Small macrocycles as highly active integrin α2β1 antagonists

Article abstract of DOI:10.1021/ml4004556

Starting from clinical candidates Firategrast, Valategrast, and AJM-300, a series of novel macrocyclic platelet collagen receptor α2β1 antagonists were developed. The amino acid derived low molecular weight 14-18-membered macrocycles turned out to be highly active toward integrin α2β1 with IC₅₀s in the low nanomolar range. The conformation of the macrocycles was found to be highly important for the activity, and an X-ray crystal structure was obtained to clarify this. Subsequent docking into the metal-ion-dependent adhesion site (MIDAS) of a β1 unit revealed a binding model indicating key binding features. Macrocycle 38 was selected for further in vitro and in vivo profiling.

Full text of DOI:10.1021/ml4004556

Letter
pubs.acs.org/acsmedchemlett
Small Macrocycles As Highly Active Integrin α2β1 Antagonists
Nis Halland,* Horst Blum, Christian Buning, Markus Kohlmann, and Andreas Lindenschmidt
Sanofi R&D, Industriepark Hochst Building G838, D-65926 Frankfurt am Main, Germany
̈
S
* Supporting Information
ABSTRACT: Starting from clinical candidates Firategrast,
Valategrast, and AJM-300, a series of novel macrocyclic platelet
collagen receptor α2β1 antagonists were developed. The
amino acid derived low molecular weight 14−18-membered
macrocycles turned out to be highly active toward integrin
α2β1 with IC50s in the low nanomolar range. The
conformation of the macrocycles was found to be highly
important for the activity, and an X-ray crystal structure was
obtained to clarify this. Subsequent docking into the metal-ion-
dependent adhesion site (MIDAS) of a β1 unit revealed a binding model indicating key binding features. Macrocycle 38 was
selected for further in vitro and in vivo profiling.
KEYWORDS: Integrin, macrocycle, platelet collagen receptor, metathesis, restricted conformation
ntegrins are a family of adhesion molecules responsible for
Itransmembrane signaling by undergoing conformational
rearrangements. They are involved in a wide range of biological
processes, e.g., angiogenesis, inflammation, cancer, and hemo-
stasis, and are therefore highly interesting drug targets.
Integrins are pairs of noncovalently bound heterodimers,
where 18α and 8β units are known to form 24 α/β
combinations, making them a functionally and structurally
diverse target class.1−4 We were particularly interested in the
biological effects of blocking the integrin α2β1 as this can lead
to several therapeutic effects, e.g., antithrombotic5,6 or
antiangiogenic.7 Our approach toward an orally available
small-molecule α2β1 antagonist started with the reported
α4β1 (VLA-4) and α4β7 dual antagonists Firategrast,8
Valategrast,9 and AJM-30010 since analogues of these
compounds exhibit high activities toward platelet collagen
receptor α2β1 (in-house data). As shown in Figure 1, these
compounds contain a common L-phenylalanine-N-aroyl motif
where the carboxylic acid is responsible for binding to the metal
ion in the metal-ion-dependent adhesion site (MIDAS).11 In
order to obtain novel compounds in this highly competitive
field we decided to prepare macrocyclic analogues of Firategrast
and Valategrast and test them for integrin α2β1 activity. Our
strategy was to prepare small macrocycles by using O-allylated
L-tyrosine analogues and benzoic acids and subjecting them to
ring closing metathesis conditions as this should afford 14−18-
membered macrocycles in a highly efficient way in a limited
number of steps. Furthermore, the planned macrocycles were
expected to have a considerable structural rigidity, improved
physicochemical properties, and be Lipinski rule-of-five
compatible and thus possibly allowing for oral application.12
The 6−7 step synthetic route to the macrocycles is exemplified
by the synthesis of compounds 6 and 7 in Scheme 1. First, the
commercially available N-Boc protected and O-allylated (s)-
tyrosine isomer was deprotected and esterified in one pot to
Figure 1. Clinical candidates Firategrast (MS, asthma, and Crohns
disease) and Valategrast (MS, RA, and asthma), both discontinued,
and AJM-300 (phase II, ulcerative colitis).
