Discovery of N-{N-[(3-cyanophenyl)sulfonyl]-4(R)-cyclobutylamino-(L)- prolyl}-4-[(3′,5′-dichloroisonicotinoyl)amino]-(L)-phenylalanine (MK-0668), an extremely potent and orally active antagonist of very late antigen-4

Article abstract of DOI:10.1021/jm900257b

Extremely potent very late antigen-4 (VLA-4) antagonists with picomolar, whole blood activity and slow dissociation rates were discovered by incorporating an amino substituent on the proline fragment of the initial lead structure. This level of potency ag

Full text of DOI:10.1021/jm900257b

J. Med. Chem. 2009, 52, 3449–3452
3449
Discovery of N-{N-[(3-Cyanophenyl)sulfonyl]-
4(R)-cyclobutylamino-(L)-prolyl}-4-[(3′,5′-
dichloroisonicotinoyl)amino]-(L)-phenylalanine
(MK-0668), an Extremely Potent and Orally
Active Antagonist of Very Late Antigen-4
Linus S. Lin,*,† Thomas Lanza, Jr.,† James P. Jewell,†
Ping Liu,† Carrie Jones,† Gerard R. Kieczykowski,†
Kelly Treonze,‡ Qian Si,‡ Salony Manior,‡ Gloria Koo,‡
Xinchun Tong,† Junying Wang,† Anne Schuelke,†
James Pivnichny,† Regina Wang,§ Conrad Raab,§
Stella Vincent,§ Philip Davies,‡ Malcolm MacCoss,†
Richard A. Mumford,‡ and William K. Hagmann†
Figure 1. Structure of a clinical VLA-4 antagonist.
Departments of Medicinal Chemistry, Immunology and
Rheumatology, and Drug Metabolism, Merck Research
Laboratories, Rahway, New Jersey 07065
ReceiVed March 1, 2009
Abstract: Extremely potent very late antigen-4 (VLA-4) antagonists
with picomolar, whole blood activity and slow dissociation rates were
discovered by incorporating an amino substituent on the proline
fragment of the initial lead structure. This level of potency against the
unactivated form of VLA-4 was shown to be sufficient to overcome
the poor pharmacokinetic profiles typical of this class of VLA-4
antagonists, and sustained activity as measured by receptor occupancy
was achieved in preclinical species after oral dosing.
Figure 2. Lead structures of tight binding VLA-4 antagonists.
Clinical proof of concept was first achieved with an anti-R4
neutralizing antibody, natalizumab, which has been shown to
be effective in human clinical trials of multiple sclerosis (MS)
and Crohn’s disease (CD) and has been approved by FDA for
both indications.8,9 At a monthly iv dose of 3 and 6 mg,
approximately 80% and 90% saturation, respectively, of R4ꢀ1
receptors (receptor occupancy or RO) on peripheral blood
lymphocytes was reported.8 Subsequently, a small molecule
VLA-4 antagonist, valategrast (R411) (Figure 1), was shown
to be beneficial for the treatment of asthma in a 12-week phase
II clinical trial.10-12 The active metabolite of valategrast
(RO0270608, Figure 1) was a dual R4ꢀ1-R4ꢀ7 antagonist with
high affinity for the activated states of both R4ꢀ1 and R4ꢀ7
integrins, but the affinity was lower and the dissociation was
rapid when the receptors were not activated.11
Previously, we identified a tight binding VLA-4 antagonist
1a (Figure 2) with picomolar affinity on the unactivated form
of VLA-4 along with a slow dissociation rate or off-rate.13
However, it suffered from poor bioavailability and a short half-
life in preclinical species. After iv dosing (0.5 mpk) in rats,
only 15% receptor occupancy was observed at 8 h postdosing.
