy that all these N-substituted
compounds had much reduced human microsomal stabilities
as compared to those free amino compounds 23, 29, and 46,
probably due to the dealkylation of tertiary amines.
To examine the selectivity profile of our tetrahydroisoqui-
noline-based ROCK inhibitors, compounds 29, 35, 46, and 48
were selected for counter screening against several protein
kinases other than PKA. As summarized in Table 5, these
compounds were generally inactive against JNK3 and p38.
The selectivity against MRCKR (belongs to the same kinase
family as ROCK) was lower but still reasonable (>55-fold).
Examination of ROCK-I activities indicated that these com-
pounds were pan-ROCK inhibitors. Inhibition of a few cyto-
chrome P450 isoforms, 1A2, 2C9, 2D6, and 3A4, was also
studied and single-dose P450 results suggested that P450 inhi-
bition was not an issue for these compounds with the highest
inhibition being ∼80% at 10 μM for 46 (CYP2D6) and ∼70%
for 48 (CYP2C9) (Table 5). Data for CYP3A4, the isoform of
highest concern, showed all compounds were clean.
human liver microsomes. Among them, compound 35, which
had an (N-methyl-piperidin-4-yl)methoxy side chain, was the
best overall with subnanomolar (IC50 < 1 nM) ROCK-II
potency, high PKA selectivity(>5000fold), goodcellpotency
(IC50 = 51 nM) as well as good microsomal stability. The
similar compound 37, which has one less methylene group,
was slightly less potent but the PKA selectivity was only 57-
fold. Compound 36, which possesses a 2-(pyrolidin-1-
yl)ethoxy side chain, was also very potent, selective, and stable
in human microsomes. Compounds 38 and 39, which had an
ether or a hydroxyl containing alkoxy side chain, were slightly
less potent against ROCK-II but still selective over PKA.
However, their cell potency was not satisfactory. The tertiary
amine containing thioether side chain analogues (compounds
40, 41, 42) were also potent and selective against PKA, yet all
these compounds showed low microsomal stability, which
might be a result of the easy oxidization of the thioether to
sulfone or sulfoxide (observed under acidic condition). The
dimethyl amide moiety on compound 42 was even less stable
potentially due to demethylation.
After the investigation of monosubstitution on the central
phenyl ring, compounds with multiple substitutions (43, 44,
and 45) were also studied. Extra fluoro substitution was intro-
duced on different positions due to the unique role of fluorine
in medicinal chemistry.36,37 As shown in Table 3, among the
three possible substitutions, the 5-F analogue 44 was the best
in terms of both enzyme and cell-based potency. However,
compared to the nonfluoro substituted compound 29, com-
pound 44 showed reduced microsomal stability (51 min vs 101
min in 29) although it had slightly improved potency.
To further investigate the general selectivity of these tetra-
hydroisoquinoline-based ROCK inhibitors, compound 35
was selected to be profiled against a large panel of kinases.
Single point profiling of 442 kinases (Ambit Screen38) at 3 μM
(a concentration more than 3000-fold of its ROCK-II IC50
value of <1 nM, Table 3) identified only seven other kinases
whereby 35 showed >65% inhibition: Clk1(86%), Clk4
The NH group on the tetrahydroisoquinoline moiety might
be a concern because it could harm the compound’s capability
for membrane penetration such as cornea penetration (an
important property for the topical antiglaucoma application).

5732 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Fang et al.
Table 5. Selectivity of the Lead Compounds over Other Kinases and Cytochrome P450 Isoforms
IC50 (nM)a
% inh at 10 μM
1A2/2C9/2D6/3A4
compd
ROCK-II
ROCK-I
MRCKR
JNK3
p38
46
29
35
48
31/62/77/18
21/31/40/-3
3/45/52/15
6
10
33
48
10
16
333
5350
669
>20000
>20000
>20000
>20000
>20000
>20000
>20000
>20000
<1
2
-24/70/52/39
810
a Average of two or more experiments, and deviation less than 50%.
Table 6. Rat Pharmacokinetic Data of Selected Compoundsa
Cl
(mL/min/kg)
Vd
(L/kg)
t1/2
(h)
Cmax
PO (nM)
oral
F (%)
compd
29
35
44
46
49
51
17
41
7.2
9.5
0.49
24
5.5
3.4
1.5
2.7
2.3
1.7
242
0
28
0
4.6
106
42
32
18
0
0.3
16
0
7
17
130
0
0
a Data is the average from three sets dosed at 1.0 mg/kg (iv) or 2.0 mg/
kg (PO).
(91%), Lats2 (75%), MRCKβ (77%), NDR1 (72%), PI3Kβ
(74%), and STK33 (66%). All other kinases showed either no
or very low inhibition (see Supporting Information). An off-
target hit rate of only 1.6% (7 out of 442) makes this
compound a remarkably selective ATP-competitive kinase
inhibitor.38 Multiple interactions between 35 and the enzyme
are believed to be the reason for this high general kinase
selectivity. In addition to the four interactions observed in the
docking pose (Figure 3), two more interactions are highly
possible: one is between the tetrahydroisoquinoline NH group
and the DFG chain, either directly or through a water
molecule; another one is an H-bond between the methoxy
group on the tetrahydroisoquinoline ring and the surrounding
residues on the enzyme, either directly or via a water molecule.
More importantly, all these interactions are believed to be
optimal between 35 and ROCK-II, but not other kinases.
The pharmacokinetic properties in rats for compounds 29,
35, 44, 46, 49, and 51 were evaluated (Table 6). All tested
compounds except 29 and 44 showed high clearance (Cl), high
volume of distribution (Vd), low oral Cmax, and minimal to low
bioavailability (F). Compound 44 also had very low oral
bioavailability (0.3%). These PK properties are desirable for
the topical applications of compounds to be used as antiglau-
coma agents, preventing systemic exposure of compound that
drains through the tear duct. Because oral exposure of com-
pounds could be achieved through drainage of compounds
through the tear duct, and ROCK inhibitors have been shown
to reduce blood pressure, PK properties such as low oral
bioavailability and high clearance might be beneficial for this
class of compounds even when formulated for topical dosing.