afford 1 in 88% isolated yield. The unprotected 2-chloro-5-
hydroxybenzoic acid was selectively allylated in 76% yield with
Received: November 12, 2013
Accepted: January 8, 2014
© XXXX American Chemical Society
A
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To our delight, the tested macrocycles showed high activities
of IC50 = 163 and 114 nM, respectively. This encouraged us to
prepare regioisomeric macrocycles 8−16 using the same
synthetic route. As shown in Table 1, attaching the linker at
Scheme 1. General Synthetic Route to Macrocycles As
Exemplified by the Synthesis of 6 and 7
a
Table 1. SAR of Macrocyclic Hydroxyphenylalanines/
Tyrosine Regioisomers 6−16
phenylalanine
linker pos.
benzamide
linker pos.
alkene/
saturated
IC50
(μM)
compd
valategrast−
0.214
OH
6
3
3
3
3
3
3
2
4
4
4
4
4
4
3
3
2
2
3
2
2
3
3
alkene
0.163
0.114
0.233
0.142
0.588
0.978
>4
7
saturated
alkene
8
9
saturated
alkene
10
11
saturated
saturated
alkene
a
12
13
14
15
16
0.178
0.106
0.101
0.731
saturated
alkene
saturated
a
Racemic compound.
a
Reactions and conditions: (a) AcCl, MeOH, reflux, 88%. (b)
the meta position of the phenylalanine and the other end to the
3 or 4 position of the benzamide afforded highly active
macrocyclic integrin α2β1 antagonists (compounds 6−9) with
activities in the IC50 = 100−200 nM range. Attachment to the
benzamide 2 position afforded somewhat less active com-
pounds (compounds 10, 11), but activity was regained by
moving the linker from the meta position to the para position
of the phenylalanine/tyrosine (macrocycles 13−16), and
compound 15 was found to be the most active macrocycle
with an activity on α2β1 of IC50 = 101 nM.
The nature of the linker, n-butyl or 2-butenyl, seemed only to
have a minor effect on the α2β1 activity in most macrocycles,
except for the pair 15 and 16, where a 7-fold decrease in activity
was observed upon saturating the linker. It has previously been
reported that at least one ortho substituent is required on the
benzamide moiety in order to obtain activity on integrins α4β1
and α4β7 as the substituent twists the conformation of the
amide out of the plane and thus structurally mimics the natural
RGD peptide substrates.9,15,16 This turned out to be true for
integrin α2β1 as well, as all attempts to replace the chlorine
atom in the 6-position afforded much less active compounds as
shown in Table 2. Replacing the chlorine atom with smaller
substituents, e.g., a hydrogen or fluorine atom as in Firategrast
afforded poorly active compounds with IC50 > 1 μM
(compounds 17, 20−23). Methyl substitution as in Valategrast
afforded moderately active macrocycles 18 and 19 that were 3−
7 times less potent than their corresponding chloro substituted
analogues 8 and 9. The 6-nitro substituted macrocycle 25
afforded a moderate activity of IC50 = 667 nM, whereas the 6-
methoxy substituted macrocycle 24 and the CF3-substituted
nicotinamide 26 were only weakly active. Because of the
unsuccessful attempt to introduce other substituents all further
Allylbromide, Bu4POH (40% aq), THF, rt, 76%. (c) HOBt, EDC,
DIPEA, DMF, rt, 72%. (d) Hoveyda−Grubbs second generation
catalyst, DCM, reflux, 3 h, 87%. (e) PtO2, H2 (1 bar), THF-MeOH, rt,
99%. (f) NaOH, THF−water, rt, 20 h, 78%.
allylbromide using tetrabutylphosphonium hydroxide as the
base.13
The two allylated fragments were condensed using standard
peptide coupling procedures to obtain the precursor 3 for the
ring closing metathesis step. After screening a number of
catalysts it was found that the relatively stable second
generation Hoveyda−Grubbs catalyst in refluxing CH2Cl2
afforded the cyclized metathesis product 4 in 87% yield when
the concentration was kept below 2 mM. The cis/trans
selectivity was very high as expected, favoring formation of the
trans double bond. Hydrolysis of the methyl ester provided the
unsaturated macrocyclic carboxylic acid 6.
