The synthesis and structure-activity relationship of substituted N-phenyl anthranilic acid analogs as amyloid aggregation inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.12.049

It is believed that β-amyloid aggregation is an important event in the development of Alzheimer's disease. In the course of our studies to identify β-amyloid aggregation inhibitors, a series of N-phenyl anthranilic acid analogs were synthesized and studied for β-amyloid inhibition activity. The synthesis, structure-activity relationship, and in vivo activity of these analogs are discussed.

Full text of DOI:10.1016/j.bmcl.2008.12.049

Bioorganic & Medicinal Chemistry Letters 19 (2009) 654–657
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The synthesis and structure–activity relationship of substituted
N-phenyl anthranilic acid analogs as amyloid aggregation inhibitors
Lloyd J. Simons a, Bradley W. Caprathe a, Michael Callahan a, James M. Graham a, Takenori Kimura b,
Yingjie Lai a, Harry LeVine III a, William Lipinski a, Annette T. Sakkab a, Yoshikazu Tasaki b, Lary C. Walker a,
Tomoyuki Yasunaga b, Yuyang Ye a, Nian Zhuang a, Corinne E. Augelli-Szafran a,
*
a Pfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Rd., Ann Arbor, MI 48105, USA
b Yamanouchi Pharmaceutical Co., Ltd., Tsukuba Research Center, 21 Miyukigaoka, Tsukuba, Ibaraki 305, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
It is believed that b-amyloid aggregation is an important event in the development of Alzheimer’s dis-
ease. In the course of our studies to identify b-amyloid aggregation inhibitors, a series of N-phenyl
anthranilic acid analogs were synthesized and studied for b-amyloid inhibition activity. The synthesis,
structure–activity relationship, and in vivo activity of these analogs are discussed.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 14 October 2008
Revised 10 December 2008
Accepted 11 December 2008
Available online 24 December 2008
Keywords:
Beta-amyloid
Alzheimer’s disease
N-Phenyl anthranilic acid
Analogs
Amyloid plaques
Amyloid aggregation
Tg2576 mice
Hsiao mouse model
Amyloid aggregation inhibitors
Genetic, pathological and biochemical evidence implicates
aggregation of b-amyloid (Ab) peptide in the pathogenesis of Alz-
heimer’s disease.1 Current marketed therapies for Alzheimer’s dis-
In Scheme 1, olefin 3 was generated from heating a pyridine
solution of p-nitrophenylacetic acid 1 and substituted aldehyde
2. Hydrogenation of 3 gave amine 4 which was then coupled with
ease are palliative and only modestly effective.
A disease-
modifying drug would be a major therapeutic breakthrough to-
wards the treatment of this disease.
NH2
N
H2N
N
Our initial attempts at generating small molecule inhibitors for
b-amyloid aggregation were based on Congo Red, a well-known
stain for amyloid protein fibrils.2 Figure 1 depicts the refinement
of the structural class from a diazo-linked anthranilic acid deriva-
tive to an ethyl-linked moiety. The addition of a single methylene
unit in the linker generated compounds with increased in vitro po-
tency. Based on the promising in vitro and in vivo results3 of initial
leads PD 0118057 (14) and PD 0202091 (57), additional synthesis
was pursued to generate a structure–activity relationship within
this series of compounds.
N
N
NaO3S
SO3Na
Congo Red
N
N
N
N
H
H
CO2H
CO2H
CO2H
N
N
H
H
CO2H
* Corresponding author at present address: Laboratory for Experimental Alzhei-
mer Drugs (LEAD), Harvard Medical School and Brigham & Women’s Hospital, 77
Avenue Louis Pasteur, HIM 756, Boston, MA 02115, USA. Tel.: +1 617 525 5230; fax:
+1 617 525 5252.
"Ethyl-linked"
"Propyl-Linked"
Figure 1. Evolution of N-phenyl anthranilic acid analogs as amyloid aggregation
E-mail address: caugelli@rics.bwh.harvard.edu (C.E. Augelli-Szafran).