The IOP lowering effect of these tetrahydroisoquinoline-
based ROCKinhibitors was investigated on the eyes of Brown
Norway rats (n = 7/group) housed under constant low-light
conditions. Thus, compound 35 was applied at 40 μg/dose
(2 ꢀ 20 μL drops of a 0.1% solution) to rat eyes of an elevated
IOP model39 (initial IOP was 29 mmHg). As shown in Figure 4,
significant decreases in IOP were detected from around 2 h,
maximized at or after 8 h, and IOP returning to baseline at
24 h as compared to the vehicle. Data in Figure 4 also indicated
that the duration of action for 35 on IOP lowering was long
enough to possibly warrant aqd dosing regimen even at a
concentration of 0.1%. These results demonstrated that tetra-
Figure 4. Rat IOP effects of 35 (IOP decreases relative to vehicle).
hydroisoquinoline-based ROCK inhibitors are promising drug
candidates as antiglaucoma therapeutics. Future investigation
for these compounds will be focused on improved IOP lowering
(g20%) at low concentration (e0.1%), ocular stability, ocular
pharmacokinetics, and toxicity studies such as the hyperemia
effect, which was observed in rabbits and monkeys after long-
term dosing of other ROCK compounds.40
Conclusion
In this study, we have developed a series of tetrahydroiso-
quinoline-based amides as ROCK inhibitors starting from the
initial lead 18. SAR investigation and optimization success-
fully provided potent compounds with high selectivity as well
as pharmacokinetic properties that might be desirable for
antiglaucoma applications. SAR features attributed to this
high potency, selectivity, and low oral bioavailabilityincludes:
a tertiary amine containing alkoxy side chain on the central
phenyl ring, interactions of the tetrahydroisoquinoline moiety
in the hydrophobic pocket of ROCK-II, and methoxy sub-
stitution on the tetrahydroisoquinoline. Compound 35, a
highly potent and selective inhibitor with ROCK-II potency
in the subnanomolar range and an off-target hit rate of only
1.6% against a panel of 442 kinases, was demonstrated to be
effective in reducing intraocular pressure (IOP) on rat eyes.
Future optimizations for these tetrahydroisoquinoline-based
ROCK inhibitors will be mainly focused on toxicity issues,
physicochemical stabilities, and the pharmacokinetic proper-
ties that might be dedicated to nontopical administration for
other ROCK-related indications. These studies will be re-
ported in due course.
Experimental Section
Chemical Materials and Methods. Proton nuclear magnetic
resonance (1H NMR, 13C NMR, 19F NMR) spectra were
recorded at 400 MHz (Bruker), and chemical shifts are reported

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5733
in parts per million downfield (δ) from Me4Si (δ = 0.0).
Analytical HPLC data were generated by injecting 10 μL of
very dilute sample solution in methanol or acetonitrile to a
reverse phase HPLC system run over 14 min (5-95% acetoni-
trile/water with 0.1% TFA in each solvent). The products were
detected by UV in the detection range of 215-310 nm. LC-MS
data were collected from Thermo or Angilent instrument.
HRMS (electrospray ionization) experiments were performed
with a Thermo Finnigan orbitrap mass analyzer. Data were
acquired in the positive ion mode at a resolving power of 100000
at m/z 400. Calibration was performed with an external calibra-
tion mixture immediately prior to analysis. Final products were
all purified by reverse-phase preparative HPLC to provide a
purity of >95% based on UV detection (at 254 nm) in analytical
HPLC traces.
General Procedure A: (R)-N-(4-(1H-Pyrazol-4-yl)phenyl)-2-
amino-3-(3-fluorophenyl)propanamide (18). To a stirred solution
of N-Boc protected D-3-fluoro-phenylalanine (62 mg, 0.22
mmol) in dry DMF (2 mL) was added 4-bromoaniline (34 mg,
0.2 mmol), DIPEA (0.174 mL, 1.0 mmol), and HATU (91 mg,
0.24 mmol) consequently. The solution was stirred at ambient
temperature for 4 h until LC-MS indicated that all aniline was
consumed. The reaction mixture was concentrated, and the
residue was diluted with EtOAc. The resulting solution was
then washed with saturated NaHCO3 solution and brine and
dried over anhydrous Na2SO4. The solvent was evaporated to
provide the crude bromo-amide, which was used in the next step
without further purification.
The crude amide (0.2 mmol), pyrazole boronic pinacol ester
(54 mg, 0.28 mmol), K2CO3 (111 mg, 0.8 mmol), and palladium
tetrakis(triphenylphosphine) (23 mg, 0.02 mmol) was suspended
in 4:1 dioxane/water (2 mL). The resulting mixture was sealed in
a microwave tube. The suspension was degassed under vacuum
and charged with argon. It was then heated on microwave oven
at 140 °C for 30 min. After the completion of reaction as
indicated by LC-MS, the reaction mixture was cooled to room
temperature. After concentration, the residue was suspended in
EtOAc. The mixture was washed with saturated NaHCO3
solution and brine. It was filtered and concentrated to provide
the crude intermediate, which was treated with 1:1 TFA/CH2Cl2
in the presence of 5% of triisopropylsilane as scavenger. The
reaction was stirred at ambient temperature for 30 min to
remove the N-Boc group. After the solvent was removed under
reduced pressure, the residue was subjected to preparative
HPLC to provide 18 (24 mg, 37% over 3 steps) as a fine powder
of its TFA salt. 1H NMR (DMSO-d6, 400 MHz) δ 3.18 (m, 2H),
4.19 (bs, 1H), 7.11 (m, 2H), 7.36 (m, 1H), 7.54 (m, 5H), 8.01 (s,
2H), 8.36 (s, 2H), 10.44 (s, 1H). Single peak in analytical HPLC.
LC-MS (ESI): m/z 325 [M þ 1]þ. HRMS (ESI), MHþ calcd for
C18H17FN4O, 325.1486; found, 325.1456.
J = 8.3 Hz, 1H), 8.01 (s, 2H). Single peak in analytical HPLC.
LC-MS (ESI): m/z 414 [M þ 1]þ. HRMS (ESI-Orbitrap), MHþ
calcd for C21H24ClN5O2, 414.1718; found, 414.1689.
N-(2-(2-(Dimethylamino)ethoxy)-4-(1H-pyrazol-4-yl)phenyl)-
1,2,3,4-tetra-hydroisoquinoline-1-carboxamide (22). H NMR
1
(MeOH-d4, 400 MHz) δ 3.06 (s, 6H), 3.50-3.58 (m, 2H), 3.68-
3.79 (m, 2H), 4.52-4.64 (m, 5H), 4.75 (s, 1H), 7.30-7.38 (m, 4H),
7.40-7.44 (m, 2H), 8.06-8.10 (m, 3H). 13C NMR (CH3CN-d3,
100 MHz) δ 31.0, 48.1, 56.9, 57.7, 68.2, 110.7, 119.9, 124.8,
125.3, 127.9, 128.6, 129.0, 129.2, 130.1, 132.6, 150.1, 168.1.
Single peak in analytical HPLC. LC-MS (ESI): m/z 406 [M þ 1]þ.
HRMS (ESI-Orbitrap), MHþ calcd for C23H27N5O2, 406.2264;
found, 406.2235.
(R)-N-(2-(2-(Dimethylamino)ethoxy)-4-(1H-pyrazol-4-yl)-
phenyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (23).1HNMR
(CH3CN-d3, 400 MHz), δ 2.80-2.95 (m, 1H), 3.29-3.39 (m, 1H),
3.48-3.64 (m, 2H), 4.30-4.50 (m, 3H), 4.62 (dd, J = 11.3, 4.8 Hz,
1H), 5.73 (bs, 1H), 7.05-7.45 (m, 5H), 7.77-8.29 (m, 2H), 9.54 (bs,
1H), 10.18 (bs, 1H). 13C NMR (CH3CN-d3, 100 MHz) δ 30.6, 45.2,
56.7, 57.4, 63.2, 110.6, 119.5, 124.5, 125.3, 127.6, 128.3, 129.0, 129.1,
129.7, 132.1, 149.9, 167.9. Single peak in analytical HPLC. LC-MS
(ESI): m/z 406 [M þ 1]þ. HRMS (ESI-Orbitrap), MHþ calcd for
C23H27N5O2, 406.2264; found, 406.2238.
