Synthesis and SAR of N-benzoyl-L-biphenylalanine derivatives: Discovery of TR-14035, a dual α4β7/α4β1 integrin antagonist

Article abstract of DOI:10.1016/S0968-0896(02)00021-4

α₄β₁ and α₄β₇ integrins are key regulators of physiologic and pathologic responses in inflammation and autoimmune disease. The effectiveness of anti-integrin antibodies to attenuate a number of inflammatory/immune conditions provides a strong rationale to target integrins for drug development. Important advances have been made in identifying potent and selective candidates, peptides and peptidomimetics, for further development. Herein, we report the discovery of a series of novel N-benzoyl-L-biphenylalanine derivatives that are potent inhibitors of α4 integrins. The potency of the initial lead compound (1: IC₅₀ α₄β₇/α₄β₁ =5/33 μM) was optimized via sequential manipulation of substituents to generate low nM, orally bioavailable dual α₄β₁/α₄β₇ antagonists. The SAR also led to the identification of several subnanomolar antagonists (134, 142, and 143). Compound 81 (TR-14035; IC₅₀ α₄β₇/α₄β₁ =7/87 nM) has completed Phase I studies in Europe. The synthesis, SAR and biological evaluation of these compounds are described.

Full text of DOI:10.1016/S0968-0896(02)00021-4

Bioorganic & Medicinal Chemistry 10 (2002) 2051–2066
Synthesis and SAR of N-Benzoyl-L-Biphenylalanine
Derivatives: Discovery of TR-14035,
A Dual ꢀ4ꢁ7/ꢀ4ꢁ1 Integrin Antagonisty
Ila Sircar,a,* Kristjan S. Gudmundsson,a Richard Martin,a Jimmy Liang,a
Sumihiro Nomura,a Honnappa Jayakumar,a Bradley R. Teegarden,a
Dawn M. Nowlin,b Pina M. Cardarelli,b Jason R. Mah,b Samuel Connell,b
Ronald C. Griffitha and Elias Lazaridesb
aDepartment of Chemical Science, Tanabe Research Laboratories, USA, Inc.,
4540 Towne Centre Court, San Diego, CA 92121, USA
bDepartment of Biological Science, Tanabe Research Laboratories, USA, Inc.,
4540 Towne Centre Court, San Diego, CA 92121, USA
Received 26 September 2001; accepted 15 November 2001
Abstract—a4b1 and a4b7 integrins are key regulators of physiologic and pathologic responses in inflammation and autoimmune
disease. The effectiveness of anti-integrin antibodies to attenuate a number of inflammatory/immune conditions provides a strong
rationale to target integrins for drug development. Important advances have been made in identifying potent and selective candi-
dates, peptides and peptidomimetics, for further development. Herein, we report the discovery of a series of novel N-benzoyl-l-
biphenylalanine derivatives that are potent inhibitors of a4 integrins. The potency of the initial lead compound (1: IC50 a4b7/
a4b1=5/33 mM) was optimized via sequential manipulation of substituents to generate low nM, orally bioavailable dual a4b1/a4b7
antagonists. The SAR also led to the identification of several subnanomolar antagonists (134, 142, and 143). Compound 81 (TR-
14035; IC50 a4b7/a4b1=7/87 nM) has completed Phase I studies in Europe. The synthesis, SAR and biological evaluation of these
compounds are described. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
sequence of FN have also been shown to block VCAM/
The a4 integrins, a4b1 (VLA-4) and a4b7 are hetero-
dimeric cell surface proteins expressed on most leuko-
cytes that are involved in cell–cell and cell–matrix
interactions.2 These leukocytes play a key role in
inflammation and autoimmune diseases and a4b1 is
believed to be involved in the cell adhesion, migration
and activation of these cell types at sites of inflamma-
tion.3 Their central role in animal models of chronic
inflammatory diseases, including asthma, rheumatoid
arthritis and multiple sclerosis has been extensively
documented using a4-specific monoclonal antibodies
and inhibitory peptides.4À8 The primary ligands for a4b1
are the endothelial surface protein vascular cell adhe-
sion molecule (VCAM-1)9 and the alternatively spliced
region of the extracellular matrix protein fibronectin
connecting segment 1 (FN, CS-1).10 Peptides and pepti-
domimetics derived from the connecting segment 1
VLA-4 interactions and to block allergen-induced air-
way responses in a sheep model of asthma.11À13 The
a4b7 integrin is widely expressed on leukocyte subtypes,
although its distribution is more restricted than that of
a4b1. In addition to the two ligands that are shared by
both a4b1 and a4b7 (VCAM-1 and CS-1), the ligand
mucosal addressin cell adhesion molecule (MAdCAM)
shows unique specificity for the integrin a4b7. MAd-
CAM is highly expressed on Peyer’s patch high endo-
thelial venules, in mesenteric lymph nodes, gut lamina
propria and mammary gland venules.14,15 Integrin a4b7
and MAdCAM have been shown to be important in
regulating lymphocyte trafficking to normal intes-
tine.16,17 Using a panel of monoclonal antibodies, it was
shown that the a4b7 interaction with its three ligands
involves distinct, but overlapping epitopes.18
Based on the promising integrin biology, these adhesion
molecules have received considerable attention as drug
targets. An intense effort by multiple biopharmaceutical
ySee ref 1.
*Corresponding author. Fax: +1-858-558-9383; e-mail: sircar@trlusa.
com
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00021-4

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I. Sircar et al. / Bioorg. Med. Chem. 10 (2002) 2051–2066
companies has resulted in very rapid progress in small
molecule development.9,19 Although several highly
potent compounds including a series of aryl diamide
analogues of p-aminophenylalanine have been repor-
ted,20 oral bioavailability was an issue with these early
leads. In order to reduce the peptidic nature of these
inhibitors the biphenylalanine scaffold was designed.
Initial efforts in our laboratories led to the identification
of compound 1 (unpublished results). Herein, we report
the discovery of a series of potent N-benzoyl-l-biphenyl-
alanine compounds (Fig. 1) that led to the first orally
bioavailable small molecule a4 antagonist. Compound
81, TR-14035, has completed phase 1 studies in Europe.
Chemistry
Schemes 1 and 2 were utilized for the synthesis of the N-
benzoyl biphenyl scaffold. Schemes 3–9 illustrate reac-
tions for functional group transformations for addi-
tional targets. For A-ring modified compounds, N-boc-
tyrosine triflate methyl ester21 or N-boc-p-BrPhe methyl
ester was used as the starting material. These were
reacted with the requisite boronic acids under Suzuki
reaction22,23 to give the desired N-boc-biphenylalanine
derivatives. The boc group was removed and the amine was
reacted with requisite benzoic acids (or chlorides) to give
the coupled products, which were saponified to produce the
Figure 1.
Scheme 3. Reagents: (a) R0COCl; (b) (R0SO2)2O, DIEA or R0SO2Cl,
DIEA; (c) R0NCO; (d) R0NCS; (e) R0OCOCl; (f) LiOH.
Scheme 1. Reagents: (a) (CF3SO2)2O, pyridine; (b) K2CO3, Pd(PPh3)4,
DME; (c) TFA; (d) EDC, HOBT (R5=OH); (e) DIEA, DCM
(R5=Cl); (f) LiOH.
Scheme 4. Reagents: (a) LiOH; (b) TFA; (c) CDI, NH4OH; (d) CDI,
MeSO2NH2, DBU.
Scheme 2. Reagents: (a) ROH, HCl; (b) EDC, HOBT (R6=OH);
(c) DIEA, DCM (R6=Cl); (d) (CF3SO2)2O, pyridine; (e) K2CO3,
Pd(PPh3)4, DME; (f) LiOH.
Scheme 5. Reagents: (a) KI, KCN, Pd(PPh3)4; (b) LiOH.

I. Sircar et al. / Bioorg. Med. Chem. 10 (2002) 2051–2066
2053
target compounds (Scheme 1). For B-ring modified
compounds, the reaction sequences were reversed. Tyro-
sine or p-bromophenylalanine methyl ester was reacted
with requisite benzoic acids (or chlorides) followed by
Suzuki coupling of the resultant benzoyl derivatives with
desired boronic acids. The resulting biphenyl compounds
were saponified to yield the target acids (Scheme 2).
For the carboxylic acid derivatives (14 and 15), the dif-
ferentially-protected diester was utilized (Scheme 4).
The carboxylic acid analogues in the dichloro series (133
and 134) were prepared from the corresponding nitrile
132, which in turn was synthesized from the bromide
130 by treatment with KCN24 in the presence of
Pd(PPh3)4 (Scheme 5).
The amine derivatives (20–43 and 136–143) were pre-
pared from the amine-ester via diverse transformations
followed by saponification (Scheme 3).
Compounds 58–60 were prepared via reductive amin-
ation of 55 with requisite amines (Scheme 6). Additional
functional group transformations in ring B( 62–66 and
68–77) are also depicted in Scheme 6. The synthesis of
85 and 87–93 was accomplished from 81 methylester as
outlined in Scheme 7.
Scheme 8 illustrates the synthesis of compounds 99–102.
The bis–MOM ether compound was deprotected with
HCl to give the bis–OH compound, which was alkyl-
ated with the requisite alkyl halide in the presence of
base to generate target compounds as methyl esters.
Scheme 6. Reagents: (a) TFA; (b) R0COCl; (c) ClSO2NCO;
(d) (RSO2)2O, DIEA; (e) R0OCOCl, DIEA; (f) HCHO, NaCNBH4;
(g) RNH2, NaCNBH4; (h) NaBH4; (i) KMnO4; (j) CDI, R0R00N;
(k) LiOH; (l) Cs2CO3, MeI.
Scheme 8. Reagents: (a) HCl; (b) Cs2CO3, R3X; (c) LiOH.
.
Scheme 7. Reagents: (a) (Bu)4NBr Br2; (b) tertrabutyl fluoropyr-
idinium tetrafluoroborate; (c) HNO3; (d) Na2S2O4; (e) Ac2O;
(f) MeNCO; (g) HCl; (h) ClSO2NCO; (i) LiOH.
Scheme 9. Reagents: (a) H2, Raney Ni; (b) Ac2O, DIEA; (c) EtO-
COCl, DIEA; (d) MeSO2Cl; (e) NaBH4; (f) Et3SiH, BF3.Et2O;
(g) LiOH.