The saturated macrocyclic carboxylic acid 7 was obtained by
reducing the alkene with hydrogen (1 bar) and a PtO2 or Rh/C
catalyst after the ring closing metathesis step. The macrocycles
6 and 7 were tested in a collagen-interaction solid phase assay,
mimicking the adhesion of α2β1-expressing cells with the
ECM, for activity on integrin α2β1. The recombinantly
expressed extracellular part of the collagen-binding integrin
α2β1, was coated to 96-well plates and incubated with
biotinylated collagen in the presence or absence of serial
dilutions of the macrocycles.14 The readout was via a
peroxidase reaction with the substrate ABTS. The absorbance
was determined in a microplate reader at 405 nm. The
percentage of inhibition of the macrocycles was calculated and
reported as IC50 values (μM).
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Table 2. Replacement of 6-Chloro Substituent in Meta-
Linked Phenylalanines
Table 3. Heteroaromatic Replacement of Phenyl in
Phenylalanine
compd
R
benzamide linker pos. alkene/saturated IC50 (μM)
17
18
19
20
21
22
23
24
25
H
3
3
3
4
4
2
2
2
3
4
saturated
saturated
alkene
>10
0.745
1.05
>1
Me
Me
F
alkene
F
saturated
alkene
>4
F
>1
F
saturated
saturated
saturated
alkene
>1
OMe
NO2
CF3
>4
0.667
1.9
a
26
a
Nicotinamide (N in 3-position) was used instead of benzamide.
macrocycles were prepared with a 6-chloro substituent on the
benzamide moiety. We then focused on reducing lipophilicity
and plasma protein binding by replacing the phenylalanine with
other amino acids carrying heteroaromatic rings such as
tryptophan or histidine as well as synthetic amino acids.
The macrocycles 29−39 were synthesized according to the
general synthetic route described in Scheme 1, and their
structure and α2β1 inhibitory activity is shown in Table 3. The
novel 1,2,3-triazole α-amino acids used in the synthesis of
macrocycles 34−37 were prepared using click chemistry
(Scheme 2).
a
E/Z ≈ 3:1.
a
Scheme 2. Synthesis of Novel 1,2,3-Triazole α-Amino Acids
By linking directly through the 5-membered heterocycle, the
ring size is reduced by one oxygen atom and thereby changing
the conformation of the macrocycle, possibly explaining the
increased activity. This positive effect was confirmed by the
macrocycles 34 and 36 having an 1,2,3-triazole α-amino acid
and an alkene linker displayed activities of IC50 = 38 and 34
nM, respectively. Interestingly, the unreduced 2-butenyl linkers
generally displayed superior activities of more than a factor 10
compared to the corresponding n-butyl linkers when attached
to a heteroaromatic amino acid, a trend that is not obvious for
macrocycles 6−16 (Table 1). Replacing the phenyl group with
2-aminopyridine as in compounds 29 and 30 only afforded
poor α2β1 inhibitors, whereas replacement with a pyridine as in
compounds 31 and 32 yielded similar activities compared to
macrocycles 15 and 16 (Table 1). Using tryptophan, linked
through the indole nitrogen, instead of phenylalanines afforded
the very potent α2β1 inhibitor 33 with an IC50 = 12 nM. This
result demonstrated that a highly active macrocycle con-
formation could be achieved when a 1,3-substituted 5-
membered heteroaromatic ring was used instead of a 6-
membered (hetero)aromatic ring.
a
Reactions and conditions: (a) CuSO4, L-ascorbic acid sodium salt,
tBuOH−water, rt, 79%. (b) NaN3, CuSO4, L-ascorbic acid sodium salt,
tBuOH−water, rt, 13% (regioisomer 28).
tendency to racemize, the optical purity of 38 was determined
by chiral stationary phase HPLC to be >95% and thus not
compromised during preparation. We decided to expand the
series of histidine derived macrocycles by making various
analogues as depicted in Table 4.