(S)-N-(2-(2-(Dimethylamino)ethoxy)-4-(1H-pyrazol-4-yl)phenyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (24). 1H NMR
(CH3CN-d3, 400 MHz) δ 2.97-2.88 (b, 1H), 3.36-3.27 (m,
1H), 3.66-3.44 (m, 2H), 4.53-4.31 (m, 3H), 4.62 (dd, J =
11.27,4.84 Hz, 1H), 5.77 (b, 1H), 7.43-7.01 (m, 5H), 8.29-7.77
(m, 2H), 9.66 (bs, 1H), 10.28 (bs, 1H). 13C NMR (CH3CN-d3,
100 MHz) δ 31.0, 45.1, 56.9, 57.7, 63.2, 110.7, 119.9, 124.8,
125.3, 127.9, 128.6, 129.0, 129.2, 130.1, 132.6, 150.1, 168.1.
Single peak in analytical HPLC. LC-MS (ESI): m/z 406 [M þ 1]þ.
HRMS (ESI-Orbitrap), MHþ calcd for C23H27N5O2, 406.2264;
found, 406.2235.
General Procedure B: (R)-N-(2-(2-(Dimethylamino)ethoxy)-
4-(1H-pyrazol-4-yl)phenyl)-6-fluoro-1,2,3,4-tetrahydroisoquino-
line-3-carboxamide (25). Aqueous formaldehyde (37%, 1.49 g,
20 mmol) was added to a suspension of D-3-fluoro-phenylala-
nine (930 mg, 5 mmol) in 6 M hydrochloric acid (6 mL). The
mixture was stirred at ambient temperature overnight and a
white precipitate appeared. The precipitate was filtered off and
washed with 1 M HCl solution. The solid was collected and dried
under vacuum to provide crude (R)-6-fluoro-1,2,3,4-tetrahy-
droisoquinoline-3-carboxylic acid HCl salt as white solid. This
intermediate was then dissolved in 1:1 1 M NaOH/dioxane (25
mL), and the solution of di-tert-butyl dicarbonate (958 mg, 5.5
mmol) in 12 mL of dioxane was added. The mixture was stirred
at ambient temperature for 2 h. After the completion of reaction,
the reaction mixture was poured into 100 mL of 1 M aqueous
citric acid solution, which was extracted three times with EtOAc.
The combined organic layer was washed with brine and dried
over anhydrous Na2SO4. After filtration, all solvent was eva-
porated and the residue was subjected to flash chromatography
(0-15% of MeOH in CH2Cl2) to provide the intermediate (R)-
2-(tert-butoxycarbonyl)-6-fluoro-1,2,3,4-tetrahydroisoquino-
line-3-carboxylic acid. General procedure A was then employed
to finish the synthesis of 25. 1H NMR (MeOH-d4, 400 MHz) δ
2.99-3.06 (m, 8H), 3.43-3.61 (m, 1H), 3.61-3.79 (m, 2H), 4.41-
4.62 (m, 4H), 7.04-7.42 (m, 5H), 7.97-8.05 (m, 3H), 9.87-9.97
(m, 3H), 10.01 (b, 1H). Single peak in analytical HPLC. LC-MS
(ESI): m/z 424 [M þ 1]þ; HRMS (ESI-Orbitrap), MHþ calcd for
C23H26FN5O2, 424.2170; found, 424.2141.
Compounds 18-46 were prepared according to general pro-
cedure A:
N-(4-(1H-Pyrazol-4-yl)phenyl)-2-amino-3-(3-chlorophenyl)-
propanamide (19). 1H NMR (DMSO-d6, 400 MHz) δ 2.98-3.03
(dd, J = 7.6, 13.6 Hz, 1H), 3.08-3.13 (dd, J = 6.0, 13.6 Hz, 1H),
4.05-4.06 (m, 1H), 7.21-7.23 (m, 2H), 7.33-7.35 (m, 2H),
7.41-7.44 (m, 2H), 7.51-7.53 (m, 2H), 7.95 (s, 2H), 8.23 (m,
3H), 10.32 (s, 1H). Single peak in analytical HPLC. LC-MS
(ESI): m/z 341[M þ 1]þ. HRMS (ESI-Orbitrap), MHþ calcd for
C18H17ClN4O, 341.1191; found, 341.1162.
N-(4-(1H-Pyrazol-4-yl)phenyl)-2-amino-2-(4-chlorophenyl)-
acetamide (20). 1H NMR (DMSO-d6, 400 MHz) δ 5.30 (m, 1H),
7.45-7.52 (m, 8H), 7.93 (s, 2H), 8.67 (m, 3H), 10.51 (s, 1H).
Single peak in analytical HPLC. LC-MS (ESI): m/z 327 [M þ
1]þ. HRMS (ESI-Orbitrap), MHþ calcd for C17H15ClN4O,
327.1034; found, 327.1005.
The 1,2,3,4-tetrahydroisoquiniline moiety of compounds 26, 29-
31, and 33-46 were prepared according to general procedure B:
(S)-N-(2-(2-(Dimethylamino)ethoxy)-4-(1H-pyrazol-4-yl)-
phenyl)-6-fluoro-1,2,3,4-tetrahydroiso-quinoline-3-carboxamide
(26). 1H NMR (MeOH-d4, 400 MHz) δ 2.99-3.03 (m, 2H), 3.05
(s, 6H), 3.43-3.61 (m, 1H), 3.61-3.79 (m, 2H), 4.41-4.48 (m,
1H), 4.51-4.64 (m, 3H), 7.04-7.24 (m, 2H), 7.28-7.42 (m, 3H),
7.99-8.04(m, 3H), 9.87 (b, 1H), 9.90-9.98 (m, 3H), 10.01 (b, 1H).
2-Amino-2-(4-chlorophenyl)-N-(2-(2-(dimethylamino)ethoxy)-
4-(1H-pyrazol-4-yl)phenyl)aceta-mide (21). H NMR (MeOH-
1
d4, 400 MHz), δ 2.86 (s, 6H), 3.59-3.40 (m, 2H), 4.51-4.31 (m,
2H), 5.42 (s, 1H), 7.30-7.24 (m, 2H), 7.64-7.53 (m, 4H), 7.89 (d,

5734 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Fang et al.
Single peak in analytical HPLC. LC-MS (ESI): m/z 424 [M þ 1]þ.
HRMS (ESI-Orbitrap), MHþ calcd for C23H26FN5O2, 424.2170;
found, 424.2139.
