Discovery and development of LX7101, a dual LIM-kinase and ROCK inhibitor for the treatment of glaucoma

Article abstract of DOI:10.1021/ml500367g

The structure of LX7101, a dual LIM-kinase and ROCK inhibitor for the treatment of ocular hypertension and associated glaucoma, is disclosed. Previously reported LIM kinase inhibitors suffered from poor aqueous stability due to solvolysis of the central urea. Replacement of the urea with a hindered amide resulted in aqueous stable compounds, and addition of solubilizing groups resulted in a set of compounds with good properties for topical dosing in the eye and good efficacy in a mouse model of ocular hypertension. LX7101 was selected as a clinical candidate from this group based on superior efficacy in lowering intraocular pressure and a good safety profile. LX7101 completed IND enabling studies and was tested in a Phase 1 clinical trial in glaucoma patients, where it showed efficacy in lowering intraocular pressure. (Figure Presented).

Full text of DOI:10.1021/ml500367g

Letter
pubs.acs.org/acsmedchemlett
Discovery and Development of LX7101, a Dual LIM-Kinase and ROCK
Inhibitor for the Treatment of Glaucoma
Bryce A. Harrison,*,†,⊥ Zheng Y. Almstead,† Hugh Burgoon,† Michael Gardyan,† Nicole C. Goodwin,†
Jason Healy,† Ying Liu,† Ross Mabon,† Brett Marinelli,† Lakshman Samala,† Yulian Zhang,†
Terry R. Stouch,† N. Andrew Whitlock,∥ Suma Gopinathan,§ Beth McKnight,∥ Shuli Wang,‡ Nita Patel,‡
Alan G. E. Wilson,§ Brian D. Hamman,‡ Dennis S. Rice,∥ and David B. Rawlins†
†Department of Medicinal Chemistry, Lexicon Pharmaceuticals, 350 Carter Road, Princeton, New Jersey 08540, United States
‡Department of Pharmaceutical Discovery, §Department of Drug Metabolism, Pharmacokinetics, and Toxicology and ∥Department of
Ophthalmology, Lexicon Pharmaceuticals, 8800 Technology Forest Place, The Woodlands, Texas 77381, United States
S
* Supporting Information
ABSTRACT: The structure of LX7101, a dual LIM-kinase and
ROCK inhibitor for the treatment of ocular hypertension and
associated glaucoma, is disclosed. Previously reported LIM kinase
inhibitors suffered from poor aqueous stability due to solvolysis of
the central urea. Replacement of the urea with a hindered amide
resulted in aqueous stable compounds, and addition of
solubilizing groups resulted in a set of compounds with good
properties for topical dosing in the eye and good efficacy in a
mouse model of ocular hypertension. LX7101 was selected as a
clinical candidate from this group based on superior efficacy in
lowering intraocular pressure and a good safety profile. LX7101
completed IND enabling studies and was tested in a Phase 1
clinical trial in glaucoma patients, where it showed efficacy in lowering intraocular pressure.
KEYWORDS: LX7101, glaucoma, kinase, ROCK inhibitor, LIM-kinase
IM-kinases 1 and 2 (LIMK1 and LIMK2) regulate
Lcytoskeletal dynamics by phosphorylating and deactivating
cofilin, a protein that depolymerizes actin filaments.1 As
cytoskeletal regulators, LIM-kinases have been investigated
for their role in a variety of cellular processes2 including cell
barrier integrity,3 axonal elongation,4 cell migration,5 and cell
cycle arrest6 as they pertain to various disease states such as
HIV infection7 and cancer.8 Along with growing interest in
these kinases, a number of LIMK inhibitors have also been
reported.9−11 We recently reported the discovery of a novel
class of pyrrolopyrimidine LIMK inhibitors12 and demonstrated
their use for the reduction of intraocular pressure (IOP) in a
mouse model of ocular hypertension.13
Ocular hypertension is associated with open-angle glaucoma,
a neurodegenerative disease of the inner retina and optic nerve
head and a leading cause of blindness worldwide.14 Although
many therapies exist for the treatment of glaucoma through the
lowering of IOP, many patients fail to adequately control IOP
or experience significant side effects with current medications,
indicating a need for new, more efficacious therapies.15
body, flows through the anterior chamber, and exits through
trabecular meshwork. In patients suffering from ocular
hypertension, structural changes in the trabecular meshwork
can result in decreased outflow facility, resulting in an increase
in pressure.16 Relaxation of the trabecular meshwork with
LIMK inhibitors could counteract this effect, directly reversing
the cause of ocular hypertension.
