Bioorganic & Medicinal Chemistry Letters 23 (2013) 1036–1040
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of N-acyl 4-(3-pyridonyl)phenylalanine derivatives and
their orally active prodrug esters as dual acting a4b1 and a4b7
receptor antagonists
Jefferson W. Tilley a,⇑, Achyutharao Sidduri a, Jianping Lou a, Gerry Kaplan a, Nadine Tare b, Gary Cavallo b,
Karl Frank c, Anjula Pamidimukkala c, Duk Soon Choi d, Louise Gerber d, Aruna Railkar d, Louis Renzetti b
a
Discovery Chemistry, Pharmaceutical Research and Early Drug Development, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, USA
Discovery Inflammation and Respiratory Diseases, Pharmaceutical Research and Early Drug Development, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, USA
c
Drug Metabolism and Pharmacokinetics, Pharmaceutical Research and Early Drug Development, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, USA
d
PARD, Pharmaceutical Research and Early Drug Development, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, USA
b
a r t i c l e
i n f o
Article history:
Received 5 October 2012
Revised 30 November 2012
Accepted 10 December 2012
Available online 20 December 2012
a b s t r a c t
From a series of N-acyl 4-(3-pyridonyl)phenylalanine derivatives of 4, the trifluoromethyl derivative 28
was identified as a potent, dual acting alpha4 integrin antagonist with activity in primate models of allergic asthma. Investigation of a series of prodrug esters led to the discovery of the morpholinopropyl derivative 48 that demonstrated good intestinal fluid stability, solubility and permeability. Compound 48 gave
high blood levels of 28 when dosed orally in cynomolgus monkeys. Surprisingly, hydrolysis of 48 was
rapid in liver microsomes from the pharmacological species, mouse, rat and monkey, but slow in dog
and human; in vivo studies also indicated there was prolonged exposure to unchanged prodrug in dogs.
Ó 2012 Elsevier Ltd. All rights reserved.
Alpha4 integrins are expressed on a variety of leukocytes,
including B-cells, T-cells, basophils and eosinophils, and are involved in the recruitment, activation and survival of these cell
types. Data supporting a role for alpha4 integrins in a number of
inflammatory diseases, including multiple sclerosis, inflammatory
bowel diseases, asthma, rheumatoid arthritis, and atherosclerosis,
have emerged and are summarized in recent reviews.1 A humanized mouse anti-alpha4 antibody, Natalizumab, has been approved
for the treatment of multiple sclerosis2 and Crohn’s disease.3 More
recently a small molecule alpha4 integrin antagonist was shown to
be effective for the treatment of MS,4 fully validating alpha4 integrins as targets for human disease and increasing interest in the
discovery of small molecules with superior activities.
We previously reported that the potent, dual-acting antagonist
of a4b1 and a4b7, 1, is effective for the treatment of asthma in
man5 and sought additional compounds that might have lower
clearance and greater selectivity for a4b7 to further assess their
potential as alpha4 integrin antagonists. Inspired by reports of
the alpha4 integrin antagonist activity of members of the biphenylalanine class such as 2,6 as well as the interesting, selective integrin
antagonist activity of members of the pyridizinone family represented by 3,7 we report here on a new series of potent alpha4 integrin antagonists, N-acyl 4-(3-pyridonyl)-phenylalanine derivatives
4 and their orally active prodrug esters.
The compounds reported in Table 1 were typically prepared
by a palladium-catalyzed coupling reaction of the 4-iodophenylalanine derivative 17 with an in situ generated organozinc
intermediate derived from the appropriate 4,6-disubstituted
3-iodo-pyridone intermediate such as 11 and 16 (Scheme 3).
Cl
OCH3
O
Cl HN
OCH3
O
E-mail address: tilleyjk@optonline.net (J.W. Tilley).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.019
O
O
O
Cl
CH3
1
α4β1 IC50 10 nM
α4β7 IC50 43 nM
2
R1
CH3
N
N
R2
O
N
O
R3
OCH3
OH
Cl HN
O
⇑ Corresponding author.