J = 17.0, 4.3 Hz, 1H), 3.58 (s, 1H), 3.76 (s, 3H), 3.78-3.87 (m,
1H), 4.25-4.54 (m, 4H), 6.86-7.01 (m, 3H), 7.13-7.40 (m, 4H),
7.73-7.80 (m, 1H), 8.10 (s, 2H). 13C NMR (CH3CN-d3, 100 MHz)
δ 34.1, 45.1, 54.8, 56.9, 57.7, 63.2, 110.8, 114.2, 114.9, 119.4, 121.2,
124.0, 125.5, 127.7, 131.1, 134.7, 146.9, 156.2, 167.0. Single peak
in analytical HPLC. LC-MS (ESI): m/z 436 [M þ 1]þ. HRMS
(ESI-Orbitrap), MHþ calcd for C24H29N5O3, 436.2370; found,
436.2340.
(S)-N-(2-(2-(Dimethylamino)ethoxy)-4-(1H-pyrazol-4-yl)-
phenyl)-6-methoxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxamide
(30). 1H NMR (DMSO-d6, 400 MHz) δ 2.65-2.74 (m, 1H),
2.82-2.89 (m, 1H), 2.94 (s, 6H), 3.04-3.17 (m, 1H), 3.30-3.35
(m, 1H), 3.59 (m, 1H), 3.74 (s, 3H), 3.77-3.88 (m, 1H), 4.24-
4.55 (m, 4H), 6.86 (m, 3H), 7.10-7.39 (m, 4H), 7.70-7.80 (m,
1H), 8.11 (s, 2H). 13C NMR (CH3CN-d3, 100 MHz) δ 34.9, 45.8,
54.9, 57.5, 58.3, 63.2, 111.0, 114.3, 115.2, 119.9, 121.7, 124.4,
126.5, 127.9, 131.5, 135.0, 147.2, 155.9, 168.0. Single peak in
analytical HPLC. LC-MS (ESI): m/z 436 [M þ 1]þ.
(R)-N-(2-(2-(Dimethylamino)ethoxy)-4-(1H-pyrazol-4-yl)-
phenyl)-6-chloro-1,2,3,4-tetrahydroiso-quinoline-3-carboxamide
(27). To 30 mL of anhydrous MeOH was slowly added thionyl
chloride (1.5 g, 12.5 mmol) at 0 °C. After stirring for additional
10 min, D-3-chloro-phenylalanine (1 g, 5 mmol) was added in
several portions. The reaction mixture was then warmed to
room temperature and stirred overnight until reaction was
complete. The solvent was removed by evaporation, and the
residue was diluted with 200 mL of EtOAc. The resulting
mixture was washed with saturated NaHCO3 solution, brine,
and dried over anhydrous Na2SO4. Filtration followed by
concentration provided the crude amino acid methyl ester.
The crude ester was then dissolved in 50 mL of dichloro-
methane followed by the addition of pyridine (0.809 mL, 10
mmol). The reaction mixture was cooled to 0 °C, and ethyl
chloroformate (0.540 mL, 5.5 mmol) was then added carefully.
The reaction mixture was warmed to room temperature and
stirred overnight until the reaction was complete. Solvent was
evaporated, and the residue was diluted with EtOAc. The
resulting mixture was washed with 1 M HCl, brine, saturated
NaHCO3 solution, and brine consequently, and dried over
anhydrous Na2SO4. After filtration and concentration, the
residue was dried under vacuum to provide crude (R)-6-chloro-
2-(ethoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid, methyl ester (1.17 g, 4.1 mmol), which was used for follow-
ing transformations.
N-(2-(2-(Dimethylamino)ethoxy)-4-(1H-pyrazol-4-yl)phenyl)-
6-ethoxy-1,2,3,4-tetrahydroiso-quinoline-3-carboxamide (31).
1H NMR (DMSO-d6, 400 MHz) δ 1.36-1.26 (m, 3H), 2.55 (t,
J = 5.6 Hz, 2H), 2.89 (s, 6H), 3.16-3.04 (m, 1H), 2H), 3.41-
3.32 (m, 1H), 3.67-3.49 (m, 2H), 4.06-3.97 (m, 1H), 4.54-4.24
(m, 4H), 7.02-6.85 (m, 3H), 7.37-7.12 (m, 4H), 7.80-7.75 (m,
1H), 8.16-8.06 (m, 2H). 13C NMR (CH3CN-d3, 100 MHz) δ
29.6, 34.3, 43.6, 56.0, 63.2, 65.0, 109.2, 111.9, 113.7, 114.8, 117.7,
119.5, 121.2, 123.1, 124.1, 127.5, 130.7, 132.2, 148.5, 158.4,
160.0, 166.7. Single peak in analytical HPLC. LC-MS (ESI): m/z
450 [M þ 1]þ.
(R)-N-(2-(2-(Dimethylamino)ethoxy)-4-(1H-pyrazol-4-yl)-
phenyl)-7-methoxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxamide
(32). 1H NMR (DMSO-d6, 400 MHz) δ 2.86-2.92 (m, 1H), 2.82
(s, 6H), 3.04-3.17 (m, 1H), 3.27-3.30 (m, 1H), 3.49 (s, 1H), 3.64
(s, 3H), 3.78-3.87 (m, 1H), 4.23-4.38 (m, 2H), 6.71-6.81 (m,
2H), 7.10-7.12 (m, 1H), 7.58-7.37 (d, J = 6.8 Hz, 1H), 7.74-7.80
(m, 2H), 7.99 (s, 2H), 9.49 (b, 1H), 9.59 (b, 1H), 9.82 (m, 2H).
Single peak in analytical HPLC. LC-MS (ESI): m/z 436 [M þ 1]þ.
HRMS (ESI-Orbitrap), MHþ calcd for C24H29N5O3, 436.2370;
found, 436.2340.
N-(2-(2-(Dimethylamino)ethoxy)-4-(1H-pyrazol-4-yl)phenyl)-
8-methoxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxamide (33).
1H NMR (MeOH-d4, 400 MHz) δ 2.86-2.92 (m, 1H), 2.99 (s,
6H), 3.04-3.17 (m, 1H), 3.52 (dd, J = 19.0, 4.0 Hz, 1H), 3.67-
3.79 (m, 1H), 3.82 (s, 3H), 4.37-4.45 (m, 1H), 4.53-4.62 (m,
3H), 6.88-6.94 (m, 3H), 7.20-7.25 (m, 1H), 7.40-7.44 (m, 1H),
8.05-8.09 (m, 3H). Single peak in analytical HPLC. LC-MS
(ESI): m/z 436 [M þ 1]þ. HRMS (ESI-Orbitrap), MHþ calcd for
C24H29N5O3, 436.2370; found, 436.2341.