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I. Sircar et al. / Bioorg. Med. Chem. 10 (2002) 2051–2066
Saponification resulted in the desired acids. Similar
procedures were utilized for compounds 94, 96 and 97
starting from the 2-OMe, 6-MOM compound. Com-
pounds in Table 7 (104–115) were prepared in a similar
manner.
Results and Discussion
Among the ring A substitutions studied in the des-
methoxybiphenylalanine series (e.g., halogens, CN,
OMe, SMe, NO2, CO2H, NH2, and OCF3), 2-halo
compound showed the best potency (unpublished data,
Tanabe Research laboratories). The SAR was extended
in the 2-OMe series (Table 1). A halogen substitution in
the ortho position enhanced a4b7 potency by 7- to 9-
fold (e.g., 2 and 3) as seen with the des-methoxy series.
The effects of additional substitution on the 2-chloro
compound 2 were evaluated. Compound 9, the 4-amine
derivative, retained potency similar to 2, whereas intro-
duction of an acidic group at the 4-position increased
potency. The di-acid 13 reached 15 and 200 nM potency
for a4b7 and a4b1, respectively. In addition, a 6-halo
substitution improved potency (16, a4b7/a4b1: 77/850
nM). The rank order of potency for 2,6-disubstitution
was: 2,6-diCl >2-Cl,6-F>2,6-diF=2,6-diMe. In gen-
eral, these compounds showed 10- to 60-fold selectivity
for a4b7 over a4b1.
The synthesis of C-ring modified compounds is shown
in Scheme 9. The hydroxyalkyl compounds 118–121
were prepared from the corresponding carbonyl com-
pounds via NaBH4 reduction. Compound 122 was pre-
pared from 118 via reduction with (Et)3SiH and
.
BF3 Et2O. The amine derivatives 123–125 were pre-
pared from the corresponding amine which was
obtained from the nitro compound via reduction with
Raney–Ni.
The synthesis of the requisite 2,6-di-chlorobenzoic acids
(ring A) is shown in Scheme 10. 4-Amino-2,6-dichloro-
benzoic acid was diazotized and subsequently converted
to the corresponding 4-OH, 4-Br, and 4-F derivatives.
Schemes 11 and 12 outline the synthesis of the starting
boronic acids.
Several derivatives of 9 were synthesized in order to
enhance its potency (Table 2). The acetamide 21, car-
bamate 22, urea 23 and thiourea 24 maintained a similar
potency. Further exploration of a small series of
thiourea derivatives resulted in the methyl urea 25 being
the most potent and the benzyl urea 29 being the least
potent. The methanesulfonamide 30 showed a 10-fold
Biology
All compounds were tested for a4b7 and a4b1 activity in
the RPMI and Jurkat cell adhesion assays (Tables 1–9).
IC50 values were generated by nonlinear regression from
titration curves of compounds from 10 doses and
reported as the average of a minimum of two experi-
ments as described in the Experimental. For the Jurkat-
CS1 assay, the standard error of the mean was typically
<10% for each experiment. For the RPMI assay, the
IC50 values were normalized against an internal standard.
Table 1. Inhibitory effects of the ring A modified compounds
Compd
X
Y
Z
a4b7-CS1
a4b1-CS1
IC50, mMa,b
IC50, mMa,c
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
H
Cl
Br
CF3
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
F
H
H
H
5.08
0.56
0.77
1.1
0.3
0.31
1.4
0.27
0.31
0.15
0.14
0.46
0.015
0.12
0.060
0.077
0.17
0.33
0.34
33
13
16
6.9
36
15
64
17
15
2.6
9
H
OMe
Cl
Br
NO2
NH2
SO2Me
OH
Scheme 10. Reagents: (a) Cl2, SbCl3; (b) PhCO2H, Cu; (c) HNO3,
160 ꢀC; (d) SOCl2; (e) MeOH; (f) Na2S2O4; (g) HBr, NaNO2; (h) Cu,
heat; (i) LiOH; (j) HCl, NaNO2; (k) H2O, reflux; (l) NaOH, reflux;
(m) HBF4, heat; (n) TMSI.
COt2Bu
CO2H
CONH2
CONHSO2Me
H
26
0.20
1.1
0.456
0.85
2.9
6.6
1.9
Scheme 11. Reagents: (a) H2O2, H2SO4, MeOH; (b) K2CO3, MeI,
Bu4NI, DMF; (c) n-BuLi, B(OMe)3.
H
H
H
F
Me
Me
aIC50 values were generated by nonlinear regression from titration
curves of compounds from seven doses and reported as the average of
a minimum of two experiments.
bIC50 values were normalized against an internal standard.
cStandard error of the mean was typically <10% for each experiment.
Scheme 12. Reagents: (a) H2O2, H2SO4; (b) MOMCl, K2CO3;
(3) n-BuLi, B(OMe)3.

I. Sircar et al. / Bioorg. Med. Chem. 10 (2002) 2051–2066
Table 3. Inhibitory effects of the ring Bmodified compounds
2055
improvement in potency, whereas the corresponding
N-methyl compound 31 showed a 5-fold reduction in
potency implicating the need for an acidic proton for
optimum potency. Compound 33, a trifluoro derivative
of 30, showed improved a4b7 potency (3-fold), but
reduced a4b1 potency (3-fold). Replacing the methyl
group with a phenyl group in 30 produced 34 that
approached a potency similar to 33. The potency could
not be enhanced further with additional substitution on
the phenyl moiety. Both compounds 33 and 34 dis-
played improved a4b7 selectivity by 246- and 271-fold,
respectively. The 2-thienyl compound 40 showed even
greater a4b7 potency (11 nM) and selectivity (645-fold)
relative to 34. It should be noted that a negative charge
at the 4-position improved both a4b7/a4b1 adhesion,
whereas a large hydrophobe adjacent to the acidic moi-
ety did not support a4b1 adhesion (compare 13 to 34
and 40, respectively). These data collectively implicate
the divergence of a4b7/a4b1 SAR at this site. It is
important to note that regioisomers of compound 33
lost potency in the order of 4->5->3->6-.
Compd
R
a4b7-CS1
a4b1-CS1
IC50, mMa,b
IC50, mMa,c
16
44
45
46
47
48
49
50
51
52
53
54
55
56
57
2-OMe
H
0.077
2.3
6.2
0.85
36
49
3-OMe
4-OMe
2-OCF3
2-OisoPr
2-OBu
2-OisoBu
2-Me
2-CF3
2-OH
2-CN
1.1
26
0.42
1.17
1.6
10.4
5.4
5.1
8.4
11.0
3.4
1.9
7.0
3.7
1.5
1.1
2.1
0.75
0.13
0.33
0.13
0.16
0.14
0.34
2-CHO
2-CH2OH
2-CH2OMe
Having established the optimum substitutions for ring
A, attention was turned to ring B. For ease of synthesis,
aIC50 values were generated by nonlinear regression from titration
curves of compounds from seven doses and reported as the average of
a minimum of two experiments.
bIC50 values were normalized against an internal standard.
cStandard error of the mean was typically <10% for each experiment.
Table 2. Inhibitory effects of 4-aminoderivatives of the ring A
Table 4. Inhibitory effects of the ring Bmodified compounds
Compd
Z
a4b7-CS1
a4b1-CS1 a4b1/a4b7
IC50, mMa,b IC50, mMa,c
9
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
4-NH2
4-NHMe
4-NHCOMe
4-NHCOOEt
4-NHCONH2
4-NHCSNH2
4-NHCSNHMe
4-NHCSNHEt
4-NHCSNHisoPr
4-NHCSNHPh
4-NHCSNHBn
4-NHSO2Me
4-NMeSO2Me
4-NHSO2NMe2
4-NHSO2CF3
0.31
0.49
0.33
0.20
0.14
0.14
0.030
0.12
0.32
0.31
1.5
0.041
0.20
0.052
0.013
0.021
0.049
0.073
0.15
15
49
5.7
9.6
2.4
3.3
0.59
5.7
48
100
17
48
17
24
20
48
39
77
15
32
15
Compd
R
a4b7-CS1
a4b1-CS1
IC50, mMa,b
IC50, mMa,c
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
2-CH2NH2
2-CH2NHPh
2-CH2NHCH2Ph
2-COOH
2-CONH2
2-CONHMe
2-CONMe2
2-CONHBu
2-CONHCH2Ph
2-SO2NHtBu
2-SO2NH2
2-SO2NHCOPh
2-SO2NHCOMe
2-NHCOOtBu
2-NH2
0.30
0.28
1.66
6.14
0.51
0.79
0.79
1.44
1.93
0.15
0.10
0.35
1.6
1.7
2.8
8.1
5.3
1.4
0.92
1.0
2.4
2.9
0.55
0.59
3.1
3.7
4.0
0.94
1.9
15
24
27
1.3
3.0
3.1
3.2
5.7
11
9.8
26
13
15
7.1
34
4.9
65
60
246
271
224
134
173
155
125
645
82
4-NHSO2Ph
4-NHSO2(4-FPh)
4-NHSO2(4-MePh)
4-NHSO2(4-CF3Ph)
4-NHSO2(4-OMePh)
4-NHSO2(2-MePh)
4-NHSO2(2-thienyl)
3-NHSO2CF3
2.7
0.45
0.40
0.27
0.78
0.57
0.20
0.084
0.12
0.011
0.361
0.060
2-NHCONH2
2-NHSO2Me
2-NHCOMe
2-NHCOOMe
2-NMe2
1.7
0.88
1.3
5-NHSO2CF3
6-NHSO2CF3
82
<3
0.72
>20
aIC50 values were generated by nonlinear regression from titration
curves of compounds from seven doses and reported as the average of
a minimum of two experiments.
bIC50 values were normalized against an internal standard.
cStandard error of the mean was typically <10% for each experiment.
aIC50 values were generated by nonlinear regression from titration
curves of compounds from seven doses and reported as the average of
a minimum of two experiments.
bIC50 values were normalized against an internal standard.
cStandard error of the mean was typically <10% for each experiment.