OH
Cl HN
OH
Cl HN
Cl
3
OH
Cl HN
O
O
R4
O
4
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J. W. Tilley et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1036–1040
Table 1
VCAM/VLA-4 binding inhibition of N-acyl 4-(3-pyridonyl)-L-phenylalanine derivatives
R2
R3
N
O
R4
OH
HN
R1
No.
R1
O
O
R2
R3
R4
a4b1 Ramos cells/VCAM IC50 nM (Rel Potency)a
a4b7 RPMI cells/MadCAM IC50 nM (Rel Potency)a
CH3
H
H
419
ND
CH3
H
H
582 (0.03)
687 (0.46)
CH3
H
CH3
226 (0.23)
363 (0.68)
CH3
H
CH3
90 (0.37)
179 (1.6)
CH3
CH3
CF3
48 (0.47)
154 (3.5)
CH3
CH3
CF3
32 (0.81)
42 (6.5)
H
CH3
CF3
820 (0.08)
810 (0.65)
C2H5
CH3
CF3
112 (0.40)
61 (1.9)
CH3
CF3
463 (0.03)
1230 (0.34)
CH2–C5H6
CH3
CF3
560 (0.12)
600 (0.96)
CH3
CH3
CF3
225 (0.41)
767 (0.70)
CH3
CH3
CF3
366 (0.36)
1,290 (0.23)
CH3
CH3
CF3
140 (0.75)
671 (0.54)
CH3
CF3
CH3
269 (0.11)
126 (2.2)
CH3
CF3
CH3
267 (0.06)
160 (2.6)
H3CO
23
Br
Cl
24
CH3
Cl
25
CH3
Cl
26
Cl
Cl
27
CH3
Cl
28
Cl
Cl
29
Cl
Cl
30
Cl
Cl
31
Cl
Cl
32
Cl
CH3
33
C2H 5
CH 3
34
CH(CH3)2
35
CH 3 O
Cl
36
Cl
Cl
37
CH 3
a
Rel potency refers to activity relative to the reference standard RO0270608 run as a positive control in each binding experiment.
1038
J. W. Tilley et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1036–1040
O
O
OMe
O
OMe
a
NH 2
N
F
F
+
b
H
N
H 3C
CH3
N
O
O
H3C
CF 3
7
6
c
a, b
I
CF3
I
CF3
8
CH3
N
O
H3 C
CO 2Me
F
O
5
O
OMe
BocHN
H
N
H 3C
c
O
d
H
N
H 3C
11
CH 3
O
CO2H
N
H3C
e
I
I
CF3
CF 3
9
10
11
H3 C
CF3
R
OMe
ClH.H2N
O
O
F
O
F
F
19
F
F
F
O
O
c
N
F3C
O
OEt
CH 3 O
O
O
X = OH or Cl
O
CH3 O
13
N
14
a, b
O
CH3
F3C
N
CH3
O
e
F 3C
N
21
O
I
CH3
15
CH3
N
16
Scheme 2. Reagents and conditions: (a) pyridine, CH2Cl2, 0 °C to rt, 15 h; (b) NaOEt,
EtOH, reflux, 15 h; (c) MeI, K2CO3, DME, reflux, 15 h; (d) LiCl, DMF, 160C, 19 h; (e)
NIS, trifluoroacetic anhydride, TFA, 70–85 °C, 2 h.
H3C
22
CH 3
N
O
H 3C
c. d
CF3
OH
Cl HN
O
Cl
The 4,6-disubstituted 3-iodo-pyridone intermediates (11 and
16) were prepared using the process described in Schemes 1 and
2 while others were prepared using similar approach. In both, a
4,6-disubstituted pyridone core was built by a condensation between the mono ester malonamide and the diketone 7 or the related methoxy vinylketone 12 to give 8 and 13, respectively.