inhibitors.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.049

L. J. Simons et al. / Bioorg. Med. Chem. Lett. 19 (2009) 654–657
R1
655
The in vitro data were obtained from two primary assays, BASSR
and BASST, which were designed to determine the inhibition of
amyloid fibril formation by the test compound. BASSR (Beta-Amy-
loid-Self-Seeding Radioassay) is a radiolabeled 125I-Ab(1-40) filtra-
tion assay. The BASST (Beta-Amyloid-Self-Seeding, Thioflavin T) is
an amyloid-specific fluorescence assay. The test compound was
incubated under the same conditions in both assays with Ab(1–
R1
CO2H
a
+
O2N
CHO
3
2
1
NO2
b
R1
R1
c
R2
40)/buffer (30 lM unlabeled Ab(1–40)/50 mM NaPi-150 mM NaCl,
F
1 M urea, 0.02% w/v NaN3, pH 7.5). In the BASSR assay, a tracer
amount of 125I-labeled Ab(1–40) was added to the assay. In the
N
NH2
H
4
6-42
CO2H
CO2H
R2
BASST assay, 5 lM ThioflavinT (ThT) was added after fibril forma-
5
tion.4 BASST reports on the formation of the b-sheet structure of
amyloid fibrils.5 This measure can be complicated by false positives
that result from the competition of test compounds with the
extrinsic fluorescent ThT probe for binding to amyloid fibrils.
Therefore, an additional assay, BASSR, was used to detect the inhi-
bition of peptide aggregation. BASSR measures the formation of the
Scheme 1. Reagents: (a) Pyr; (b) RaNi, H2, THF; (c) LHMDS, THF.
O
O
CHO
a
radiolabeled fibrils that do not pass through a 0.2 lm filter. These
+
two assays provided complementary information about the aggre-
gation state and fibrillar amyloid conformation with compound
present. Eight concentrations of each compound were used for
IC50 determinations that were calculated by logit transformation
of the titration curves. Variation of individual points was 10%.
The average IC50s of two separate experiments are reported. At
R3
NH2
NH2
R3
45
43
44
b
R4
c
30 lM of Ab(1–40) peptide, an inhibitor with an IC50 of 1 lM tar-
N
H
NH2
R3
R3
gets a species that is less than 3% of the total peptide concentra-
tion. These assays were designed to detect inhibitors at an early
stage in fibril formation with an amount of compound that would
be less than the Ab(1–40) peptide monomer concentration. An IC50
F
R4
CO2H
48-72
46
CO2H
47
of 20 lM is approximately equivalent to a ratio of 0.7 moles com-
Scheme 2. Reagents: (a) NaOH, EtOH or H2SO4, AcOH; (b) Et3SiH, TFA; (c) LiNH2,
pound/peptide monomer and thus, is borderline substoichiomet-
ric.4,5 (The observed IC50 is a product of the true affinity of the
compound, the stoichiometry of binding, and the concentration
of the target aggregation intermediate.)
In vivo efficacy studies were performed in Tg2576 mice that
over-express human bAPP695Swe. These mice show an age-related
increase in cerebral Ab deposition, and therefore, are suitable mod-
els for testing b-amyloid aggregation inhibitors.6 Bioavailable and
brain-penetrant compounds were delivered orally by mixture in
chow.
THF.
Table 1
Modifications of ethyl-linked derivatives
R1
6-19
N
H
CO2H
Our initial efforts in the ethyl-linked series produced a series of
compounds (Table 1) that showed acceptable activity in the BASST
assay, but were inactive in the BASSR assay.
By adding a nitro group to the anthranilic acid ring (Table 2),
potency increased significantly in both assays. However, the nitro
functionality is considered to be a risk for CNS compounds since
it may decrease brain-penetration of the compound or have poten-
tial toxicity. Therefore, other substituents on the anthranilic acid
ring were explored (Table 3).
When the carboxylic acid group was moved to the 4- and 5-
positions on the aromatic ring (entries 32 and 34, Table 3 vs entry
14, Table 1), a 5-fold decrease in potency in BASST was observed.
The methyl ester analog (entry 33) of entry 32 exhibited no activity
in either BASST or BASSR. Some of the most potent derivatives of
14 had a carboxylic acid group in the 6-position and an electron-
deficient anthranilic acid ring (see Table 2 and entries 37, 39 and
41 in Table 3).
Compound
R1
BASSR IC50
(
lM)
BASST IC50 (lM)
6
7
8
4-OMe
N-DHIQa
H
3,4-Me
2-Cl
3-Cl
4-Cl
3-F
3,4-Cl
2,4-Cl
3,5-Cl
3,4-F
4-F, 3-CF3
3-Cl, 4-Me
>100
>100
60
>100
1
10
10
5
19
3
70
2
51
15
2
2
1
9
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
88
10
11
12
13
14
15
16
17
18
19
a
N-decahydroisoquinoline.