2056
I. Sircar et al. / Bioorg. Med. Chem. 10 (2002) 2051–2066
most of the SAR on ring Bwas carried out on the 2,6-
dichlorobenzoyl compound 16. Tables 3 and 4 list
monosubstituted compounds. Moving the 2-OMe group
around the ring resulted in a loss of potency implicating
the importance of the twisted conformation of the
biphenyl system. Extension of the 2-OMe group to
higher alkyloxy and -OCF3 groups also caused a loss in
potency. Compound 51, wherein the methoxy group
was replaced with a methyl group, showed reduced
potency (10- to 16-fold) implicating the need for a H-
bond acceptor at this site. This loss was partially
regained by substituting the methyl group with a tri-
fluoromethyl group (52). Compounds 53–57 where
other H-bond accepting moieties were incorporated at
Table 5. Inhibitory effects of multisubstitution in the ring B
Table 7. Inhibitory effects of the ring B-fused bicyclic compounds
Compd
R1
R2
a4b7-CS1
a4b1-CS1
IC50, mMa,b
IC50, mMa,c
Compd
R
W
n
a4b7-CS1
a4b1-CS1
IC50, mMa,b
IC50, mMa,c
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
3-OMe
4-OMe
5-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
6-OMe
H
H
H
H
4-OMe
4-OH
3-OMOM
3-OH
3-OMe
3-Br
3-F
0.13
0.1
4.0
0.74
0.578
0.087
0.12
0.108
0.062
0.208
0.046
0.197
0.074
0.313
0.029
0.061
0.036
0.029
104
105
106
107
108
109
110
111
112
113
114
115
H
OMe
OCH2OMe
OCH2CN
OH
OCH2CH3
OCH2CH2OH
OCH2CH2OMe
O(CH2)2NMe2
OMe
O
O
O
O
O
O
O
O
O
1
1
1
1
1
1
1
1
1
2
1
2
0.928
0.013
0.068
0.069
0.137
0.109
0.065
0.45
0.225
0.006
0.233
0.07
25
0.017
0.007
0.008
0.017
0.009
0.042
0.010
0.018
0.022
0.071
0.010
0.012
0.020
0.020
0.383
1.3
2.3
3.8
1.0
0.591
0.735
0.823
0.043
13.6
1.7
O
CH2
CH2
3,5-diF
3-CONH2
3-NH2
3-NHCOMe
3-NHCONHMe
H
H
aIC50 values were generated by nonlinear regression from titration
curves of compounds from seven doses and reported as the average of
a minimum of two experiments.
bIC50 values were normalized against an internal standard.
cStandard error of the mean was typically <10% for each experiment.
aIC50 values were generated by nonlinear regression from titration
curves of compounds from seven doses and reported as the average of
a minimum of two experiments.
bIC50 values were normalized against an internal standard.
cStandard error of the mean was typically <10% for each experiment.
Table 8. Inhibitory effects of the ring C modified compounds
Table 6. Inhibitory effects of replacement of OMe group in the ring B
Compd
R4
R1
a4b7-CS1
a4b1-CS1
IC50, mMa,b
IC50, mMa,c
Compd
R
R1
a4b7-CS1
a4b1-CS1
IC50, mMa,b IC50, mMa,c
16
81
H
H
OMe
H
H
H
0.077
0.007
0.040
0.17
0.027
0.047
0.155
0.008
0.21
0.85
H
OMe
COMe
CH(OH)Me
CH(OH)Et
CH(OH)Bu
CH(OH)Me
CH2Me
0.087
0.46
0.5
0.048
0.071
0.253
0.012
0.68
116
117
118
119
120
121
122
123
124
125
94
95
96
97
98
99
100
101
102
103
OMe
OMe
OMe
OMe
OEt
OEt
OH
O(CH2)2F
O
isoPr
0.027
0.031
0.039
0.245
0.16
0.12
0.56
0.146
0.509
0.19
3.1
0.545
2.9
0.183
H
H
OEt
OH
OMe
H
OMe
OMe
OMe
OH
0.5
OCH2CH2OH OCH2CH2OH
0. 282
0.676
0.055
1.34
NHCOMe
NHCOOEt
NHSO2Me
0.030
0.123
0.010
0.026
0.068
0.022
OCH2CF3
OCH2CN
Me
OCH2CF3
OCH2CN
Me
31
aIC50 values were generated by nonlinear regression from titration
curves of compounds from seven doses and reported as the average of
a minimum of two experiments.
bIC50 values were normalized against an internal standard.
cStandard error of the mean was typically <10% for each experiment.
aIC50 values were generated by nonlinear regression from titration
curves of compounds from seven doses and reported as the average of
a minimum of two experiments.
bIC50 values were normalized against an internal standard.
cStandard error of the mean was typically <10% for each experiment.