(R)-N-(4-(1H-Pyrazol-4-yl)phenyl)-6-methoxy-1,2,3,4-tetra-
hydroisoquinoline-3-carboxamide (34). 1H NMR (MeOH-d4,
400 MHz) δ 3.29-3.20 (m, 1H), 3.46 (dd, J = 17.0, 4.8 Hz,
1H), 3.79 (s, 3H), 4.28 (dd, J = 12.1, 4.8 Hz, 1H), 4.40 (m, 2H),
6.94-6.81 (m, 2H), 7.17 (d, J = 8.5 Hz, 1H), 7.68-7.53 (m, 4H),
7.98-7.95 (m, 2H). Single peak in analytical HPLC. LC-MS
(ESI): m/z 349 [M þ 1]þ. HRMS (ESI-Orbitrap), MHþ calcd for
C20H20N4O2, 349.1686; found, 349.1656.
To the solution of the ester from last step in 3:1 AcOH/
sulfuric acid (6 mL), paraformaldehyde (0.135 g, 4.5 mmol) was
added. The solution was then stirred overnight to complete the
reaction. It was quenched by pouring into 200 mL of ice water.
The resulting mixture was extracted with EtOAc three times.
The combined organic layer was then washed with saturated
NaHCO3 solution, brine, and dried over anhydrous Na2SO4.
After filtration and concentration, the crude product was ob-
tained and was directly used in next step.
The intermediate was suspended in 6 M HCl solution and
refluxed overnight until ethoxy carbonyl and ester group were
completely removed (monitored by LC-MS). The solvents were
removed under reduced pressure, and the solid residue was
dissolved in 20 mL of 1:1 1 M NaOH/dioxane to form a solution,
to which a solution of di-tert-butyl dicarbonate (1.05 g, 4.8
mmol) in 10 mL of dioxane was added slowly. The reaction
mixture was stirred for 2 h until the reaction was complete. It
was quenched and acidified by pouring into 100 mL of 1 M citric
acid solution. The solution was extracted with EtOAc three
times, and the combined organic layer was washed with brine
and dried over anhydrous Na2SO4. After concentration, flash
chromatography was applied (3-10% of MeOH in CH2Cl2) to
provide the (R)-2-(tert-butoxycarbonyl)-6-choro-1,2,3,4-tetra-
hydroisoquinoline-3-carboxylic acid as pale-yellow syrup. Gen-
eral procedure A was then employed to provide the titled
1
compound. H NMR (MeOH-d4, 400 MHz) δ 2.76-2.80 (m,
2H), 2.82 (s, 6H), 3.22-3.28 (m, 2H), 3.85-4.10 (m, 5H),
7.32-7.60 (m, 5H), 7.76 (d, J = 6.5 Hz, 1H), 8.50 (s, 2H). Single
peak in analytical HPLC. LC-MS (ESI): m/z 440 [M þ 1]þ.
(S)-N-(2-(2-(Dimethylamino)ethoxy)-4-(1H-pyrazol-4-yl)-
phenyl)-6-chloro-1,2,3,4-tetrahydroiso-quinoline-3-carboxamide
(28). The titled compound was synthesized according to the
procedures described in the synthesis of compound 27. 1H NMR
(MeOH-d4, 400 MHz) δ 2.76-2.82 (m, 8H), 3.21-2.25 (m, 2H),
3.89-4.12 (m, 5H), 7.32-7.41 (m, 2H), 7.51-7.60 (m, 3H), 7.73
(d, J = 6.5 Hz, 1H), 8.48 (s, 2H). Single peak in analytical
HPLC. LC-MS (ESI): m/z 440 [M þ 1]þ.
(R)-6-Methoxy-N-(2-((1-methylpiperidin-4-yl)methoxy)-4-(1H-
pyrazol-4-yl)phenyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide
1
(35). H NMR (CH3CN-d3, 400 MHz) δ 1.83-2.11 (m, 4H),
2.74 (s, 3H), 2.82-2.92 (m, 1H), 3.20-3.32 (m, 1H), 3.39 (dd,
J = 16.6, 4.5 Hz, 1H), 3.44-3.53 (m, 2H), 3.78 (s, 3H), 3.95-
4.03 (m, 1H), 4.38 (q, J = 15.4 Hz, 2H), 4.50-4.62 (m, 1H), 6.80
(d, J = 2.0 Hz, 1H), 6.87 (dd, J = 2.4, 8.5 Hz, 1H), 7.05-7.21
(m, 3H), 7.83 (d, J = 8.0 Hz, 1H), 7.92 (bs, 2H), 8.95 (s, 1H),
10.61 (bs, 1H). 13C NMR (CH3CN-d3, 100 MHz) δ 26.7, 26.9,
31.0, 33.9, 44.0, 44.6, 55.1, 56.0, 56.2, 72.7, 110.2, 114.3, 114.4,
121.3, 124.1, 125.1, 128.7, 133.6, 146.8, 150.9, 160.3. Single peak
in analytical HPLC. LC-MS (ESI): m/z 476 [M þ 1]þ. HRMS
(ESI-Orbitrap), MHþ calcd for C27H33N5O3, 476.2683; found,
476.2655.
(R)-N-(2-(2-(Dimethylamino)ethoxy)-4-(1H-pyrazol-4-yl)-
phenyl)-6-methoxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxamide
(29). 1H NMR (DMSO-d6, 400 MHz) δ 2.64-2.75 (m, 1H),
2.86-2.92 (m, 1H), 2.96 (s, 6H), 3.04-3.17 (m, 1H), 3.33 (dd,

Article
(R)-N-(4-(1H-Pyrazol-4-yl)-2-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5735
J = 2.11 Hz, 1H), 7.93 (s, 2H), 8.18 (d, J = 8.53 Hz, 1H), 10.47 (s,
1H). LC-MS (ESI): m/z 480 [M þ 1]þ. HRMS (ESI-Orbitrap),
MHþ calcd for C25H29N5O3S, 480.2090; found, 480.2063.
(R)-N-(2-(2-(Dimethylamino)ethoxy)-3-fluoro-4-(1H-pyrazol-
4-yl)phenyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxa-
mide (43). 1H NMR (DMSO-d6, 400 MHz) δ 2.55-2.64 (m, 1H),
2.82-2.89 (m, 1H), 2.94 (s, 6H), 3.17-3.04 (m, 1H), 3.33 (dd, J =
17.0, 4.3 Hz, 1H), 3.58 (s, 1H), 3.69 (s, 3H), 3.67-3.78 (m, 1H),
4.24-4.55 (m, 4H), 6.86 (m, 3H), 7.10-7.19 (m, 4H), 7.70-7.73
(m, 1H), 8.11 (s, 2H). Single peak in analytical HPLC. LC-MS
(ESI): m/z 454 [M þ 1]þ. HRMS (ESI-Orbitrap), MHþ calcd for
C24H28FN5O3, 454.2276; found, 454.2246.