Treatment of the acid 9 with iodine/KI/NaHCO3 in aqueous methanol at 65 °C led to a nearly quantitative conversion to the iodide 10,
which was then alkylated to give 11.
Surprisingly, the carboxylic acid derived from 13 did not provide the
corresponding iodide under similar conditions. Thus, 13 was converted
to the N-methyl intermediate 14 which was then treated with lithium
chloride in DMF at 160 °C to effect hydrolysis and decarboxylation followed by treatment with NIS in trifluoroacetic acid in the presence of
trifluoroacetic anhydride to give 16 exclusively in 62% yield.
The iodide 11 was then reacted with an activated zinc dust in
dimethylacetamide at 70 °C to give the corresponding organozinc
intermediate.8 The coupling reaction of this organozinc intermediate with 17 proceeded smoothly in the presence of Pd(dba)2 and
trifurylphosphine (TFP) in THF at 50 °C to obtain the desired product 18 in 77% isolated yield. The remaining steps, BOC removal,
benzoylation, and hydrolysis proceeded in a straightforward manner to obtain the target compounds.
In general, the alkyl ester prodrugs were prepared by treatment
of the carboxylic acids with the appropriate alkyl iodide or alkyl
bromide in the presence of sodium bicarbonate in DMF. For example, the preparation of morpholinopropyl iodide and the corresponding ester prodrug and its HCl salt is shown in Scheme 4.
28
I
O
O
d
20
OEt
OEt
H 2N
O
O
Scheme 3. Reagents and conditions: (a) Zn dust (activated using 10 mol %
dibromoethane and 10 mol % TMSCl in THF), DMAC, 70 °C, 15 h; (b) Pd(dba)2, TFP,
THF, 50C, 15 h; (c) 4.0 N HCl in dioxane, dioxane, rt, 5 h; (d) X = OH, HBTU, DIPEA,
DMF, rt, 15 h or X = Cl, DIPEA, CH2Cl2, rt, 15 h; (e) 1.0 N NaOH, EtOH, rt, 5 h.
CH3
H
N
F 3C
OH
HN
R1
F
12
b
R
R1
F
O
a
O
CF3
X
O
O
F
CH3
N
O
H 3C
CH3
N
O
d, e
Scheme 1. Reagents and conditions: (a) TMSCl, H2O, 0 °C, 15 h; (b) piperidine,
EtOH, reflux, 15 h; (c) LiOH, THF, MeOH, reflux, 48 h; (d) NaHCO3, I2, KI, MeOH, H2O,
65–70 °C, 15 h; (e) MeI, K2CO3, DME, rt, 15 h.
+
18
17
O
CF3
O
OEt
BocHN
O
CH3
N
O
CF3
O
O
Cl HN
O
O
O
N
HCl
Cl
48
Scheme 4. Reagents and conditions: (a) LAH, THF, rt, 2 h; (b) NaI, MeSO3H, CH2Cl2,
reflux, 15 h; (c) NaHCO3, DMF, rt, 48 h; (d) TMSCl, iPrOH, rt, 2 h.
The in vitro potency of the compounds listed in Table 1 was assessed by determining the ability of serial dilutions to inhibit the
binding of RAMOS cells (a4b1-specific binding) and RPMI 8866
cells (a4b7-specific binding) with recombinant human VCAM-1
used as the counter ligand for the RAMOS cell assay and human
MAdCAM-1 for the RPMI 8866 cell assay. Cells were labeled with
Calcein AM, a fluorescent dye, and then activated with a binding
buffer containing Mn2+ to achieve maximum activation prior to assay. RO0270608 (1) was used as a positive control on each plate.
The data in Table 1 are reported both as the observed IC50 and as
fold-potency relative to 1 since the activity of the cell lines varied
over time with the number of passages.