I. Sircar et al. / Bioorg. Med. Chem. 10 (2002) 2051–2066
2057
the 2-position retained reasonable potencies. However,
compounds with similar substitutions at the 3-position
showed a significant reduction in potency (data not
shown). Additional 2-substituted compounds with
H-bond donating and accepting groups are listed in
Table 4. Although the benzylamines 58 and 59 retained
moderate a4b7 potencies, the dibenzylamine 60 lost
potency. A carboxylic acid group at this position was
detrimental, whereas the carboxamides showed
improved potency (compare 61 to 62–64). The activity
of the amides decreased with the increasing size of the
amine. The sulfonamides 67 and 68 retained dual
potencies similar to the methoxy compound 16. A small
series of amine derivatives (72–77) were evaluated that
retained reasonably good potency for a4b7. The SAR
led to the hypothesis that a H-bond accepting group
and a small hydrophobe around that region are mini-
mum requirements for activity.
proline derivatives.25 Additional modifications were
carried out with 81. Incorporation of additional alkyl-
oxy groups in the 4- and 3-position of 81 (82 and 86)
maintained potency. By contrast, compounds 83 and 85,
4- and 3-OH derivatives of 81, lost potency. The fluori-
nated compounds (88–89) were synthesized to improve
the physicochemical properties. Unfortunately, the
potencies were not maintained with these analogues.
The amine and its derivatives (91–93) retained excellent
dual potency. Our next challenge was to find replace-
ments for the metabolically labile methoxy groups of 81.
Table 6 lists compounds synthesized towards this end
without much success. In general, the SAR paralleled
the monomethoxy series (Table 3). Compounds 94, 96,
and 97, wherein one of the –OMe groups was replaced
with higher alkyloxy groups, resulted in a loss of activity
with the increasing size of the alkyl group. The potency
loss was more significant in the di-alkyloxy compounds
98, 100 and 101. The electron withdrawing nature of the
fluorines in 101 reduce the H-bonding potential of the
ether oxygens compared to 98. The loss of potency (0.16
to 0.676) further supports the role of H-bonding in this
region of the molecule. The hydroxy compounds 95 and
99 showed reduced potency relative to 81, as expected.
Compound 103 lost potency similar to the monomethyl
compound 51.
The prototype compound 16 was chosen for subsequent
modification. As evident from Table 5, the introduction
of a second methoxy group impacted on potency. A 10-
fold enhancement in potency was observed with the 2,6-
diOMe compound 81 (IC50 a4b7/a4b1=7/87 nM). This
supported the initial hypothesis that the restricted rota-
tion around the Phe–Phe bond was critical for optimum
potency. 2,6-Disubstitution increased the rotational
barrier between the phenyl groups resulting in a non-
planar configuration of the phenyl groups. Similar
potency enhancement was observed in sulfonylated
A small series of compounds were synthesized in the
bicyclic system wherein the 2- and 3-methoxy groups
were incorporated into the ring (Table 7). The parent
compound 104 in the methylenedioxy series showed a
7-fold reduction in potency relative to 78. Addition of a
second -OMe group in the 6-position improved potency
71-fold. The resulting compound 105 retained a4b7
potency similar to 86; a4b1 potency was 8-fold less,
however. The replacement of the 6-methoxy group of
105 with other alkyloxy substituents caused a reduction
in potency similar to that seen in the monocyclic series
(Table 6). Compound 113, an analogous compound in
the ethylenedioxy series, gained potency similar to 81.
Similar trends were observed with compounds in the
benzofuran and benzopyran series (115 was more potent
than 114).
Table 9. Representative derivatives of compound 81
Compd X
Y
Z
a4b7-CS1 a4b1-CS1 a4b1/a4b7
IC50, mMa,b IC50, mMa,c
81 Cl Cl
126 Br Br
H
H
H
H
F
0.007
0.046
0.059
0.018
0.023
0.087
0.171
0.505
0.158
0.098
0.377
0.044
0.019
0.012
0.003
0.17
0.314
0.11
0.19
0.083
0.152
0.125
0.192
0.063
12
9
9
9
4
13
2
1
The final strategy was to modify the middle ring (ring C,
Table 8). It was rationalized that if restricted rotation is
important for activity, then addition of a substituent in
the 3-position (ortho to ring B) should improve potency.
Indeed, compound 116 improved a4b7 potency ꢁ2-fold
relative to 16. This result encouraged us to evaluate the
effects of additional H-bond donating and/or accepting
groups at this position. The initial data indicated that a
H-bond donating group might be preferred (compare
118 to 117). The corresponding dimethoxy compound
121 showed enhanced potency (ꢁ4-fold) resulting in a
balanced a4b7/a4b1 compound with IC50 values of 8 and
12 nM, respectively. The effects of higher alkyls on the
hydroxyalkyl analogue 118 were also evaluated. Data
from compounds 119 and 120 indicated that small alkyl
groups were preferred at this site. The reduced potency
associated with the deoxygenated analogue further sup-
ported the role of the H-bond donating moieties at this
site (compare 118 to 122). It is notable that compounds
127
F
F
128 Cl Me
129 Cl Cl
130 Cl Cl
131 Cl Cl
132 Cl Cl
133 Cl Cl
134 Cl Cl
135 Cl Cl
136 Cl Cl
137 Cl Cl
138 Cl Cl
139 Cl Cl
140 Cl Cl
141 Cl Cl
142 Cl Cl
Br 0.029
OH 0.018
CN 0.013
CONH2 0.0025
CO2H 0.00033
NH2 0.007
5
10
24
22
14
16
11
23
5
NHCOMe 0.014
NHCOOMe 0.008
NHCOOEt 0.012
NHCONH2 0.0075
NHCONHMe 0.0065
NHCSNHMe 0.025
NHSO2CF3 0.0005
384
119
143 Cl Cl NHSO2(2-thienyl) 0.00053
aIC50 values were generated by nonlinear regression from titration
curves of compounds from seven doses and reported as the average of
a minimum of two experiments.
bIC50 values were normalized against an internal standard.
cStandard error of the mean was typically <10% for each experiment.

2058
I. Sircar et al. / Bioorg. Med. Chem. 10 (2002) 2051–2066
118 and 121 are diastereoisomeric mixtures that were
not resolved; therefore, the net effect of the hydro-
xyethyl at this position was to greatly enhance a4b1
potency relative to a4b7 potency. Of the three-amine
derivatives (123–125), the sulfonamide compound 125
equalled the potency of 121.
General procedures for compound synthesis
N-(2,6-Dichlorobenzoyl)-4-(2-methoxyphenyl) - L - phenyl-
alanine (16). To a mixture of 2-methoxybenzeneboronic
acid (0.446 g, 2.94 mmol) and anhydrous K2CO3 (0.84
g, 6.1 mmol) in toluene/DMF (25 mL/2.5 mL) under N2
was added
a
solution of tert-Boc-l-tyrosine(tri-
Additional analogues of 81 with variations on the A-
ring are listed in Table 9. Di-Cl substitutions seems to
be optimum for dual potency (compare 81 to 126–128).
Addition of a halogen at the 4-position (129 and 130)
reduced potency 5- to 10-fold, whereas 4-OH and 4-CN
(131 and 132) had very small effects. Compound 134,
the 4-CO2H derivative, showed a 18-fold enhancement
in potency reaching subnanomolar (0.33) and low nM
(3.0) potency for a4b7 and a4b1 adhesion, respectively. The
corresponding amide 133 also retained excellent potency
relative to 81. Also, a small series of 4-amine derivatives
were evaluated. Of those, the two sulfonamides 142 and
143 showed improved potency resulting in subnanomolar
compounds, as predicted from the SAR. The sulfonamides
142 and 143 were more selective for a4b7 (384- and
119-fold, respectively) compared to the carboxylic acid
134 (10-fold). Thus, the pharmacophore requirements
[2,6-diCl, 4-CO2H in ring A, 2,6-diOMe in ring Band
3-CH(OH)Me in ring C] for best dual potency were
established.
flate)methyl ester21 (1.0 g, 2.33 mmol) in 5 mL of tolu-
ene. The catalyst Pd(PPh3)4 (0.48 g, 0.42 mmol) was
added and the mixture was heated at 80 ꢀC for 24 h. The
reaction mixture was cooled, filtered through Celite and
evaporated to dryness. The residue was taken up in
EtOAc and the solution washed with water. It was
dried, evaporated, and the crude material was purified
via flash chromatography (hexane/EtOAc, 3/1) to yield
N-tert-butoxycarbonyl-4-(2-methoxyphenyl) - l - phenyl-
alanine methyl ester (0.64 g) as a clear oil: MS m/z 386
(M++H) and 408 (M++Na). To a solution of the tert-
Boc-derivative (2.97 g, 7.3 mmol) in CH2Cl2 (20 mL)
was added TFA (20 mL) and the mixture stirred for 1.5
h. The solution was evaporated to dryness. The residue
was re-dissolved in CH2Cl2 (20 mL) and the solution
was evaporated. This process was repeated one more
time and finally the residue was dried under high
vacuum to yield the amine (2.93 g) as the TFA salt: MS
m/z 286 (M+ + H). To a solution of the above amine
salt (2.3 g, 5.8 mmol) in CH2Cl2 (30 mL) containing
DIEA (2.24 g, 3 mL, 17 mmol) at 0 ꢀC was added a
solution of 2,6-dichlorobenzoyl chloride (0.99 mL, 6.9
mmol) with stirring. The solution was warmed to room
temperature and stirred for 24 h. The solution was
washed with water, 1 N HCl, saturated NaHCO3, brine,
dried, filtered and concentrated. The residue was puri-
fied via flash chromatography (hexane/EtOAc, 4/1) to
yield 1.64 g of N-(2,6-dichlorobenzoyl)-4-(2-methoxy-
phenyl)-l-phenylalanine methyl ester: MS m/z 458 (M+
+ H) and 471 (M+ + Na). The above ester (0.1 g, 0.22
mmol) was dissolved in a mixture of THF/MeOH
(5 mL/2 mL). A solution of LiOH (14 mg, 0.33 mmol)
in water (2 mL) was added and the mixture stirred at
room temperature for 3 h. The solution was evaporated
and the residue was treated with water. The solution
was adjusted to pH 2 and the organic material was
extracted with EtOAc. The organic layer was washed
with brine, dried and evaporated to yield 0.08 g of the
titled compound: MS m/z 444 (M+ + H) and 467 (M+
Conclusion
A novel series of small molecule non-peptide a4b7
receptor antagonists has been discovered. The potency
of the initial lead compound 1 (IC50 a4b7/a4b1=5/33
mM) was optimized to generate low nM compounds via
SAR. Compound 81 (TR-14035), a dual active antago-
nist, showed a 1000-fold enhancement in potency (IC50
a4b7/a4b1=7/87 nM) compared to 1. The SAR also led
to three subnanomolar potency compounds, 134, 142,
and 143. Of those, the sulfonamide derivatives 142
and 143 showed 100- to 400-fold selectivity for a4b7.
All these compounds are fully selective at the tested
concentrations with respect to a5b1, a6b1, avb3, and
aLb2 (IC50 >20 mM). Compound 81 has been shown
to be orally absorbed in several animal species (data
not shown). Compound 81 was selected for clinical
development and has finished Phase I testing in healthy
volunteers in Europe. The pharmacological character-
ization of 81 (TR-14035) will be reported in a separate
publication.
+ Na); mp 211 ꢀC; H NMR (CDCl3) 3.32 (dd, 1H),
1
3.37 (dd, 1H), 3.78 (s, 3H), 5.2 (m, 1H), 6.3 (d, 1H), 6.88
(m, 2H), 7.20–7.38 (m, 7H), 7.42 (d, 2H). Anal. calcd for
C23H19Cl2NOꢃ40.3H2O (449.72): C, 61.37, H, 4.36, N,
3.11; found: C, 61.08, H, 4.23, N, 3.09.
N-(2,6-Dichlorobenzoyl)-4-(2,6-dimethoxyphenyl)-L-phe-
nylalanine (81). HCl (g) was bubbled into a methanolic
(100 mL) solution of (L) 4-bromo-phenylalanine (5 g)
and the mixture was left overnight at room temperature.
The solid was filtered off, washed with ether and air-
dried to give 5.54 g of the methyl ester of (L) 4-bromo-
phenylalanine, HCl salt: MS m/z 274 (M+ + H) and
296 (M+ + Na). Et3N (8 mL, 56.7 mmol) was added to
a suspension of the above methyl ester (5.55 g, 18.9
mmol) in CH2Cl2 (40 mL) at 0 ꢀC. To this was added a
solution of 2,6-dichlorobenzoyl chloride (3.52 mL, 25
Experimental
1H NMR spectra were recorded on a Bruker 300 MHz
instrument in DMSO-d6 (unless otherwise stated) with
TMS as the internal standard. Mass spectra were
obtained by electron spray method on the SCIEX
API100. Medium pressure chromatography was con-
ducted on a Biotage (4 ꢂ 15 cm, SiO2) instrument. All
flash chromatography was done with SiO2. Melting
points were uncorrected. Solutions in organic solvents
were dried with anhydrous MgSO4.