(R)-N-(2-(2-(Dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-
4-yl)phenyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carbo-
xamide (44). 1H NMR (DMSO-d6, 400 MHz) δ 2.95 (s, 6H), 3.13
(dd, J = 16.3, 12.4 Hz, 1H), 3.39 (dd, J = 16.8, 4.4 Hz, 1H),
3.66-3.60 (m, 2H), 3.76 (s, 3H), 4.33 (q, J = 15.6 Hz, 2H), 4.51
(t, J = 5.0 Hz, 3H), 6.93-6.83 (m, 2H), 7.22 (d, J = 8.6 Hz, 1H),
7.49 (d, J = 7.0 Hz, 1H), 7.88 (dd, J = 12.5, 4.6 Hz, 1H), 8.11 (d,
J = 1.6 Hz, 2H), 9.68 (s, 1H), 10.05-9.86 (m, 2H), 10.24 (s, 1H).
Single peak in analytical HPLC. LC-MS (ESI): m/z 454 [M þ
1]þ. HRMS (ESI-Orbitrap), MHþ calcd for C24H28FN5O3,
454.2276; found, 454.2246.
6-methoxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxamide (36).
1H NMR (CH3CN-d3, 400 MHz) δ 2.00-2.11 (m, 4H), 2.74
(s, 2H), 3.24-3.34 (m, 1H), 3.43 (dd, J = 16.8, 4.8 Hz, 1H),
3.55-3.66 (m, 2H), 3.78 (s, 3H), 4.31-4.45 (m, 4H), 4.56 (dd,
J = 11.1, 4.9 Hz, 1H), 6.78-6.80 (m, 1H), 6.87 (dd, J = 2.5,
8.6 Hz, 1H), 7.13-7.21 (m, 3H), 7.85-7.93 (m, 2H), 9.72 (s, 1H).
13C NMR (CH3CN-d3, 100 MHz) δ 23.8, 30.8, 38.8, 44.7, 54.8,
56.0, 56.4, 64.7, 110.8, 114.2, 114.4, 119.4, 121.2, 124.4, 125.5,
128.7, 131.8, 133.7, 149.9, 160.2, 168.0. Single peak in analytical
HPLC. LC-MS (ESI): m/z 462 [M þ 1]þ. HRMS (ESI-Orbitrap),
MHþ calcd for C26H31N5O3, 462.2527; found, 462.2498.
(R)-6-Methoxy-N-(2-(1-methylpiperidin-4-yloxy)-4-(1H-pyrazol-
4-yl)phenyl)-1,2,3,4-tetrahydro-isoquinoline-3-carboxamide (37).
1H NMR (DMSO-d6, 400 MHz) δ 2.75-2.64 (m, 1H), 2.92-
2.86 (m, 1H), 2.96 (s, 6H), 3.17-3.04 (m, 1H), 3.33 (dd, J = 17.0,
4.3 Hz, 1H), 3.58 (s, 1H), 3.76 (s, 3H), 3.87-3.78 (m, 1H),
4.54-4.25 (m, 4H), 7.01-6.86 (m, 3H), 7.40-7.13 (m, 4H),
7.80-7.73 (m, 1H), 8.11-8.03 (m, 2H). 13C NMR (CH3CN-d3,
100 MHz) δ 27.9, 30.9, 42.6, 44.9, 50.4, 52.5, 56.0, 56.4, 67.9,
71.4, 110.9, 112.5, 114.3, 114.5, 119.5, 120.8, 124.7, 125.3, 125.6,
126.3, 128.8, 133.2, 149.4, 160.4, 167.7. Single peak in analytical
HPLC. LC-MS (ESI): m/z 462 [M þ 1]þ. HRMS (ESI-Orbitrap),
MHþ calcd for C26H31N5O3, 462.2527; found, 462.2499.
(R)-6-Methoxy-N-(2-(2-methoxyethoxy)-4-(1H-pyrazol-4-yl)-
phenyl)-1,2,3,4-tetrahydroisoquino-line-3-carboxamide (38). 1H
NMR (MeOH-d4, 400 MHz) δ 2.72-2.64 (m, 1H), 3.03 (s, 6H),
3.18-3.10 (m, 1H), 3.57 (dd, J = 17.0, 4.6 Hz, 1H), 3.83-3.66
(m, 2H), 4.48 (dd, J = 30.5, 15.5 Hz, 2H), 4.64-4.57 (m, 2H),
4.68 (dd, J = 12.1, 5.1 Hz, 2H), 7.13-7.05 (m, 2H), 7.32 (dd, J =
9.1, 5.0 Hz, 1H), 7.60 (d, J = 6.8 Hz, 1H), 8.02-7.99 (m, 2H),
8.30 (d, J = 6.8 Hz, 1H), 8.45 (d, J = 9.0 Hz, 1H). Single peak in
analytical HPLC. LC-MS (ESI): m/z 423 [M þ 1]þ. HRMS
(ESI-Orbitrap), MHþ calcd for C23H26N4O4, 423.2053; found,
423.2025.
(R)-N-(2-(2-(Dimethylamino)ethoxy)-6-fluoro-4-(1H-pyrazol-
4-yl)phenyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carbo-
1
xamide (45). H NMR (DMSO-d6, 400 MHz) δ 2.96 (s, 6H),
3.17-3.04 (m, 2H), 3.28-3.33 (m, 2H), 3.58 (s, 1H), 3.77 (s, 3H),
3.87-3.78 (m, 1H), 4.54-4.25 (m, 3H), 6.86-6.98 (m, 3H),
7.40-7.53 (m, 4H), 7.70-7.73 (m, 1H), 8.14 (s, 2H). Single peak
in analytical HPLC. LC-MS (ESI): m/z 454 [M þ 1]þ. HRMS
(ESI-Orbitrap), MHþ calcd for C24H28FN5O3, 454.2276; found,
454.2246.
(R)-N-(2-(2-(Dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-
4-yl)phenyl)-1,2,3,4-tetrahydroiso-quinoline-3-carboxamide (46).
1H NMR (DMSO-d6, 400 MHz) δ 2.93 (s, 6H), 3.14 (dd, J =
12.4, 16.8 Hz, 1H), 3.45 (dd, J = 4.4, 16.8 Hz, 1H), 3.60 (s, 2H),
4.41 (m, 2H), 4.49 (m, 3H), 7.28 (m, 4H), 7.48 (d, J = 6.8 Hz,
1H), 7.87 (d, J = 12.4 Hz, 1H), 8.10 (s, 2H), 9.62(b, 1H), 9.80 (b,
1H), 10.00 (s, 1H), 10.10 (b, 1H). Single peak in analytical
HPLC. LC-MS (ESI): m/z 424 [M þ 1]þ. HRMS (ESI-Orbitrap),
MHþ calcd for C23H26FN5O2, 424.2170; found, 424.2141.
General Procedure C: (R)-N-(2-(2-(Dimethylamino)ethoxy)-5-
fluoro-4-(1H-pyrazol-4-yl)phenyl)-2-methyl-1,2,3,4-tetrahydroi-
soquinoline-3-carboxamide (47). (R)-N-(4-bromo-2-(2-(dimethyl-
amino)ethoxy)-5-fluorophenyl)-1,2,3,4-tetrahydroisoquinoline-
3-carboxamide was synthesized according to the first step in general
procedure A. The Boc group was removed by treatment with 1:1
TFA/CH2Cl2 (10 mL) in the presence of 5% of TIS. After conce-
ntration, the residue was dissolved in EtOAc, washed with satu-
rated NaHCO3 and brine, and dried over anhydrous Na2SO4.