By increasing the length of the alkyl linker by one methylene
unit (i.e., propyl-linked, Table 4), a significant increase in potency
was observed in the BASSR assay relative to the similarly substi-
tuted inactive ethyl-linked compounds (entries 50, 52, 54–56 in
Table 4 vs 8–12 in Table 1). In addition to this observed increase
in potency, another advantage of the propyl-linked analogs was
the consistent activity in both BASSR and BASST. However, substi-
tution on the anthranilic ring of the propyl-linked analogs with
electron withdrawing groups (Table 5, entries 65, 66) did not give
2-fluoro benzoic acid 5 in the presence of a strong base to give
ethyl-linked derivatives 6–42.
Propyl-linked derivatives (Scheme 2) were prepared via an acid-
or base-catalyzed Aldol reaction. Reaction of substituted aldehydes
43 and 4-aminoacetophenone 44 generated chalcone 45. Reduc-
tion of chalcone 45 gave propyl-linked anilines 46 that were subse-
quently coupled with 2-fluoro benzoic acids 47 to yield the
corresponding propyl-linked anthranilic acids 48–72.

656
L. J. Simons et al. / Bioorg. Med. Chem. Lett. 19 (2009) 654–657
Table 2
Table 5
4-Nitro substitution of ethyl-linked derivatives
Other modifications of propyl-linked derivative
R8
R2
NO2
R7
N
H
20-31
N
H
62-72
CO2H
CO2H
Compound
R2
BASSR IC50
(
lM)
BASST IC50
(
lM)
Entry
R7
R8
BASSR IC50
(l
M)
BASST IC50 (lM)
20
21
22
23
24
25
26
27
28
29
30
31
4-N(nBu)2
DHIQb
H
3,4-Me
2-Cl
15
5
70
12
3
33
15
43
17
41
3
1
1
1.5
2
0.5
0.3
0.4
0.5
4
4
0.8
1
62
63
64
65
66
67
68
69
70
71
72
3,4-Cl
3,4-Cl
3,4-Cl
3,4-Cl
3,4-Me
3,4-Me
3,4-Me
3,4-Me
3,4-Me
4-OMe
4-OMe
4-F
86
1
1
>100
5
6
1
9
5.4
3
9
4-NO2
4-Me
2-aza
4-NO2
4-F
2-aza
3-F
4-Me
3-F
>100
>100
60
14
>100
14
12
16
14
14
3-Cl
4-Cl
3,4-Cl
2,4-Cl
3,4-F
4-F, 3-CF3
4-Cl, 3-CF3
4-F
10
16
b
N-decahydroisoquinoline.
Table 3
Other modifications of ethyl-linked derivatives
Cl
R4
Cl
R5
32-42
N
H
Compound
R4
R5
BASSR IC50
(
lM)
BASST IC50 (lM)
32
33
34
35
36
37
38
39
40
41
42
H
H
H
4-CF3
4-F
4-CO2H
4-CO2Me
5-CO2H
5-CO2H
5-CO2H
6-CO2H
6-CO2H
6-CO2H
6-CO2H
6-CO2H
6-CO2H
>100
>100
>100
>100
>100
7
>100
27
>100
70
10
>100
10
35
3
2
2
4
Figure 2. Plaque density of control versus PD 0118057-treated Tg2576 mice,
200 mg/kg/day.
2-azac
4-Me
3-CF3
2-CF3
5-CF3
4-NEt2
30
0.1
53
>100
c
substituted pyridine ring.
Table 4
Propyl-linked derivatives
Figure 3. Plaque density of control versus PD 0202091-treated Tg2576 mice,
60 mg/kg/day.
N
R6
48-61
H
CO2H
could potentially generate clinical candidates for Alzheimer’s dis-
ease therapy. Figures 2 and 3 illustrate the reduction of senile pla-
ques (black spots in cortex) in treated Tg2576 mice with PD
0118057 (14) and PD 0202091 (57), respectively, compared to an
untreated control mouse.
As further noted in Figure 4, 57 showed a significant reduction
in plaque load over a 7-month and then a 3-month treatment per-
iod at a lower dose versus 14. A 3-fold lower dose of 57 versus 14
gave the same reduction in plaque load in the Tg2576 mouse.7
Additional evidence of these types of inhibitors preventing the
aggregation of the Ab protein was provided by an atomic force
microscopy (AFM) study.8 Results indicated that smaller, frag-
mented Ab species were present after in vitro treatment of the
Ab protein with, for example, 14 (Fig. 5).