I. Sircar et al. / Bioorg. Med. Chem. 10 (2002) 2051–2066
2059
mmol) in CH2Cl2 (5 mL) and the mixture was stirred
overnight at room temperature. The reaction mixture
was diluted with CH2Cl2 and washed with water, brine,
dried, filtered and concentrated to a syrup. The residue
was purified by flash column chromatography (hexane/
EtOAc, 2/1) to yield 3.9 g of N-(2,6-dichlorobenzoyl)-
(l) 4-bromophenylalanine methyl ester: MS m/z 446
(M+ + H) and 468 (M+ + Na). This was used for the
coupling reaction.
extracts were combined, dried, filtered and evaporated.
The residue was purified by chromatotron using a gra-
dient of CHCl3/MeOH to give the sulfonylurea com-
pound (43.0 mg, 61%) as a white solid: MS m/z 560
(MÀ). The ester was saponified with 3 equiv of LiOH to
yield the titled compound (24 mg, 87%) as a white solid:
MS m/z 468 (M+ + H). 1H NMR (CD3OD) d 3.09 (dd,
1H), 3.37 (dd, 1H), 3.76 (s, 3H), 4.89 (dd, 1H), 6.97 (dt,
1H), 7.03 (dd, 1H), 7.22–7.42 (m, 6H), 7.60 (bs, 1H).
To a suspension of 2,6-dimethoxybenzeneboronic acid
(0.3 g, 1.6 mmol) and K2CO3 (0.5 g, 3.6 mmol) in DME
(10 mL) was added the above methyl ester (0.3 g, 0.7
mmol), Pd(Ph3P)4 (0.3 g, 0.26 mmol) and water (0.4
mL). The mixture was heated at 80 ꢀC for 6 h. The
reaction mixture was cooled, EtOAc and water were
added to the reaction mixture and the phases separated.
The EtOAc layer was dried, filtered and evaporated to
provide a black gum. The gum was purified by flash
column chromatography (hexane/EtOAc, 1/2) to give
0.2 g (59%) of the desired compound as a white solid:
N-[2-Chloro-4-(3-methylthioureido)benzoyl]-4-(2-methoxy-
phenyl)-L-phenylalanine (25). Methylisothiocyanate (628
mmol, 43 mL) was added to a solution of 9 methyl ester
(55.1 mg, 126 mmol) in anhydrous DMF (1 mL) con-
taining DIEA (126 mmol, 22 mL) and DMAP (cat.). The
resulting mixture was then heated at 90 ꢀC under N2 for
1 day. The mixture was taken up with EtOAc (40 mL)
and the solution was washed with 1 N HCl (3 ꢂ 20 mL),
saturated NaHCO3 (2 ꢂ 20 mL), water (1 ꢂ 20 mL),
dried, filtered and evaporated. The residue was purified
by chromatotron using CH2Cl2/MeOH (15/1) to give
the desired thiourea (22.7 mg, 35%) as a white solid:
MS m/z 512 (M+ + H). This material was saponified to
yield the titled compound (22.0 mg) as a white solid: MS m/
z 496 (M+ + H). 1H NMR (CD3OD) d 3.02 (s, 1H), 3.09
(dd, 1H), 3.38 (dd, 1H), 3.76 (s, 3H), 4.90 (dd, 1H), 6.98 (dt,
1H), 7.04 (dd, 1H), 7.23–7.43 (m, 8H), 7.60 (d, 1H).
1
MS m/z 488 (M+ + H); H NMR (CDCl3) 3.3 (dd,
2H), 3.7 (s, 6H), 3.8 (s, 3H), 5.2 (m, 1H), 6.4 (d, 1H), 6.6
(d, 2H), 7.2–7.3 (m, 8H). To a solution of the above
methyl ester (0.1 g, 0.2 mmol) in THF (5 mL) was added
aqueous LiOH (12 mg, 0.5 mmol in 0.5 mL water) and a
few drops of MeOH. The resulting mixture was stirred
at room temperature for 2 h. THF was removed, the
residue dissolved in water and the solution neutralized
with 10% citric acid to yield a white solid. The solid was
filtered, washed with water and dried to provide 80 mg
(85%) of the titled compound: MS m/z 474 (M+ + H),
N - [2 - Chloro - 4 - (methanesulfonylamino)benzoyl] - 4 - (2 -
methoxyphenyl)-L-phenyl alanine (30). MeSO2Cl (24 mL,
306 mmol) was added to a solution of 9 methyl ester
(56.0 mg, 128 mmol) in anhydrous CH2Cl2 (1 mL) con-
taining DIEA (383 mmol, 66.6 mL). The resulting mix-
ture was stirred at room temperature under N2 for 3 h
and taken up with CH2Cl2 (40 mL), washed with 1 N
HCl (3 ꢂ 20 mL), water (20 mL), dried, filtered and
evaporated. The residue was purified on a short silica
plug using CH2Cl2 as eluent to yield 59.4 mg (78%) of a
brownish solid: MS m/z 595 (M+ +H). The ester was
saponified to give the titled compound (43.4 mg, 86%)
as a white solid: MS m/z 503 (M+ +H). 1H NMR
(CD3OD) 2.99 (s, 3H), 3.08 (dd, 1H), 3.38 (dd, 1H), 3.76
(s, 3H), 4.90 (dd, 1H), 6.98 (dt, 1H), 7.03 (dd, 1H), 7.17
(dd, 1H), 7.22–7.42 (m, 8H).
472 (M- H); mp 243.6 ꢀC (dec); H NMR d 2.9 (dd, 1H),
1
3.2 (dd, 1H) , 3.7 (s, 6H), 4.72 (m, 1H), 6.7 (d, 2H), 7.1–7.5
(m, 8H), 9.1 (d, 1H). Anal. calcd for C24H21Cl2NO5: C,
60.77; H, 4.46; N, 2.95; found: C, 60.63; H, 4.44; N, 2.82.
Compound 81 was also prepared in a manner similar to 16.
N-(4-Amino-2-chlorobenzoyl)-4-(2-methoxyphenyl)-L-
phenylalanine (9). To a solution of 4-(2-methoxy-
phenyl)-l-phenylalanine methyl ester, TFA salt (1.17 g,
2.78 mmol) in DMF (110 mL) containing DIEA (2.24 g,
0.48 mL, 5.57 mmol) at 0 ꢀC was added a solution of 4-
amino-2-chlorobenzoic acid (0.48 g, 2.78 mmol) with
stirring. HOBT (0.64 g, 4.18 mmol) was added followed
by EDC (0.53 g, 2.78 mmol) and the mixture was stirred
overnight at room temperature. DMF was distilled and
the residue was dissolved in EtOAc. The solution was
washed with water, saturated NaHCO3, brine, dried,
filtered and concentrated. The residue was purified via
flash chromatography (hexane/EtOAc, 1/1) to yield 0.85
g of the titled compound methyl ester as a white solid:
MS m/z 439 (M+ + H) and 461 (M+ + Na). This was
saponified to give 0.6 g of the titled acid: MS m/z 425
(M+ + H) and 447 (M+ + Na).
N-[2-Chloro-4-(trifluoromethanesulfonylamino)benzoyl]-
4-(2-methoxyphenyl)-L-phenylalanine (33). (CF3SO2)2O
(2.91 mmol, 0.49 mL) was added dropwise to a solution
of the compound 9 methyl ester (850 mg, 1.94 mmol) in
anhydrous CH2Cl2 (20 mL) containing anhydrous pyri-
dine (5.81 mmol, 0.47 mL) at 0 ꢀC. The resulting mix-
ture was stirred at room temperature under N2 for 1 h.
The reaction mixture was taken up with CH2Cl2 (80
mL) and the solution was washed with 1 N HCl (3 ꢂ 50
mL) followed by water (50 mL), dried, filtered and eva-
porated. The residue was purified by medium pressure
chromatography (CH2Cl2/MeOH, 50/1) to afford a
mixture of mono- and di-substituted sulfonamide (750
mg) as a brownish solid: TLC (CH2Cl2/MeOH, 20/1) Rf
0.05 for mono, 0.64 for di and 0.31 for starting material.
1 N LiOH (8 mL, 8 mmol) was added to a solution of
the above mixture (750 mg) in THF/MeOH (47 mL/8 mL)
and the resulting mixture stirred at room temperature
N-(2-Chloro-4-ureidobenzoyl)-4-(2-methoxyphenyl)-L-
phenylalanine (23). Chlorosulfonylisocyanate (189
mmol, 16.4 mL) was added to a solution of 9 methyl ester
(55.2 mg, 126 mmol) in anhydrous MeCN (1 mL) and
the mixture was stirred at room temperature under N2
for 1 h. Saturated NaHCO3 (40 mL) was added slowly
and the mixture extracted with EtOAc (3 ꢂ 20 mL). The