Comparing the relative potencies of the compounds reported in
Table 1 in the cell based assay with the reference compound 1 indicates that 28 is nearly equally potent to 1 against a4b1 and is several-fold more potent as an antagonist of a4b7. The data indicate
that R2 is preferably –CH3 and that R4 is preferentially trifluoromethyl group relative to –CH3 or H. These findings are consistent
with our previously reported observation with 4-benzoylcarbonylaminophenylalanines related to 1 that an electron deficient distal
aromatic ring was favorable for potency9 and esptablished that
R1 is preferably a 2,6-dichlorobenzoyl group.10 Since 28 met our
criteria for potency and selectivity as an a4b7 antagonist, it was
1039
J. W. Tilley et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1036–1040
selected for further profiling. Intravenous PK experiments in rat
(Table 3) indicated that 28 had an acceptable PK properties.
Previous experience with related compounds indicated that the
free acids in the acylphenylalanine class have very poor bioavailability; thus, a prodrug strategy was pursued from the outset.
The ethyl ester 38 is extremely insoluble in water (6 lg/mL) and
while bioavailability in rats was a modest 18%, it was only 2% in
dogs. Other candidate prodrugs were assessed for Caco-2 permeability, solubility, and stability in simulated intestinal fluid, and
promising compounds were further profiled in rat PK (Tables 2
and 3). Most of the prodrugs have good stability and reasonable
conversion in plasma to the parent drug. Only the basic prodrugs
had good solubility and of these, the morpholinopropyl ester 48
emerged as the best candidate for further profiling in large animal
PK and efficacy experiments.
PK data for 48 in rat, dog and monkey are shown in Table 3.
Although oral bioavailability was only modest in rat and dog, the
relatively high bioavailability in monkey, combined with its good
solubility and permeability, prompted us to test this compound
further in the pivotal monkey lung inflammation model. This model was used to profile 1, which was validated clinically, and thus
became our major in vivo hurdle for taking compounds forward.
Thus cynomolgus monkeys were challenged with aerosolized
Ascaris suum extract 2 h after dosing. Inflammatory cell accumulation in the lungs was assessed by examination of bronchiolar lavage fluid withdrawn 24 h after the antigen challenge. At oral
doses of 10 and 30 mg/kg po, 48 was efficacious in blocking influx
of eosinophils, lymphocytes, neutrophils and total leukocytes into
Table 3
PK properties of compounds 28 and 48
Compd
Dose (iv) (mg/kg)
AUC (ng h/mL)
Cl (mL/min/kg)
Vss (mL/kg)
Dose (po) (mg/kg)
AUC of 28 (ng h/mL)
Cmax (mg/mL)
F (%)
28
48
Rat
Dog
Monkey
10
10,860
15.7
320
50
6100
2.5
11
10
10,880
16.5
250
50
6420
2.4
13
15
35,100
3.0
67
30
30,150
5.0
46
their lungs as shown in Figure 1. Based on its in vivo profile, 48
was selected for preclinical safety studies.
Surprisingly, although 48 was readily hydrolyzed in mouse, rat
and monkey liver microsomes, ester hydrolysis was slow in dog
and human. A graphic representation of the microsomal results
are shown in Figure 2. The significance of these findings was highlighted by our observation that there was about 10% unchanged
prodrug in dog plasma on days 1 and 6 during subchronic safety
studies. This gave rise to concerns about tissue distribution and
possible safety issues associated with significant exposure to the
unchanged prodrug, particularly as it could be anticipated to occur
in humans as well.