Several inhibitors of ROCK, a kinase upstream of LIMK in
the signaling cascade that regulates actin filament dynamics, are
being investigated in the clinic for reduction of IOP through
relaxation of the trabecular meshwork. These compounds
effectively lower IOP; however, they are associated with
significant side effects, particularly conjunctival hyperemia,
which may limit their use.17 Since LIM-kinases are downstream
from ROCK in the signaling cascade and their effects are more
specific to actin polymerization, inhibitors of LIMK may reduce
IOP with fewer side effects than inhibitors of ROCK.
Our previous report detailed the discovery of a class of
pyrrolopyrimidine LIMK inhibitors such as compound 1
LIMK inhibitors are believed to lower IOP by inducing
depolymerization of actin filaments in the trabecular meshwork
in the eye, resulting in relaxation of the tissue. Pressure in the
eye is regulated by the fluid dynamics of aqueous humor in the
anterior chamber. Aqueous humor is produced by the ciliary
Special Issue: New Frontiers in Kinases
Received: September 9, 2014
Accepted: November 24, 2014
Published: November 24, 2014
© 2014 American Chemical Society
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4 °C in 1:1 MeOH/H2O at pH 7.2, HPLC purity of 1 still
decreased 1−2% over 5 days. Since topical ophthalmic
formulations generally require 12 months stability,18 we did
not believe this level of stability to be sufficient. Removal of the
MeOH cosolvent also prevented degradation, even at 60 °C;
however, without MeOH as a cosolvent, the compound was not
soluble in water, so these conditions were not helpful for
topical ophthalmic dosing. Additionally, replacement of MeOH
with MeCN or removal of the buffer salts had only a small
stabilizing effect on the degradation of 1. Finally, the effect of
pH on compound stability was tested. Acidifying the solution to
pH 5 had minimal effect, while basifying the solution with
NaOH dramatically decreased stability. On the basis of these
results, various compounds were tested for stability in 1:1
H2O/MeOH at pH 7.2 at 60 °C for 24 h to establish the
structure−activity relationship (SAR) of the solvolysis reaction.
Modifications of the aniline portion of the molecule failed to
produce stable, active compounds (Table 2). Substitution of
Figure 1. Observed products from solvolysis of compound 1.
(Figure 1). These compounds potently and selectively inhibit
LIMK1 and LIMK2 in both in vitro and cell-based assays.
Administration of an aqueous formulation of 1 to the eyes of
ocular hypertensive mice resulted in reduced IOP.
Table 2. Modifications of Aniline
In the process of investigating aqueous formulations of 1, we
discovered that the compound slowly decomposes in aqueous
solution (Figure 1). For example, when 1 was dissolved in pH
7.2 phosphate buffered H2O/MeOH at 60 °C, high-perform-
ance liquid chromatography (HPLC) purity of the compound
decreased to 84% after 6 h and 58% after 24 h. Analysis of the
decomposition products by liquid chromatography−mass
spectrometry (LC−MS) revealed the formation of 2, 3, and
4, resulting from solvolysis of the central urea. The presence of
3 indicates reactivity of the bond between the piperazine
nitrogen and the carbonyl, and no corresponding product was
observed from the methanolysis of the aniline carbonyl bond.
Various conditions were examined to better understand this
unusual reactivity (Table 1). At lower temperatures, the
decomposition of 1 was greatly delayed; however, at 20 and
Table 1. Aqueous Instability of Compound 1 under Various
a
Conditions
a
Stability tested at 2 mg/mL in 1:1 H2O/MeOH at 60 °C for 24 h.
Purity evaluated by HPLC.
the aryl ring with electron donating groups only slightly
improved stability (compounds 5−7). Replacement of the
aniline nitrogen with a methylene produced a stable compound
(8) but resulted in a 500-fold reduction in activity for LIMK2.
Benzyl ureas 9 and 10 also showed improved stability, but lost
8−150-fold activity.
Modifications of the piperazine were examined as well (Table
3). Replacement of the piperazine nitrogen attached to the
pyrimidine with a methylene resulted in a small increase in
stability (12 compared to 11). Larger effects on the stability of
1 resulted from changes to the substitution alpha to the
piperazinyl urea nitrogen. Removal of the alpha methyl (11)
a
b
Stability of 1 tested at 2 mg/mL in 1:1 H2O/cosolvent. Purity
evaluated by HPLC using 10 mM aq. NH4OAc/MeCN gradient on a
4.6 × 50 mm 4.6 μm C18 column with 220 nm UV detection.