On the basis of the reported experience with natalizumab, we
hypothesized that a small molecule antagonist would need to
achieve g80% RO at 12-24 h to be effective in the treatment
of MS and CD. Therefore, much improvement in whole blood
activity and/or pharmacokinetics over 1a would be necessary
to achieve this level of RO. Because of the peptidic nature and
molecular weight of this lead, improving pharmacokinetic
properties was a significant challenge. However, if a compound
possessed very potent whole blood activity with a fast on-rate
and also had a very slow off-rate once bound, sustained receptor
occupancy might be achievable even after the drug was rapidly
eliminated from circulation.
VLA-4a (R4ꢀ1, CD49d/CD29) is a member of the integrin
family that is expressed on all lymphocytes.1 VLA-4 normally
exists at a resting state or unactivated state. Although the precise
mechanism of activation in vivo is not clear, VLA-4 can be
activated in vitro by divalent cation Mn2+ or activating
antibodies.2 Once activated, VLA-4 binds strongly to ligands
including (1) vascular cell adhesion molecule-1 (VCAM-1),
which is expressed on activated endothelial cells at sites of
inflammation and is produced in response to inflammatory
cytokines, and (2) the CS-1 domain of fibronectin (FN) which
arises by alternative splicing within a region of FN. The binding
interactions between VLA-4 and VCAM-1/FN are proposed to
be key mediators of cell-cell and cell-matrix adhesion. As
such, these cell adhesion interactions may be essential for the
activation, migration, proliferation, and differentiation of lym-
phocytes during normal and/or pathophysiological processes.
Thus, inhibition of VLA-4 may result in a reduction in the
migration and/or activation of cell types important to sustaining
a prolonged inflammatory response.3 Anti-R4 antibodies and
blocking peptides have been reported to be efficacious in a
number of animal models of inflammatory diseases, including
antigen-induced bronchial hyperresponsiveness,4 experimental
allergic encephalomyelitis,5 adjuvant arthritis,6 and spontaneous
chronic colitis.7
* To whom correspondence should be addressed. Phone: 732-594-8391.
Fax: 732-594-5966. E-mail: linus_lin@merck.com.
†
Department of Medicinal Chemistry.
Department of Immunology and Rheumatology.
Department of Drug Metabolism.
‡
§
a Abbreviations: VLA-4, very late antigen-4; VCAM-1, vascular cell
adhesion molecule-1; FN, fibronectin; MS, multiple sclerosis; CD, Crohn’s
disease; RO, receptor occupancy; LOQ, limit of quantitation; TDI, time
dependent inhibition.
To test this hypothesis, we sought to improve the whole blood
activity and the off-rate of lead structure 1a. Compound 1a is
a carboxylic acid and is highly bound to human plasma proteins
10.1021/jm900257b CCC: $40.75
2009 American Chemical Society
Published on Web 05/14/2009

3450 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Letters
Table 1. In Vitro Potency of VLA-4 Antagonists14
Scheme 1a
a Data expressed as mean ( SD (n g 3 independent experiments) or
single value (n ) 1).
slightly diminished off-rate (4 vs 1b). Alkyl substitutions on
the amino group dramatically enhanced the off-rate while
maintaining potent whole blood activity (9a vs 4). This effect
appeared to be sensitive to the stereochemistry at C-4 as the
other diastereomer 9b had poor affinity and a faster off-rate,
suggesting that the 4-alkylamino substituent was involved in
specific interactions with VLA-4. Slight increases in off-rate
were observed by increasing the ring size of the cyclic amino
group with minimal impact on the whole blood activity (9c,d
vs 9a). Monosubstituted amino group was also effective in
enhancing VLA-4 affinity and off-rate, as reflected by 9e.
Compound 9a was selected to be profiled in vivo. Thus, 9a
was dosed iv (0.5 mg/kg) in rats and blood samples were
collected at different time points postdosing. The RO of VLA-4
on the circulating lymphocytes was assessed by competitive
binding with a radiolabeled VLA-4 ligand.16 In the same study,
the plasma concentration of 9a was also determined by LC-MS.