These concerns were validated when, metabolism studies of
radiolabeled 28 and 48 in liver microsomes from rat, dog, monkey
and human revealed that significant covalent protein binding occurred with an unknown metabolite derived from the unchanged
Table 2
Prodrug esters of the lead compound 28
CH3
H3C
N
O
CF3
O R
Cl HN
O
O
Cl
Compd
28
38
39
40
41
42
43
44
45
R
–H (Parent)
–CH2CH3
–CH2CH2CH3
–CH2CH(CH3)2
–CH2CH2OCH3
–(CH2)3OCH3
O
N(CH 3)2
CH 2
O
CH 2
OH
OH
N
46
Caco-2 Papp
10 7 cm/s
Sol. SGFa lg/
mL
Sol. SIFb lg/
mL
Stability SIFb t1/2
min
Bioavail rat F
(%)
Comment
lM
1.4
271
526
325
499
228
—
BQL
BQL
BQL
BQL
nd
—
BQL
BQL
BQL
BQL
nd
—
Stable
Stable
Stable
Stable
nd
—
2.7
nd
nd
nd
nd
—
18
nd
nd
nd
2
—
Dog F = 2%
63
0.8
0.7
Stable
nd
nd
764
0.03
0.03
Stable
nd
nd
18
190
140
Stable
nd
6
Unchg prodrug in
plasma
162
>13,000
200
240
11
15
Unchg prodrug in
plasma
96
>5000
Decomp
30
11.7
12
165
3900
330
Stable
2.9
11
4
Stable
hERG IC20
O
N
47
O
48
49
N
O
CH3
275
O
a
b
SGF is simulated gastric fluid, pH 2.0.
SIF is simulated intestinal fluid, pH 7.5.
17
Mixture of
diastereomers
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J. W. Tilley et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1036–1040
Allergen-induced Inflammatory Cell Influx in the Atopic Primate
35
Vehicle
10 mg/kg
30 mg/kg
30
Cells X 104/ml
25
20
15
10
5
0
MAC
NEU
EOS
LYM Total Cells
Cell Type
Figure 1. Accumulation of inflammatory cells in atopic non-human primate BAL
fluid 24 h after challenge with Acaris suum 2 h after oral treatment with 48.
100
90
% of 24 Remaining
80
the inhibition of a4b7. Compound 28 stood out among those investigated and a series of prodrugs was investigated to find a suitable
derivative for oral delivery of the otherwise poorly absorbed carboxylic acid. Most compounds investigated were rejected due to
poor solubility, permeability or oral bioavailability and attention
was focused on the morpholinopropyl ester 48 as having the best
combination of in vitro properties and reasonable bioavailability,
particularly in monkeys. The unanticipated species selective
hydrolysis of 48 led to the abandonment of this compound as a
lead candidate and raises a significant cautionary flag for others
considering a prodrug approach to solving drug delivery issues.
Scientists interested in pursuing prodrug strategies should verify that any prodrugs they contemplate undergo sufficiently rapid
conversion in all species used in pharmacological and safety studies. A similar incidence of species-selective prodrug ester hydrolysis was observed in a series of structurally similar amino acid
derived beta2 integrin antagonists.11 Even though many esters undergo rapid hydrolysis and are rarely employed as drugs per se for
this reason, their consistent hydrolysis cannot be taken as a given.
It would be interesting to investigate more systematically, how
common similar species effects occur. One could, for example
examine the time course of hydrolysis of a series of known prodrug
esters in liver microsomes and plasma from several species seeking
structural patterns. In a forthcoming paper, we will describe a successful prodrug strategy with a series of closely realted pyrimidine-diones in which this phenomena did not occur.12
70
References and notes
60
50
40
30
20
10
0
0
10
30
90
Time (min)
Control
Mouse
Rat
Dog
Monkey
Human
Figure 2. Percent of ester hydrolysis of 48 versus time in liver microsomal
preparations from various species. Control refers to incubation of 48 in buffer with
no microsomes added.
prodrug, but not the active pyridone acid 28 in dog and human, but
not rat or monkey. It is likely that exposure to 48 in the latter was
too transient to observe this metabolite or that the metabolic pathway simply was not operable in these species.
In the work presented in this manuscript, a series of pyridones
was prepared seeking alpha4 integrin antagonists with a bias for
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