2060
I. Sircar et al. / Bioorg. Med. Chem. 10 (2002) 2051–2066
under N2 for 2 h. The solution was concentrated and the
residue was taken up with water (75 mL). The aqueous
solution was acidified with 1 N HCl and extracted with
EtOAc (3 ꢂ 40 mL). The extract was dried, filtered and
evaporated. The residue was purified by HPLC on a C18
column with a flow rate of 90 mL/min with a gradient of
CH3CN in 0.1% aq TFA (35–60%) to yield the desired
compound (388 mg, 36% for two steps) as a beige solid:
MS m/z 557 (M+ + H), 579 (M+ + Na), 555 (MÀ); mp
134.3 ꢀC. 1H NMR d 2.96 (dd, 1H), 3.19 (dd, 1H), 3.74 (s,
3H), 4.62 (m, 1H), 7.00 (dt, 1H), 7.09 (dd, 1H), 7.22–7.41
(m, 10H), 8.85 (d, 1H).
layer was dried, filtered and evaporated. The residue
was purified through a short silica plug using hexane/
EtOAc (1/1) as eluent to yield the desired amide ester
(59.3 mg, 96%) as a white solid: MS m/z 561 (M+ +H).
The ester was saponified to yield the titled compound
(34.2 mg, 89%) as a white solid: MS m/z 547 (M+ +H).
1H NMR (CD3OD) d 3.07 (dd, 1H), 3.32 (dd, 1H), 4.32
(m, 2H), 4.98 (dd, 1H), 7.04–7.50 (m, 16H).
N-(2,6-Dichlorobenzoyl)-4-[2-(sulfamoyl)phenyl]-L-phenyl-
alanine (68). Anisole (20 mM) was added to a solution
of N - (2,6 - dichlorobenzoyl) - 4 - [2 - (N - tert - butyl-
sulfamoyl)phenyl]-l-phenylalanine methyl ester (67, 130
mg, 0.2 mmol) in TFA (2 mL) and the resulting solution
stirred at room temperature for 6 h. TFA was removed
under reduced pressure to yield 100 mg (84%) of the
desired compound methyl ester: MS m/z 507 (M+ +H).
1H NMR d 3.2 (dd, 1H), 3.3 (dd, 1H), 3.8 (s, 3H), 4.2 (s,
2H), 5.2 (m, 1H), 6.5 (d, 1H), 7.2–7.6 (m, 10H), 8.1 (d,
1H). The methyl ester (100 mg, 0.2 mmol) was saponi-
fied to provide 80 mg (83%) of the titled compound as a
N-(2,6-Dichlorobenzoyl)-4-(2-carboxyphenyl)-L-phenyl-
alanine (61). The compound 55 methyl ester (104 mg,
228 mmol, prepared by following the Scheme 1) was
dissolved in acetone (700 L) by warming to ꢁ40 ꢀC. A
warm (40 ꢀC) solution of KMnO4 in a mixture of ace-
tone/and water (900 mL/130 mL) was then added over a
1 h period and the resulting mixture was stirred at that
temperature for an additional 2 h. The MnO2 was fil-
tered over Celite and the cake washed with acetone (5
mL). The filtrate was taken up with water (30 mL) and
acidified to pH ꢁ2 with 1N HCl. The solution was
extracted with EtOAc (3 ꢂ 20 mL), the combined
extract dried, filtered and evaporated. The residue was
purified on a short silica plug using toluene followed by
a gradient of toluene/EtOAc (20:1 to 3:1) to yield the
1
white solid: MS m/z 493 (M+ +H) and 491 (MÀ). H
NMR 3.0 (dd, 1H), 3.2 (dd, 1H), 4.7 (m, 1H), 7.1 (s,
2H), 7.2–7.6 (m, 10H), 8.0 (d, 1H), 9.1 (d, 1H).
N-(2,6-Dichlorobenzoyl)-4-[2-(N-benzoylsulfamoyl)phenyl]-
L-phenylalanine (69). Benzoyl chloride (50 mL, 0.4
mmol) was added to a solution of 68 methyl ester (100
mg, 0.2 mmol) in anhydrous pyridine (5 mL) and the
reaction stirred at room temperature under N2 for 12 h.
EtOAc and saturated NaHCO3 were added to the reac-
tion mixture and the layers were separated. The EtOAc
phase was washed with 1 N HCl, dried, filtered and evap-
orated to give a gum which was purified by flash column
chromatography (hexane/EtOAc, 2/1) to yield an oil.
The ester was hydrolyzed to provide 80 mg (68%) of the
desired acid ester (85.0 mg, 79%): MS m/z 472 (M+
+
H). The ester was saponified as usual to provide the
desired dicarboxylic acid (34.1 mg, 96%) as a white
solid: MS m/z 458 (M+ + H). 1H NMR (CD3OD) 3.09
(dd, 1H), 3.33 (dd, 1H), 4.99 (dd, 1H), 7.24–7.44 (m,
9H), 7.53 (dt, 1H), 7.77 (dd, 1H).
N - (2,6 - Dichlorobenzoyl) - 4 - [2 - (carbamoyl)phenyl] - L -
phenylalanine (62). Carbonyl diimidazole (223 mmol,
36.1 mg) was added to a solution of 61 methyl ester (111
mmol, 52.6 mg) in anhydrous THF (1 mL) and the mixture
stirred at room temperature under N2 for 2 h. NH4OH
(1.11 mmol, 135 mL of 29% in water) was added and the
mixture stirred for an additional 22 h. The mixture was
then taken up with EtOAc (40 mL) and washed with
1 N HCl (3 ꢂ 20 mL), saturated NaHCO3 (3 ꢂ 20 mL)
and brine (20 mL), dried, filtered and evaporated. The
residue was purified on a short silica plug using toluene/
EtOAc (1/1) to yield the desired amide ester (48.1 mg,
92%) as a white solid: MS m/z 471 (M+ +H). The ester
was saponified to provide the titled compound (41.6 mg,
89%) as a white solid: MS m/z 457 (M+ +H). 1H NMR
(CD3OD) 3.08 (dd, 1H), 3.32 (dd, 1H), 4.98 (dd, 1H),
7.33–7.54 (m, 11H).
1
desired acid as a white solid: MS m/z 595 (MHÀ). H
NMR d 3.0 (dd, 1H), 3.2 (dd, 1H), 4.7 (m, 1H), 7.2–7.7
(m, 15H), 8.2 (d, 1H), 9.2 (d, 1H).
N-(2,6-Dichlorobenzoyl)-4-[2-(N,N-dimethylamino)phenyl]-
L-phenylalanine (77). Formalin (96 mL) and 1 N HCl (234
mL) was added to a solution of 72 methyl ester23 (100
mg, 0.2 mmol) in EtOH (5 mL). NaCNBH3 (36 mg, 0.6
mmol) was added and the resulting mixture was stirred
for 0.5 h. A mixture of EtOH and 1 N HCl (1 mL each)
were added dropwise and the reaction was stirred over-
night. Additional HCl was added and the reaction stir-
red for 0.5 h. The mixture was neutralized with
NaHCO3 and extracted with EtOAc (3 ꢂ 20 mL). The
combined organic extracts were dried, filtered and evap-
orated to give the desired ester as an oil. The ester was
saponified to give 70 mg (68%) of the titled compound
as a white solid: MS m/z 457 (M+ + H) and 455 (M).
1H NMR d 2.5 (s, 6H), 3.0 (dd, 1H), 3.2 (dd, 1H), 3.5 (s,
3H), 4.7 (m, 1H), 7.0–7.5 (m, 11H), 9.1 (d, 1H).
N-(2,6-Dichlorobenzoyl)-4-[2-(N-benzylcarbamoyl)phenyl]-
L-phenylalanine (66). To a solution of 61 methyl ester
(51.9 mg, 110 mmol) in anhydrous DMF (1 mL) was
added successively EDC (132 mmol, 25.3 mg), HOBt
(132 mmol, 20.2 mg), DIEA (165 mmol, 28.7 mL) and
benzylamine (132 mmol, 14.4 mL). The resulting mixture
was stirred at room temperature under N2 for 20 h.
EtOAc (40 mL) was added and the solution was washed
with 1 N HCl (3 ꢂ 20 mL), saturated NaHCO3 (2 ꢂ 20
mL), water (20 mL) and brine (20 mL). The organic
N-(2,6-Dichlorobenzoyl)-4-(2,6-dimethoxy-4-hydroxy-
phenyl)-L-phenylalanine (83). 3,5-Dimethoxyphenol (1 g,
6.5 mmol) was dissolved in DMF (7 mL). To this solu-
tion was added chloro-tert-butyldiphenylsilane (2 mL)
and imidazole (0.7 g) and the mixture was stirred over-
night. Water was added and the mixture was extracted