As summarized in Table 2, administration of 9a resulted in 64
( 2% RO at 8 h postdosing.15,16 As expected on the basis of
experience with this class of compounds,13 9a exhibited rapid
plasma clearance and a short plasma half-life (t1/2 < 1 h). No
9a was detectable beyond 2 h (LOQ < 0.1 nM). Compound 9a
was further evaluated in dogs and rhesus monkeys using a
similar protocol. In dogs, RO of 72 ( 6% was observed at 8 h
postdosing (0.75 mg/kg iv) while no compound was detectable
beyond 1 h. Similarly, RO of 87 ( 6% was observed at 8 h
postdosing in rhesus monkeys (0.75 mg/kg iv) while the
concentration of 9a dropped below the limit of detection at 6 h.
The peak RO of 9a reached 100% in all three species. These
results suggested that sustained RO can be achieved despite the
high clearance and low plasma concentration when the com-
pound possesses exceptional affinity to the receptor.15
a (a) HCl, dioxane; (b) 3,5-Cl2-PhSO2Cl, Et3N, DMAP (cat.), CH2Cl2/
THF; (c) LiOH, MeOH/THF/H2O; (d) P3, PyBOP, i-Pr2NEt, CH2Cl2; (e)
Me3P, THF; (f) Tf2O, i-Pr2NEt, CH2Cl2, -40 °C, 1 h; R2NH, -20 °C to
room temp, 16 h; (g) LiOH, CH3CN, H2O; (h) P3, EDC, HOBt, CH2Cl2,
H2O; (i) ArSO2Cl, Et3N, DMAP (cat.), CH2Cl2/THF.
(i.e., albumin), so the introduction of an amino group was
pursued. The syntheses of these amino substituted compounds
were well precedented, and the synthetic sequence is outlined
in Scheme 1. The key transformation was from 5 to 6 via a
stereospecific SN2 displacement of the triflate of readily available
(4S)- or (4R)-hydroxyproline by an amine nucleophile.
The affinity of these new compounds was assessed by
competitive binding to human VLA-4 in buffer and in whole
blood without addition of activating reagents. The dissociation
kinetics of these tight binding antagonists was also evaluated
at 37 °C and was expressed as % bound at a specified time.
The data are summarized in Table 1.13,14 As reported previ-
ously,13 removing the R-methyl group of the proline fragment
reduced protein binding and improved whole blood activity, but
it had little effect on the off-rate (1b vs 1a). Incorporation of
an unsubstituted amino group at the C-4 position of proline
further improved the whole blood activity as expected, presum-
ably because of reduced plasma protein binding, but it led to a

Letters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3451
Table 2. Receptor Occupancy and Pharmacokinetic Parameters of VLA-4 Antagonists16
compd
species
dose (mg/kg)
route
RO at X h (%)a
X (h)b
Clp ((mL/min)/kg)c
t1/2 (h)c
tmin (h)d
9a
9a
9a
9a
9a-Et
9e-Et
9f
9f
9f
rat
dog
monkey
rat
rat
rat
rat
dog
monkey
rat
0.5
0.75
0.75
1
1
1
5
5
5
5
iv
iv
iv
64 ( 2
72 ( 6
87 ( 6
46 ( 12
62 ( 16
51 ( 5
67 ( 11
83 ( 8
88 ( 1.8
77 ( 13
97 ( 7
95 ( 4
8
8
8
8
8
8
12
12
8
12
12
8
65
29
33
ND
ND
ND
65
29
33
1.0
0.5
1.1
ND
ND
ND
1.0
0.5
1.1
ND
ND
ND
2
1
6
ND
ND
ND
0
6
0
po
po
po
po
po
po
po
po
po
9f-Et
9f-Et
9f-Et
ND
ND
ND
0
12
0
dog
monkey
5
5
a % receptor occupancy at X h. b Time when receptor occupancy was measured. c Clp and plasma half-life were determined after iv dose. d Latest time
point when 9a or 9f could be detected (LOQ ≈ 0.1 nM).