Filtration and removal of solvent under reduced pressure pro-
vided the crude bromo-amide.
To the solution of the intermediate from last step (87 mg, 0.2
mmol) in CH2Cl2, formaldehyde (6 mg, 0.2 mmol) was added
and the reaction mixture was stirred for 5 min. Then, sodium
triacetoxyborohydride (64 mg, 0.3 mmol) was added, and the
reaction was stirred overnight. Extra NaBH4 (7 mg, 0.2 mmol)
was added to complete the reduction of imine. The reaction was
quenched by addition of methanol. After concentration, the residue
was dissolved in EtOAc, washed with 1 M NaOH solution,
brine, saturated NaHCO3 solution, and brine, and dried over
anhydrous Na2SO4. It was filtered and concentrated to provide
the crude product amide, which was used in the next step without
purification.
(R)-N-(2-(2-Hydroxyethoxy)-4-(1H-pyrazol-4-yl)phenyl)-6-
methoxy-1,2,3,4-tetrahydroisoquino-line-3-carboxamide (39). 1H
NMR (MeOH-d4, 400 MHz) δ 2.72-2.64 (m, 1H), 3.03 (s, 6H),
3.18-3.10 (m, 1H), 3.57 (dd, J = 17.0, 4.6 Hz, 1H), 3.83-3.66
(m, 2H), 4.48 (dd, J = 30.5, 15.5 Hz, 2H), 4.64-4.57 (m, 2H),
4.68 (dd, J = 12.1, 5.1 Hz, 2H), 7.13-7.05 (m, 2H), 7.32 (dd, J =
9.1, 5.0 Hz, 1H), 7.60 (d, J = 6.8 Hz, 1H), 8.02-7.99 (m, 2H),
8.30 (d, J = 6.8 Hz, 1H), 8.45 (d, J = 9.0 Hz, 1H). Single peak in
analytical HPLC. LC-MS (ESI): m/z 409 [M þ 1]þ.
(R)-N-(2-(2-(Dimethylamino)ethylthio)-4-(1H-pyrazol-4-yl)-
phenyl)-6-methoxy-1,2,3,4-tetra-hydroisoquinoline-3-carboxamide
(40). 1H NMR (MeOH-d4, 400 MHz) δ 2.93 (s, 6H), 3.07-3.18
(m, 1H), 3.37-3.45 (m, 1H), 3.59 (dd, J = 16.9, 4.7 Hz, 1H), 3.84
(s, 3H), 4.47 (s, 2H), 4.50-4.57 (m, 1H), 6.91-6.98 (m, 1H), 7.24
(d, J = 8.2 Hz, 1H), 7.59-7.69 (m, 2H), 7.87-7.91 (m, 1H), 8.09
(s, 2H). Single peak in analytical HPLC. LC-MS (ESI): m/z 452
[M þ 1]þ. HRMS (ESI-Orbitrap), MHþ calcd for C24H29N5O2S,
452.2142; found, 452.2111.
(R)-N-(2-(2-(Dimethylamino)ethylthio)-4-(3-methyl-1H-pyrazol-
4-yl)phenyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carbo-
1
xamide (41). H NMR (MeOH-d4, 400 MHz) δ 2.35 (s, 3H),
2.79-2.81 (m, 6H), 3.05-3.01 (m, 1H), 3.40-3.26 (m, 1H), 3.46
(dd, J = 16.7, 4.6 Hz, 1H), 3.72 (s, 3H), 4.31-4.44 (m, 3H),
6.79-6.86 (m, 2H), 7.12 (d, J = 8.3 Hz, 1H), 7.40 (dd, J = 8.4,
2.0 Hz, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.61 (d, J = 1.7 Hz, 1H),
7.70 (s, 1H). LC-MS (ESI): m/z 466 [M þ 1]þ. HRMS (ESI-
Orbitrap), MHþ calcd for C25H31N5O2S, 466.2298; found,
466.2269.
(R)-N-(2-(4-(Dimethylamino)-4-oxobutylthio)-4-(3-methyl-1H-
pyrazol-4-yl)phenyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-
Thus, pyrazole boronic pinacol ester (54 mg, 0.28 mmol), K2CO3
(111 mg, 0.8 mmol), and palladium tetrakis(triphenylphosphine)
(23 mg, 0.02 mmol) were added consequently to a solution of the
amide from the last step in 4:1 dioxane/water (2 mL). The re-
action mixture was sealed in a microwave vial, degassed under
vacuum, and charged with argon. It was then heated in a
1
carboxamide (42). H NMR (MeOH-d4, 400 MHz) δ 2.65-2.58
(m, 2H), 2.73 (s, 3H), 2.95 (s, 3H), 3.10-3.03 (m, 2H), 3.34-3.26
(m, 1H), 3.54 (dd, J = 16.66, 5.07 Hz, 1H), 3.78 (s, 3H), 4.45-
4.37 (m, 2H), 4.75 (dd, J = 10.44, 5.04 Hz, 1H), 6.90-6.83 (m, 2H),
7.18 (d, J = 8.48 Hz, 1H), 7.59 (dd, J = 8.49, 2.12 Hz, 1H), 7.85 (d,

5736 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Fang et al.
microwave oven at 140 °C for 30 min. All solvent was removed
and the residue was diluted with EtOAc, which was washed with
NaHCO3 solution and brine. After concentration, the residue
was subjected to preparative HPLC to provide 47 as a fine
powder of its TFA salt. 1H NMR (DMSO-d6, 400 MHz) δ 2.39
(s, 1H), 2.93 (s, 6H), 3.12 (dd, J = 11.4, 16.8 Hz, 1H), 3.45 (dd,
J = 4.4, 16.8 Hz, 1H), 3.60 (s, 2H), 4.40-4.42 (m, 2H), 4.45-
4.48 (m, 3H), 7.26-7.28 (m, 4H), 7.58 (d, J = 5.8 Hz, 1H), 7.81
(d, J = 12.4 Hz, 1H), 8.01 (s, 2H), 9.60 (b, 1H), 9.81 (b, 1H),
10.00-10.10 (m, 2H). Single peak in analytical HPLC. LC-MS
(ESI): m/z 438 [M þ 1]þ. HRMS (ESI-Orbitrap), MHþ calcd for
C24H28FN5O2, 438.2326; found, 438.2297.