I. Sircar et al. / Bioorg. Med. Chem. 10 (2002) 2051–2066
2061
with CH2Cl2. The organic phase was dried, filtered and
concentrated to a solid. This was converted to the cor-
responding boronic acid for use in the coupling pro-
cedure (similar to 81) to yield 83 methyl ester.
Saponification gave the titled compound as a white
solid: MS m/z 581 (M+ + H). 1H NMR d 2.9 (dd, 1H),
3.2 (dd, 1H), 3.64 (s, 6H), 4.7 (m, 1H), 5.25 (s, 2H), 6.4
(s, 2H), 7.0–7.5 (m, 9H), 8.6 (d, 2H), 9.1 (d, 1H).
(10 mL) was added 2,6-dichloro-1-fluoropyridinium
tetrafluoroborate (0.353 mg, 0.95 mmol) and the mix-
ture was refluxed under N2 for 24 h. Additional quan-
tities of the above reagent (0.175 mg, 0.47 mmol) were
added and the mixture was refluxed for 24 h. The mix-
ture was concentrated, diluted with water and extracted
with CH2Cl2. The extract was washed with saturated
NaHCO3, water, dried and evaporated. The residue was
purified by chromatotron (hexane/EtOAc, 5/1 to 2/1) to
give 0.109 g of the monofluoro- and 0.011 g of the
difluoro- compounds, respectively. Upon saponifica-
tion, 0.091 g of the acid 88 [MS m/z 492 (M++ H); mp
228–229 ꢀC] and 0.008 g of the acid 89 [MS m/z 510
(M+ + H); mp 201–202 ꢀC] were obtained.
N-(2,6-Dichlorobenzoyl)-4-[2,6-dimethoxy-3-(methoxy-
methoxy)phenyl]-L-phenyl alanine (84). A solution of
2,4-dimethoxyphenol26 (3.3 g, 21.4 mmol) in acetone (20
mL) was added to a suspension of anhydrous K2CO3
(3.55 g, 25.7 mmol) in acetone (10 mL) under N2.
Chloromethyl methyl ether (1.79 mL, 23.5 mmol) was
then added dropwise and the mixture was stirred at room
temperature for 18 h followed by heating at 50 ꢀC for 24
h. Additional chloromethyl methyl ether (1.79 mL, 23.5
mmol) was added and the mixture was stirred for
another day at 50 ꢀC. The volatiles were evaporated and
the residue taken up with water (75 mL) and extracted
with EtOAc (3 ꢂ 50 mL). The combined extracts were
dried, filtered and evaporated. The residue was purified
by flash chromatography using hexane/EtOAc (20/1 to
10/1) to give the MOM-protected phenol (1.18 g, 28%)
as a colorless oil. The oil was converted to the desired
boronic acid and coupled in the usual manner to give
84 methyl ester. Saponification of the ester gave 84:
MS m/z 534 (M+ + H); mp 156–157 ꢀC.
N-(2,6-Dichlorobenzoyl)-4-(3-carbamoyl-2,6-dimethoxy-
phenyl)-L-phenylalanine (90). Chlorosulfonyl isocyanate
(45 mL, 0.517 mmol) was added to a solution of 81
methyl ester (150 mg, 0.307 mmol) in MeCN (6 mL)
under N2. The mixture was stirred at room temperature
for 2.5 h. The mixture was concentrated, 1 N HCl (8
mL) was added, and the resulting mixture was stirred at
room temperature overnight. The reaction mixture was
extracted with EtOAc (3 ꢂ 10 mL), and the extract was
dried, filtered and evaporated. The residue was purified
by preparative TLC using EtOAc to give the titled pro-
duct methyl ester (156 mg, 96%). The ester was hydro-
lyzed as usual to yield 0.14 g of 90: MS m/z 517(M+
H); mp 227–228 ꢀC.
+
N-(2,6-Dichlorobenzoyl)-4-(2,6-dimethoxy-3-hydroxyphe-
nyl)-L-phenylalanine (85). A solution of HCl in dioxane
(4 M, 1 mL) was added to a solution of 84 methyl ester
(165 mg, 0.3 mmol) in MeOH (5 mL) and the mixture
was stirred at room temperature for 3 h. The solution
was evaporated and the residue taken up with water (40
mL) and extracted with CH2Cl2 (3 ꢂ 20 mL). The
combined extracts were dried, filtered and evaporated.
The residue was purified by preparative TLC (hexane/
EtOAc, 3/1 to 1/1) to give the desired phenol methyl
ester (145 mg, 96%) as a white solid. The ester was
hydrolyzed as before to give 120 mg of 85: MS m/z 490
(M+ + H); mp 164–165 ꢀC.
N-(2,6-Dichlorobenzoyl)-4-(3-amino-2,6-dimethoxyphenyl)-
L-phenylalanine (91). HNO3 (70%, 4 mL) was added to
a solution of 81 methyl ester (1.59 g, 3.25 mmol) in THF
(4 mL) under N2 and the resulting mixture was stirred at
50 ꢀC overnight. The reaction mixture was taken up in
EtOAc (150 mL) and the solution washed with water
(100 mL). The organic phase was dried, filtered and
evaporated. The residue was dissolved in anhydrous
MeOH (100 mL) and dry HCl (g) was bubbled through
the mixture at 0 ꢀC for a few min and the solution was
stirred at room temperature overnight. The mixture was
concentrated, the residue was taken up with EtOAc (150
mL) and the solution washed with 1 N HCl (100 mL),
saturated NaHCO3 (100 mL) and brine (100 mL). The
organic layer was dried, filtered and evaporated. The
crude product was then purified by flash chromatography
(hexane/EtOAc) to give 1.1 g (58%) of the desired
nitrated methyl ester. A solution of Na2S2O4 (2.6 g, 14.9
mmol) in water (5 mL) was added dropwise to a solution
of the above nitro-compound in EtOH (40 mL) and the
reaction mixture was refluxed for 2 h and concentrated.
The residue was taken up in EtOAc (100 mL) and the
solution washed with brine (2 ꢂ 50 mL). The organic
layer was dried, filtered and evaporated. The product
was purified by preparative TLC (hexane/EtOAc) to
give 0.31 g (30%) of the desired aniline. The ester was
saponified as usual to give 0.24 g of the titled acid: MS
m/z 489 (M+ + H); mp 209–210 ꢀC.
N-(2,6-Dichlorobenzoyl)-4-(3-bromo-2,6-dimethoxyphe-
nyl)-L-phenylalanine (87). Tetrabutylammonium tri-
bromide (1.21 g, 2.51 mmol) was added to a solution of
81 methylester (1.01 g, 2.07 mmol) in CH2Cl2 (40 mL)
under N2 and the reaction stirred at room temperature
overnight. Additional tetrabutylammonium tribromide
(0.55 g, 1.14 mmol) was added and the mixture was
stirred for 1 day. The reaction mixture was washed with
water (25 mL) and the organic layer was dried, filtered
and evaporated. The crude product was purified by
flash chromatography using hexanes/EtOAc to give a
light-yellow oil (1.17 g). The ester was hydrolyzed as
usual to yield 1 g of the titled compound: MS m/z 555
(M++ H); mp 205–206 ꢀC.
N-(2,6-Dichlorobenzoyl)-4-(3-fluoro-2,6-dimethoxyphe-
nyl)-L-phenylalanine (88) and N-(2,6-dichlorobenzoyl)-4-
(3,5-difluoro-2,6-dimethoxyphenyl)-L-phenylalanine (89).
To a solution of 81 (0.232 g, 0.475 mmol) in CH3CN
N-(2,6-Dichlorobenzoyl)-4-(2,6-dihydroxyphenyl)-L-phe-
nylalanine (99). To a solution of N-(2,6-dichloro-
benzoyl)-4-[2,6-di(methoxymethoxy)phenyl]-l-phenyl-
alanine ethyl ester (0.0766 mg, 0.3 mmol) in EtOH (5 mL)