Attaching the linker to the benzamide 4-position as in
compound 40 (IC50 = 470 nM) and thereby expanding the
macrocycle to a 16-membered ring led to a >100-fold loss of
activity compared to the 3-substituted regioisomer 38. As
expected, macrocycles prepared from D-histidine, D-38 and D-
This illustrates that even small conformational changes can
lead to large changes in inhibitory activity toward integrin
α2β1. We also prepared histidine derived macrocycles employ-
ing a novel N(τ) selective allylation procedure to afford
protected N(τ)-allyl histidine as the starting material.17 This
provided us with our so far most active macrocycle 38 having
an IC50 = 4 nM, a molecular weight of 362 Da, and a very good
ligand efficiency of LE = 0.46 kcal/mol. As histidine has a
C
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Table 4. Histidine Derived Heterocycles
macrocycle
size
IC50
(μM)
compd
X
n
linker
40
CH
CH
CH
CCl
N
1
1
1
1
1
1
2
2
1
1
1
4-OCH2(CH)2CH2
3-OCH2(CH)2CH2
3-O(CH2)4
16
15
15
15
15
15
16
16
16
16
14
0.470
0.909
>4
D-38
D-39
41
3-OCH2(CH)2CH2
3-OCH2(CH)2CH2
3-OCH2(CH)2CH2
3-OCH2(CH)2CH2
3-O(CH2)4
0.869
0.036
>4
Figure 2. (A) X-ray structure of compound 49. TFA salt, ethanol, and
water removed for clarity.
42
a
43
CH
CH
CH
CH
CH
CH
b
44
>5
b
45
>5
c
46
3-OCH2(CH)2(CH2)2
3-O(CH2)5
0.270
0.731
0.176
47
48
3-CH2(CH)2CH2
a
b
c
5-Chlorohistidine. Racemic homohistidine. E/Z ≈ 3:1.
39, exhibited decreased activities compared to macrocycles
synthesized using L-histidine. Adding a second ortho-chloro
substituent to the benzamide moiety as in AJM-300 also led to
a strong decrease in activity with 41 having an IC50 = 869 nM.
However, picolinic acid derived macrocycle 42 having a
nitrogen atom in the same position displayed an excellent
activity of IC50 = 36 nM. We also attempted some
modifications on the histidine part of the molecule by
introducing a chlorine atom in the 5-position of the histidine
ring 43 or enlarging the ring by using homohistidine 44 and 45,
but these efforts only yielded inactive compounds. On the
contrary, expanding the macrocycle to a 16-membered ring by
increasing the length of the linker between the histidine and the
benzamide moiety still delivered active compounds 46 and 47
with IC50 = 270 and 731 nM, respectively. Contracting the ring
to a 14-membered heterocycle by removing the oxygen atom in
the linker also led to a decrease in activity with 48 having an
activity of IC50 = 176 nM. Unfortunately, none of the modified
histidine containing macrocycles in Table 4 were superior to
macrocycle 38, and we therefore decided to focus our further
profiling on this macrocycle. Since it was obvious that even
small conformational changes in the macrocycles could have
dramatic effects on integrin α2β1 activity, we attempted to
obtain an X-ray crystal structure of compound 38 to determine
the exact configuration of the compound. This was unsuccessful
but high quality crystals were obtained by recrystallizing the
TFA salt of the cyclopropylmethylester 49 from ethanol−water
(Figure 2).18 This revealed a rigid stair-like structure with an E-
alkene linker and a benzamide that is twisted 116° out of the
plane.
Figure 3. Histogram of the torsion angle distribution of the amide
bond in the benzamide fragment as depicted on the basis of an analysis
of the CSD.
the twisted amide conformation, facilitated by the ortho-
substituent, is required for obtaining activity on integrin α2β1
as this is very sensitive to the nature of the substituent as
demonstrated by the compounds in Table 2. A twisted
benzamide conformation of 87° is also observed in a
Valategrast intermediate and thus also seems to be required
for integrin α4β1 and α4β7 binding.9 To rationalize this
observation, a docking model of 38 to the extracellular domain
(ECD) of α5β1 was generated as depicted in Figure 4.