FBS. Following attachment, cells were serum starved for 4 h and
treated with inhibitor in 0.25% DMSO (final concentration) for 1 h
at 37 °C. Cells were then treated with 10 μM LPA for 10 min, fixed
with 4% paraformaldehyde for 30 min, washed in PBS þ 0.1 M
glycine, and permeabilized in 0.2% Triton X for 10 min. Cells were
then washed once in PBS and blocked in LI-COR blocking buffer
(LI-COR Biosciences) for 1 h at 25 °C. Cells were probed for
phosphorylated myosin light chain 20 using 55 ng/mL of primary
rabbit antibody and incubated overnight at 4 °C. Following three
washes, cells were probed with goat-antirabbit IR800 antibody
(2 μg/mL in LI-COR blocking buffer þ0.025% Tween-20) for 1 h
at 25 °C. Nuclei were stained with TO-PRO-3 iodide (642/661)
(Invitrogen, Carlsbad, CA) (1:4000) for 20 min, washed twice in
PBS/0.05% Tween-20, and read with an Odyssey infrared imaging
system (LI-COR Biosciences).
Determination of IOP in a Rat Model. An elevated rat IOP
model was used to evaluate the IOP lowering effects of com-
pounds. We have previously utilized this method for evaluating
IOP lowering in rats to assess other ROCK inhibitors.26 Initial
IOP was 29 mmHg. The IOP was measured in Brown Norway
rats that were housed in constant low-light conditions to reduce
circadian IOP changes. Measurements were made using a re-
bound tonometer with n = 7 in the vehicle and drug treated
groups. The reported ΔIOP (IOP decrease relative to vehicle) at
each time point is the average of five readings. Compounds were
formulated in 50 mM citrate buffer, pH = 5.5 w:v.
Compounds 47-50 were prepared according to General
Procedure C:
(R)-N-(2-(2-(Dimethylamino)ethoxy)-5-fluoro-4-(1H-pyrazol-
4-yl)phenyl)-2-ethyl-1,2,3,4-tetra-hydroisoquinoline-3-carboxamide
1
(48). H NMR (DMSO-d6, 400 MHz) δ 1.18 (t, 3H), 2.39 (m,
2H), 2.87 (s, 6H), 3.10-3.14 (m, 1H), 3.45 (dd, J = 4.0, 14.8 Hz,
1H), 3.61 (s, 2H), 4.41-4.43 (m, 2H), 4.39-4.42 (m, 3H), 7.20-
7.24 (m, 4H), 7.48 (d, J = 5.8 Hz, 1H), 7.87 (d, J = 12.1 Hz, 1H),
8.12 (s, 2H), 9.52 (b, 1H), 9.90-9.92 (m, 2H), 10.10 (b, 1H).
Single peak in analytical HPLC. LC-MS (ESI): m/z 452 [M þ 1]þ.
HRMS (ESI-Orbitrap), MHþ calcd for C25H30FN5O2, 452.2483;
found, 452.2454.
(R)-2-(Cyclopropylmethyl)-N-(2-(2-(dimethylamino)ethoxy)-
5-fluoro-4-(1H-pyrazol-4-yl)phenyl)-1,2,3,4-tetrahydroisoquino-
line-3-carboxamide (49). 1H NMR (MeOH-d4, 400 MHz) δ 0.49-
0.53 (m, 5H), 2.41 (m, 1H), 2.98 (s, 6H), 3.14 (m, 1H), 3.45 (m, 1H),
3.60 (s, 2H), 4.41-4.43 (m, 2H), 4.48-4.50 (m, 3H), 7.25-7.28 (m,
4H), 7.48 (d, J = 6.8 Hz, 1H), 7.87 (m, 1H), 8.10 (s, 2H), 10.00 (s,
1H). Single peak in analytical HPLC. LC-MS (ESI): m/z 478
[M þ 1]þ. HRMS (ESI-Orbitrap), MHþ calcd for C27H32FN5O2,
478.2639; found, 478.2613.
Acknowledgment. We thank Professors Patrick Griffin and
William R. Roush for their support. We are grateful to Dr.
Derek Duckett and Weimin Chen for p38 and JNK assays,
and to Dr. Michael Chalmers for HRMS. We acknowledge
the resources from the University of Miami Center for Compu-
tational Science.
(R)-N-(2-(2-(Dimethylamino)ethoxy)-4-(1H-pyrazol-4-yl)-
phenyl)-2-ethyl-1,2,3,4-tetrahydroiso-quinoline-3-carboxamide
Supporting Information Available: Compound purity data by
analytical HPLC for key compounds, chirality assignments by
1H NMR of Mosher amides for those pairs of R and S
enantiomers, and the kinase panel screening data for compound
35. This material is available free of charge via the Internet at
http://pubs.acs.org.
1
(51). H NMR (DMSO-d6, 400 MHz) δ 1.10 (t, 3H), 2.40 (m,
1H), 2.98 (s, 6H), 3.04 (dd, J = 11.4, 15.9 Hz, 1H), 3.35 (dd, J =
4.4, 15.9 Hz, 1H), 3.60 (s, 2H), 4.38-4.40 (m, 2H), 4.52-4.54 (m,
3H), 7.21-7.25 (m, 4H), 7.48 (d, J = 6.5 Hz, 1H), 7.87 (d, J =
12.0 Hz, 1H), 8.10 (s, 2H), 9.62 (b, 1H), 9.80 (b, 1H), 9.90-10.00
(m, 2H). Single peak in analytical HPLC. LC-MS (ESI): m/z 464
[M þ 1]þ. HRMS (ESI-Orbitrap), MHþ calcd for C26H33N5O3,
464.2683; found, 464.2655.
References
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ROCK-I/II Assays. Assays were performed using the STK2
kinase system from Cisbio. A 5 μL mixture of a 1 μM STK2
substrate and ATP (ROCK-I: 4 μM, ROCK-II: 20 μM) in STK-
buffer was added to the wells using a BioRAPTR FRD Work-
station (Aurora Discovery). Then 20 nL of test compounds was
dispensed. Reaction was started by the addition of 5 μL of 2.5
nM ROCK-I (Upstate no. 14-601) or 0.5 nM ROCK-II in STK-
buffer. After 4 h at rt, the reaction was stopped by addition of
10 μL of 1ꢀ antibody and 62.5 nM Sa-XL in detection buffer.
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PKA Assays. First, 5 μL mixture of a 60 μM kemptide and
20 μM ATP in kinase buffer (50 mM Hepes pH 7.3, 10 mM
MgCl2, 0.1% BSA, 2 mM DTT) were added to the wells using a
BioRAPTR FRD Workstation (Aurora Discovery). Then 20 nL
of test compounds was dispensed. Reaction was started by
addition of 5 μL of 0.5 nM PKA (Upstate no. 14-440) in kinase
buffer (5 μL of kinase buffer for high wells). After 70 min at rt,
the reaction was stopped by addition of 10 μL of Kinase-Glo
reagent and the plate was read after 10 min incubation time at rt
on the Viewlux in luminescence mode.
ppMLC Cell-Based Assays. Detection of Bisphospho-MLC2-
Modulation by In-cell Western using the LI-COR Odyssey Infrared
Imaging System. Bisphosho-MLC2 levels in A7r5 cells (purchased
from ATCC, Manassas, VA) was detected and quantified as pre-
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