Compound
R6
BASSR IC50
(l
M)
BASST IC50 (lM)
48
49
50
51
52
53
54
55
56
57
58
59
60
61
4-NEt2
4-OMe
H
4-Me
3,4-Me
3-Br
2-Cl
3-Cl
4-Cl
3,4-Cl
2,4-Cl
3,5-Cl
3,4-F
3
30
14
9
6
2
6
4
3
25
2
1
17
5
1
3
3
2
3
2
1
2
3
5
3
8
5
4-F, 3-CF3
3
This AFM result correlated with the smaller size Ab peptide spe-
cies observed by analytical ultracentrifugation (fibrils > 200S, fi-
brils treated with 14 = 15S).9 These Ab species, although
multimeric, seem to be non-toxic based on results from cell culture
studies and the lack of cell loss in the mice. In contrast to amyloid
fibrils, the fragmented Ab species were protease-sensitive and thus
could be cleared from the brain.9 The size of these accumulated Ab
the same boost in potency as observed in the ethyl-linked series
(Table 2).
The in vivo and pharmacokinetic data of 14 and 57, pre-clinical
chemical leads from the ethyl- and propyl-linked series, respec-
tively, provided convincing evidence that this series of compounds

L. J. Simons et al. / Bioorg. Med. Chem. Lett. 19 (2009) 654–657
657
forms may indicate the inhibition site in the process of amyloid fi-
bril formation, perhaps at an early, non-toxic pre-protofibril stage.
Further study of the interruption of amyloid formation would be
needed to establish the relationship of fibril deposition per se to
the progression of Alzheimer’s disease. Compounds such as 14
and 57 with brain penetration and significant in vivo activity could
be useful in probing the role of fibril formation in disease
progression.
15-month-old APPtg mice
8
3 month treatment
7 month treatment
Mean
6
Following the discovery of 14 and 57, many amyloid aggrega-
tion inhibitors with low micromolar inhibition and in vivo efficacy
were synthesized. However, 14 and 57 possessed the most accept-
able drug-like properties, such as brain penetration, bioavailability,
no toxicity and in vivo efficacy. Thus, the in vitro and in vivo effects
of 14 and 57 indicate that in vitro inhibition of amyloid aggrega-
tion translated to efficacy in the Tg2576 mouse model as measured
by the reduction in senile plaque load. These results suggest that
agents limiting the self-polymerization of Ab in brain and main-
taining the Ab in a metabolizable form can impede senile plaque
formation, and hence, could lead to a disease-modifying drug for
the treatment of Alzheimer’s disease.
4
2
P = 0.016
0
Control
PD0202091
(60 mg/kg/day)
Figure 4. 3- and 7-month treatment of Tg2576 mice with PD 0202091.
References and notes
1. Selkoe, D. J.; Haass, C. Nat. Rev. Mol. Cell Biol. 2007, 8, 101.
2. Augelli-Szafran, C. E.; Walker, L. C.; LeVine, H., III Annu. Rep. Med. Chem. 1999, 34,
21.
3. Augelli-Szafran, C. E.; Bian, F.; Callahan, M. J.; Feng, R.; Iwai, A.; Lai, Y., LeVine, III,
H.; Lipinski, W.; Walker, L. C.; Yasunaga, T. Abstracts of Papers, 228th National
Meeting of the American Chemical Society, Philadelphia, PA; American Chemical
Society: Washington, DC, 2004; Abstract MEDI-315.
4. LeVine, H., III; Scholten, J. D. Meth. Enzymol. 1999, 309, 467.
5. LeVine, H., III Meth. Enzymol. 1999, 309, 278.
6. Callahan, M. J.; Lipinski, W. J.; Bian, F.; Durham, R. A.; Pack, A.; Walker, L. C. Am. J.
Pathol. 2001, 158, 1173.
7. Plaque reduction for 14 and 57 seems to be at maximum effect expected in this
model: 14 (200 mg/kg/day, PO: 7 months (n = 15, 44% reduction); 57 (60 mg/kg/
day, PO: 7 months (n = 14, 47% reduction) and 3 months (n = 16, 45% reduction).
8. Thank you to P. Lansbury/J. Harper for AFM studies and J. Kelly/H. Lashuel for the
analytical ultracentrifugation studies.
Figure 5. AFM results with PD 0118057 (14).
9. Unpublished results.