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a
Table 3. Modifications of Piperazine
Table 4. Aqueous Stable Amides
a
Stability tested at 2 mg/mL in 1:1 H2O/MeOH at 60 °C for 24 h.
b
Purity evaluated by HPLC. Not calculated.
improved stability to 88%, and addition of a second methyl
(13) decreased stability to 0.4%. Interestingly, replacement of
the methyl with tert-butyl (14) had a small stabilizing effect on
the compound, perhaps due to steric hindrance of nucleophilic
attack. The effect of alpha substitution on compound stability
could be attributed to steric destabilization of the planar,
conjugated conformation of the piperazinyl N with the urea
carbonyl, rendering the C−N bond susceptible to solvolysis.
This hypothesis was supported by molecular modeling done
on compound 7.19 Although many conformations of similar
energy were observed, the lowest energy conformation of the
piperazine places the methyl substituent in an axial con-
formation that accommodates the planar, conjugated con-
formation of the nitrogen and carbonyl. We hypothesize that
the energetic cost of this axial orientation might provide a
driving force for breaking conjugation, resulting in compound
instability.
To test the hypothesis empirically, we prepared compound
15. This compound was linked from the piperazine ring to the
aniline nitrogen enforcing a planar, conjugated conformation.
Not surprisingly, this compound showed no degradation in the
stability assay, but it was also completely inactive at LIMK2.
This result suggested that the planar conformation of the
urea was not the active one. Consequently, we returned to
looking at amides of the type found in Table 4, in which the
piperazine nitrogen was replaced by a carbon. With
replacement of the labile nitrogen carbonyl bond with a stable
carbon carbonyl bond, all amides of this form showed complete
stability in our assay for up to 3 days. We reported in our
previous communication amides of this type but had not
evaluated them more fully because unsubstituted analogues
such as 16 had low activity.
a
All compounds stable at 2 mg/mL in 1:1 H2O/MeOH at 60 °C for
24 h. Purity evaluated by HPLC.
the analogous position to compound 1 did not sufficiently
improve potency (17 and 18). However, substitution alpha to
the carbonyl did dramatically improve potency with a variety of
substituents at the apha position and on the aniline phenyl ring
(19−22). These compounds were aqueous stable and showed
in vivo activity in the mouse IOP assay, but poor aqueous
solubility made it difficult to achieve topical formulation.
The addition of solubilizing groups to the position alpha to
the carbonyl improved solubility while generally maintaining
good LIMK2 activity (23−28). We compared many com-
pounds of this type formulated as 0.1% HPMC based aqueous
solutions in the ocular hypertensive mouse model. Results for
compounds 25−28, along with selectivity for ROCK, are
shown in Table 5. Compound 25 had the best selectivity for
ROCK, but only moderate efficacy in lowering IOP.
Compounds 26 and 27 had greater efficacy, perhaps due to
improved aqueous solubility. Following this trend, compound
28, the most soluble compound (up to 10 mg/mL in HPMC
based vehicle) exhibited the best efficacy at lowering IOP,
although it was only moderately selective versus ROCK at low
ATP concentrations. However, when the in vitro binding assays
Consequently, we prepared analogues with substitution on
the piperidine hoping to improve the potency. Substitution in
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Table 5. Profiles of Key Compounds
Figure 2. Change in intraocular pressure in dexamethasone induced
ocular hypertensive mice following topical installation of 3 μL of 5
mg/mL HPMC based aqueous solution of 28, a 5 mg/mL formulation
of timolol, or a 0.05 mg/mL formulation of latanoprost.
a
b
IC50 values at 2 μM ATP, in parentheses at 200 μM ATP. Reduction
in intraocular pressure at 2 h postdose in dexamethasone induced
ocular hypertensive mice following topical instillation of 3 μL of 1 mg/
mL HPMC based aqueous solution of compound, compared to
vehicle.
additional reduction of IOP (5.0 mmHg total reduction)
compared to the 0.1% formulation and demonstrated a long
duration of action, with IOP not returning to baseline until
more than 8 h postdose. More critically, at these doses, 28
produced a significantly greater reduction of IOP than either
timolol or latanoprost.
On the basis of these results, we selected compound 28 as
clinical candidate LX7101. IND enabling studies were
completed for the compound, and it was evaluated in a phase
1 clinical trial in glaucoma patients. The compound was well
tolerated and showed significant reductions in IOP in these
patients. The results of these studies will be published at a
future time.