The coordinates from the X-ray structure of 49 have been
used as the starting geometry for a docking study for
compound 38. Generally, compound 38 shows high activity
on α2β1 (IC50 = 4 nM) as well as on α5β1 (IC50 = 74 nM) (see
Table 5). Additionally, due to highly conserved β1 subunits,
α5β1 is a valid surrogate for α2β1. The coordinates for the
α5β1 ECD were taken from the PDB entry 3vi4.20 GLIDE XP
(Schrodinger Inc.) was used for the docking studies.21 The
̈
The classification of the torsion angle has been done in the
context of published X-ray structures available in the CSD
database. The statistical distribution of benzamide torsion
angles from representative molecules is depicted in Figure 3.
Obviously, the amide torsion angle of macrocycle 49 falls into a
sparsely populated region emphasizing its exceptional geometry
and a comparable analysis of ortho-substituted benzamides
leads to the same conclusion (see Supporting Information).
Previously, an exhaustive investigation of fragment geometries
in the PDB and CSD from Stahl et al. already indicated this
observation for the benzamide torsion angle.19 It is likely that
docking mode of 38 in α5β1 indicates that the carboxylic acid
of 38 binds to the buried Mg2+ ion in the MIDAS in the β1
domain. The macrocycle is rigidly fixed to the MIDAS by H-
bond interactions from the carboxylate group and the amide of
38 to the β1 subunit backbone amino acids ASN224 and
TYR133. Furthermore, the N3 nitrogen of the imidazole forms
a weak H-bond interaction with the hydroxyl group of SER227.
Interestingly, only due to the unusual amide geometry of the
benzamide torsion the 6-chloro substituent is in a position to fit
into a small lipophilic pocket in close proximity to the Mg2+ ion
completing the binding pharmacophore of 38. This observation
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explain the fact that the lack of a substituent in this position
leads to weakly active compounds.
The hydrochloride salt of macrocycle 38 was selected for
further in vitro profiling in order to assess its properties and
potential as preclinical candidate, and the results are presented
in Table 5. As expected, 38 also showed high activity toward
integrins α1β1 and α5β1 with an IC50 = 33 and 74 nM,
respectively in ELISA-based protein−protein interaction assays.
The compound was found to be very polar with a LogD = 0.05
and therefore possessed an excellent solubility of 1.54 mg/mL.
Furthermore, it was completely stable when incubated with
human, mouse, or rat microsomes or in plasma. Unfortunately,
the Caco-2 permeability of 38 was practically nonexistent with a
value of 1 × 10−7cm/s, but methyl, ethyl, and other ester
prodrugs displayed a high Caco-2 permeability (>20 × 10−7cm/
s) although they were found to hydrolyze to some extent under
the assay conditions.
Furthermore, compound 38 was found to have no inhibitory
activity neither on the hERG channel nor on cytochrome P450
CYP3A4 as well as no relevant induction effects on isozymes
CYP1A1, CYP1A2, CYP3A4. Because of this promising in vitro
profile, compound 38 was further profiled in an in vivo
pharmacokinetic study in rat to determine important
pharmacokinetic parameters. After intravenous bolus admin-
istration of 4.8 mg/kg, blood and urine samples were collected
for up to 24 h, and the key pharmacokinetic parameters are
shown in Table 6. It was found that 24% of the administered
Figure 4. Docking model of the binding of 38 to the MIDAS of α5β1.
The van der Waals surface is shown for the extracellular domain of
α5β1 (α5, gray; β1, pink). The MIDAS is shown as a close-up
including the van der Waals surface and the docked ligand in capped
sticks. The coloring of the surface is by element. Hydrogen bonds are
displayed as dotted lines. The Mg2+ ion is depicted as a pink sphere
(MG).