Table 3. Cytochrome P450 Inhibition and clogP of VLA-4
0.13 ( 0.03 nM, dog 0.19 ( 0.01 nM, rhesus 0.21 nM, rat 0.4
( 0.1 nM; off-rate at 37 °C on human VLA-4, 64 ( 10% bound
Antagonists17
at 3 h). When evaluated in vivo (5 mg/kg po), 9f-Et demon-
strated sustained RO of 77-97% at 8-12 h postdosing across
three species (Table 2). As a comparison, the acid 9f was also
studied and afforded somewhat lower levels of RO (67-88%
at 8-12 h postdosing). Analogous to other compounds in the
series, 9f/9f-Et displayed poor pharmacokinetic properties in
the preclinical species (F < 1%, t1/2 e 1 h). Nevertheless, RO
was maintained even after no compound was detectable in the
plasma, consistent with slow dissociation rate of 9f from VLA-4
receptors. If similar PK/PD relationship holds in humans, with
a twice-daily oral dosing schedule, 9f or its prodrug 9f-Et would
compd
X, Y
CYP3A4 IC50 (µM) TDI rate (min-1
)
clogP a
be expected to achieve RO coverage comparable to that of
intravenously dosed natalizumab.
9e-Et
9f-Et
Cl, Cl
CN, H
<0.4
0.05
0.04
5.5
3.5
1.7, 2.4
In summary, extremely potent VLA-4 antagonists with
picomolar, whole blood activity and slow off-rates were
discovered by incorporating an amino substituent on the proline
fragment of the initial lead structure. This level of potency was
shown to be sufficient to overcome the poor pharmacokinetic
profiles typical of this class of VLA-4 antagonists, and sustained
activity as measured by receptor occupancy was achieved after
oral dosing.22
a Calculated by ACD8.1 software.
Compound 9a and its ethyl ester 9a-Et were then studied via
oral dosing in rats. Receptor occupancies of 46 ( 12% and 62
( 16%, respectively, were observed at 8 h postdosing (1 mg/
kg). The ethyl ester of 9e (9e-Et) was also studied by oral dosing
(1 mg/kg) in rats and afforded comparable RO (51 ( 5% at 8 h
postdosing). Although still moderate in comparison with clinical
observations of natalizumab, the level of oral activity of 9a/
9a-Et and 9e-Et as assessed by receptor occupancy represented
a breakthrough in the search for an orally active small molecule
antagonist of the unactivated form of VLA-4.
Acknowledgment. We acknowledge the contributions of
many members of the Labeled Synthesis Group and the
Laboratory of Animal Resources of Merck Research Laboratories.
Note Added after ASAP Publication. The paper was published
on May 14, 2009 with typographical errors. The revised version
was published on May 20, 2009.
Because the azetidine ring of 9a/9a-Et was found to be acid
labile, 9e/9e-Et was selected for additional characterization
toward the identification of a development candidate. Metabo-
lism studies revealed that 9e-Et is a potent and time-dependent
inhibitor (TDI) of CYP3A4 with a TDI rate of 0.05 min-1 (Table
3).17 The parent acid 9e also displayed a propensity for
bioactivation upon incubation with liver microsomes, and
covalent adducts were formed with microsomal proteins after
incubation for 1 h (rat, 108 pmol/mg; human, 150 pmol/mg).18
Because CYP inhibition and covalent adduct formation pose
significant risks of drug-drug interactions and idiosyncratic
toxicities, respectively19,20 alternative structures were sought.
It is well-known that CYP inhibition is often associated with
high lipophilicity.21 Replacing the 3,5-dichlorophenyl group of
9e-Et with a 3-cyanophenyl group (9f-Et) resulted in significant
reduction in CYP3A4 inhibition, while the clogP value de-
creased from 5.5 to 3.5. Furthermore, 9f also demonstrated
decreased levels of covalent adduct formation when incubated
with microsomal preparations (rat, 39 ( 2 pmol/mg; human,
56 ( 2 pmol/mg).
Supporting Information Available: Synthetic procedures and
analytical data of VLA-4 antagonists and conditions for all the
biological assays. This material is available free of charge via the
Internet at http://pubs.acs.org.
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JM900257B