In conclusion, we have disclosed the structure of clinical
compound LX7101 (28) for the treatment of ocular hyper-
tension and associated glaucoma. This compound is a potent
inhibitor of LIM kinase 2, although we cannot rule out the
possibility that some of the observed activity is due to weak
inhibition of ROCK. LX7101 was derived from compound 1 by
replacing the central piperazine ring of 1 with an amino-
methylene substituted piperidine. The modifications were
necessary to rectify the unusual aqueous instability of the
central urea of 1 and to increase aqueous solubility. These
changes produced a compound with high efficacy in a mouse
model of ocular hypertension that, when progressed to phase 1
clinical trials, lowered IOP in glaucoma patients.
were repeated at high ATP concentrations, compound 28
proved significantly more selective for LIMK2 (300-fold vs
ROCK1 and 45-fold vs ROCK2). As ATP has a relatively weak
affinity for LIMK2 (Km = 85 μM) compared to ROCK 1 and 2
(Km = <20 μM), it is not surprising that the overall selectivity of
compound 28 for LIMK2 increases at the higher physiological
ATP concentrations.20 Consequently, we believe that under
physiological conditions, the activity of 28 is primarily due to
inhibition of LIMK2. The activity of 28 was slightly less than
that observed with ROCK inhibitor Y-39983 in the same
assay.13
In addition, compound 28 was screened against a panel of
403 kinases at Ambit Biosciences (now DiscoverRx). Moderate
selectivity was observed in this screen (34 assays including
LIMK2 and ROCK2 indicated that the Kd is most likely <1 μM;
data not shown). Compound 28 was also evaluated in a panel
of binding assays including 78 different receptors and
transporters at Cerep where no significant cross reactivity was
observed (only 5 assays had >60% inhibition at 10 μM; data
not shown). On the basis of these results, we were unable to
rule out the possibility that the improved efficacy of 28 relative
to other LIMK inhibitors was due to inhibition of ROCK or
other kinases in addition to LIMK2. However, the improved
efficacy of 28 could also be from improved solubility, resulting
in increased exposure on topical dosing (see Supporting
Information for topical PK of 28). Unfortunately, we were not
able to find equally soluble compounds with high LIMK2
selectivity and so were unable to resolve this question.
However, we considered that tolerability was more critical
than selectivity, and consequently, topical doses of 28 were
evaluated for tolerability on the eyes of mice, rats, and rabbits.
The compound was found to be well tolerated at doses up to
0.5% in non-GLP single dose studies. In rats, systemic exposure
following topical application of 28 was below limits of
quantization for a single dose. Repeat topical dosing of 28
gave a systemic Cmax of 12−19 nM, which was well below the
exposure of the NOAEL oral dose (500 mpk). Stability testing
of 28 showed 99% stability in an aqueous solution formulation
after 14 days at 60 °C.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details and analytical data of the compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone 617-252-5328. E-mail: bharrison@forumpharma.com.
■
Present Address
⊥Department of Medicinal Chemistry, FORUM Pharmaceut-
icals, 500 Arsenal Street, Watertown, Massachusetts 02472,
United States.
Finally, a well-tolerated dose of compound 28 (0.5%) was
compared with the standard clinical doses of commercial
glaucoma drugs timolol (0.5%) and latanoprost (0.005%) in the
mouse IOP assay (Figure 2). At this dose, 28 achieved
Notes
The authors declare no competing financial interest.
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of Drug Substances and Products. Questions and Answers (FDA
Maryland).
(19) The modeling was done with the CFF98 Force Field within the
Discover and Insight programs (Biovia, Inc. (previously Accelrys, Inc.),
San Diego, CA) and confirmed with ICM (Molsoft, LLC, San Diego,
CA) employing the MMFF force field.
(20) Goodwin, N. C.; Cianchetta, G.; Burgoon, H. A.; Healy, J.;
Mabon, R.; Strobel, E. D.; Allen, J.; Wang, S.; Hamman, B. D.;
Rawlins, D. B. Discovery of a type III inhibitor of LIM kinase 2 that
binds in a DFG-out conformation. ACS Med. Chem. Lett. 2014,
DOI: 10.1021/ml500242y.
ABBREVIATIONS
■
LIMK2, LIM-kinase 2; ROCK, Rho-associated protein kinase;
IOP, intraocular pressure; HPMC, hydroxypropyl methylcellu-
lose; NOAEL, no observable adverse event level; AUC, area
under the curve; Cmax, maximum concentration; IND,
investigational new drug application
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