Table 5. Physicochemical, in Vitro, and ADMET Properties
of 38
Table 6. Pharmacokinetic Parameters in Rat after
Intravenous Bolus Administration of 4.8 mg/kg
t1/2 (hr)
0.24
1.7
3.6
24
C0 (μg/mL)
Vss (L/kg)
% of dose excreted into urine (24 h)
% of dose excreted into bile (2 h)
50
MW
362 (parent)
0.46
ligand efficacy
LogD (pH 7.4, 25 °C)
CLogP
0.05
1.9
PSA (Å2)
93.5
dose of compound 38 was excreted unchanged into the urine
after 24 h, and in an additional study, approximately 50% was
going unchanged into bile after 2 h. This resulted in a very
short half-life of approximately 15 min and plasma level <10
ng/mL 1 h after administration. The PK profile was found to be
unacceptable due to the very short half-life and extensive
excretion and no further studies were performed on compound
38.
In conclusion, we have developed highly active low molecular
weight macrocyclic integrin α2β1 antagonists from acyclic
precursors. These small macrocycles constitute a novel class of
integrin inhibitors with improved and attractive properties such
as high solubility and metabolic and plasma stability in human,
mouse, and rat. Unfortunately the compounds exhibited poor
cellular permeability and were extensively cleared from plasma
and thus exhibited a very short half-life in vivo.
H-bond donors
2
H-bond acceptors
7
rotatable bonds
1
IC50 α2β1
4 nM
33 nM
74 nM
1.54 mg/mL
3% (low)
0%
IC50 α1β1
IC50 α5β1
solubility (pH =7.4, 25 °C)
metabolic lability in human microsomes
metabolic lability in rat microsomes
metabolic lability in mouse microsomes
plasma stability (human, mouse, and rat)
Caco2 permeability (× 10−7cm/s)
0%
a
stable
1 (low)
>30 μM
<10%
>10 μM
b
CYP3A4 inhibition IC50
CYP1A1, CYP1A2, and CYP3A4 induction
c
hERG channel inhibition IC50
a
The compound was spiked to each of the blank plasmas at a
concentration of 100 ng/mL. The spiked plasma samples were
b
ASSOCIATED CONTENT
incubated in a water bath at 37 °C for up to 4 h. Incubated at 37 °C
■
c
for 10−30 min at 0.3−30 μM.22 Patch-clamp technique in the whole-
S
* Supporting Information
cell configuration on recombinant chinese hamster ovary (CHO) cells.
Representative experimental procedures for synthesis of
macrocycles, biochemical assays, and analytical data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
is in line with the SAR of the 6-chloro substituent in meta
linked phenylalanines as discussed earlier (Table 2) and might
E
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dual α4β7/α4β1 integrin antagonist. Bioorg. Med. Chem. 2002, 12,
2051−2066.
AUTHOR INFORMATION
Corresponding Author
*(N.H.) Phone: +49-69305-36193. E-mail: nis.halland@sanofi.
com.
■
(17) Halland, N. An atom-efficient direct regioselective N(τ)-
allylation of histidine derivatives. Synlett 2012, 2969−2971.
(18) The TFA salt of compound 49 was recrystallized from ethanol−
water. Crystal size (mm) 0.5 × 0.4 × 0.07. Space group P21/n.
Deposited in the Cambridge crystallographic database under number
CCDC 978145.
Notes
The authors declare no competing financial interest.
(19) Grameld, K. A.; Kuhn, B.; Reuter, D. C.; Stahl, M. Small
molecule conformational preferences derived from crystal structure
data. A medicinal chemistry focused analysis. J. Chem. Inf. Model. 2008,
48, 1−24.
(20) Nagae, M.; Nogi, T.; Takagi, J. Crystal structure of α5β1
integrin ectodomain: Atomic details of the fibronectin receptor. J. Cell.
Biol. 2012, 197 (1), 131−140.
ACKNOWLEDGMENTS
■
Thanks are expressed to DI Winfried Heyse and Harold
Schweitzer for obtaining the X-ray crystal structure of
compound 49 and Dr. Silke Haag-Diergarten for performing
the PK study.
(21) Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.;
Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T.
Extra precision glide: docking and scoring incorporating a model of
hydrophobic enclosure for protein-ligand complexes. J. Med. Chem.
2006, 49 (21), 6177−6196.
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