2062
I. Sircar et al. / Bioorg. Med. Chem. 10 (2002) 2051–2066
was added a solution of HCl in dioxane (4 M, 1.2 mL) and
the mixture was stirred at room temperature for 4 h. The
solvents were evaporated and the residue taken up with
ether and concentrated to yield a foam (61.6 mg): MS m/z
474 (M+ + H), 472 (MÀ H). Saponification gave 58.3 mg
of 99: MS m/z 446 (M+ + H); mp 238 C. H NMR
(400 MHZ) 2.93 (dd, 1H) , 3.12 (dd, 1H), 4.68 (m, 1H),
6.35 (d, 2H), 6.86 (t, 1H), 7.18 (d, 2H), 7.23 (d, 2H), 7.38–
7.43 (m, 3H), 9.01 (s, 2H), 9.11(d, 1H), 12.72 (br s, 1H).
3-N-Acetylamino-N-(2,6-dichlorobenzoyl)-4-(2,6-dime-
thoxyphenyl)-L-phenylalanine (123). Raney-Ni (100 mg)
was added to a degassed solution of N-(2,6,-dichloro-
benzoyl)-3-nitro-4-(2,6-dimethoxyphenyl)-l -phenylala-
nine methyl ester (1.07 g, 2.01 mmol) in MeOH (15 mL)
and H2 gas was bubbled through the mixture for 15
min. Stirring under H2 was continued for 6 h. The mix-
ture was filtered through Celite and the filter pack was
washed with MeOH. MeOH was removed and the resi-
due was purified via chromatography (hexane to hex-
ane/EtOAc, 1/1 gradient elution) to provide the desired
aniline (845 mg, 84%) as an off white solid: MS m/z 503
(M+ + H). Ac2O (45 mL, 0.472 mmol) was added to a
solution of the above aniline (119 mg, 0.236 mmol) in
CH2Cl2 (1 mL) and pyridine (57 mL, 0.708 mmol) and the
reaction was stirred at room temperature for 18 h. The
solvent was removed under reduced pressure. Chroma-
tography of the residue (hexane to EtOAc gradient elu-
tion) provided 123 methyl ester (127 mg, 99%) as a
white solid: MS m/z 545 (M+ + H). The methyl ester
was hydrolyzed to give the titled acid (98 mg, 80%) as a
white solid: MS m/z 531 (M+ + H); mp 142–144 ꢀC.
ꢀ
1
N-(2,6-Dichlorobenzoyl)-4-[2,6-di(2-hydroxyethoxy)phe-
nyl]-L-phenylalanine (100). A mixture of 99 ethyl ester
(0.39 g, 0.82 mmol), 2-bromoethyl acetate (2.72 mL,
2.47 mmol) and anhydrous K2CO3 (1.6 g, 4.93 mmol) in
DMF (3 mL) was maintained @ 90 ꢀC under stirring for
4 h. The reaction mixture was cooled to room tempera-
ture and diluted with EtOAc and water (20 mL, each).
The EtOAc layer was separated and the aqueous layer
was extracted with additional EtOAc (3 ꢂ 20 mL). The
combined extracts were dried, filtered and evaporated.
The residue was purified by flash chromatography using
hexane/EtOAc (1/1) to give 0.423 g (79.6%) of the
desired ethyl ester: MS m/z 647 (M+ + H). Saponifica-
tion gave 0.298 g (85.8%) of 100: MS m/z 534 (M+
H); mp 124–125 ꢀC.
+
N-(4-Carboxy-2,6-dichlorobenzoyl)-4-(2,6-dimethoxyphe-
nyl)-L-phenylalanine (134). Pd(PPh3)4 (0.204 mg, 2.64
mmol) was added, under Ar, to a mixture of N-(4-bromo-
2,6-dichlorobenzoyl)-4-(2,6-dimethoxyphenyl)-l-phenyl-
alanine methyl ester (130 methyl ester, 0.5 g, 0.88
mmol), KI (0.366 g, 2.2 mmol), KCN (0.172 mg, 2.64
mmol) in HMPA (5 mL) and the mixture was heated @
100 ꢀC overnight.24 The mixture was allowed to cool
and diluted with EtOAc (75 mL). The solution was
washed with saturated LiCl (15 mL) followed by brine
(50 mL), dried and evaporated. The residue was purified
via chromatotron using hexane/EtOAc (10:1 to 2:1) to
yield 0.240 mg of N-(4-cyano-2,6-dichlorobenzoyl)-4-
(2,6-dimethoxyphenyl)-l-phenylalanine methyl ester as a
yellow solid: MS m/z 513 (M+ + H). LiOH (2 mL) was
added to a mixture of the above cyano compound (0.237
mg, 0.46 mmol) in THF (2 mL) and the mixture stirred
overnight. The mixture was concentrated, taken up with
water and acidified to pH 2. The precipitated solid was
filtered and purified via HPLC to give 0.162 mg of the
titled compound as a white solid: MS m/z 518 (M+ + H);
mp 294 ꢀC. The corresponding carboxamido compound,
N-(4-Aminocarbonyl-2,6-dichlorobenzoyl)-4-(2,6-dimeth-
oxy phenyl)-l-phenylalanine (133, 31 mg) was obtained
as a side product: MS m/z 517 (M+ + H); mp 266.7 ꢀC.
N-(2,6,-Dichlorobenzoyl)-3-methoxy-4-(2-methoxyphe-
nyl)-L-phenylalanine (116). This was prepared in a man-
ner similar to 16 starting from 3-methoxy-l-tyrosine
ethyl ester: MS m/z 474 (M+ + H); mp 100–102 ꢀC.
3-Acetyl-N-(2,6-dichlorobenzoyl)-4-(2-methoxyphenyl)-L-
phenylalanine (117). This was prepared in a manner
similar to 16 starting from 3-acetyl-l-tyrosine ethyl-
ester:27 MS m/z 486 (M+ + H); mp 87–89 ꢀC.
N - (2,6 - Dichlorobenzoyl) - 3 - (1 - hydroxyethyl) - 4 - (2 -
methoxyphenyl)-L-phenylalanine (118). NaBH4 (12 mg,
0.31 mmol) was added to a solution of 117 ethyl ester
(0.1 g, 0.21 mmol) in MeOH (3 mL) and the mixture
was stirred at room temperature for 2 h. The reaction
was quenched with 1 N HCl and extracted with CH2Cl2.
The extract was washed successively with 1 N HCl and
brine, dried and concentrated. Chromatography of the
residue (hexane/EtOAc, 3/1) gave 45 mg of the desired
ester: MS m/z 516 (M+ + H). Saponification gave 28
mg of the titled acid as a white solid: MS m/z 488 (M+
+ H); mp 103–105 ꢀC.
N-(2,6,-Dichlorobenzoyl)-3-ethyl-4-(2-methoxyphenyl)-L-
phenylalanine (122). To a solution of 118 ethyl ester
(0.23g, 0.446 mmol) in CH3CN ( 10 mL) at 0 ꢀC was
added Et3SiH (0.156 mg, 1.34 mmol) followed by
3-Methoxy-L-tyrosine. 3,4-Dihydroxy-l-phenylalanine
methyl ester was prepared by bubbling HCl (g) into a
solution of the corresponding acid (10 g) in methanol
(100 mL). tert-Boc-anhydride (12.1 g, 55.84 mmol) was
added to a solution of the ester in THF (250 mL) and
DIEA (35.4 mL, 203 mmol). The mixture was warmed
for 5 min and stirred for 1 h at room temperature. THF
was removed and the residue was partitioned between
water and EtOAc. The organic layer was separated,
washed with 1 N HCl, brine and dried (MgSO4) and
stripped. The residue was purified by chromatography
(Biotage, hexane/EtOAc, 1/1) to yield the desired tert-
boc-methylester derivative (13.4 g): MS m/z 312 (M+ +H)
and 334 (M+ + Na). 2,6-Dichlorobenzyl chloride
.
BF3 Et2O (0.064 mg, 0.445 mmol) and the mixture was
warmed to room temperature. The reaction was stirred
for 1 h and quenched with MeOH/water. The mixture
was diluted with CH2Cl2 and the layers separated. The
aqueous solution was extracted with additional CH2Cl2
and the organic layers were combined. The combined
solution was dried, evaporated and the residue purified
by chromatotron (hexanes/EtOAc, 2/1) to give 0.117 mg
of the desired ester. Saponification of the ester gave 0.1
g of 122: MS m/z 472 (M+ + H); mp 105–107 ꢀC.

I. Sircar et al. / Bioorg. Med. Chem. 10 (2002) 2051–2066
2063
(1.73 g, 8.83 mmol), was added with stirring to a sus-
pension of the above phenylalanine derivative (2.5 g,
8.03 mmol), K2CO3 (2.22 g, 16.06 mmol) and n-Bu4NI
(0.297 g, 0.803 mmol) in DMF (15 mL) at room tem-
perature. The reaction mixture was stirred overnight at
room temperature. The mixture was diluted with water
(ꢁ20 mL) and the solution was extracted with ether (30
mL). The organic layer was separated and the aqueous
phase was extracted with additional ether (2 ꢂ 30 mL).
The combined ether extract was dried, filtered and the
solution was stripped to a light yellow oil. The oil was
purified by chromatography (Biotage, hexane/CH2Cl2/
EtOAc. 5/5/1) to yield three products: tert-Boc-3,4-di-
O-(2,6-dichloro)benzyl [2.0 g; MS m/z 630 (M+ + H)],
tert-Boc-4-hydroxy, 3-O-(2,6-dichloro)benzyl [0.39 g;
MS m/z 470 (M+ + H)] and tert-Boc-3-hydroxy-4-O-
(2,6-dichloro)benzyl [0.45. g; MS m/z 470 (M+ + 1)]
phenylalanine derivatives, respectively. CH3I (0.072 mL,
1.15 mmol) was added with stirring to a suspension of
above tert-boc-3-hydroxy-4-O-(2,6-dichlorobenzyl)phenyl-
alanine methyl ester (0.45 g, 0.96 mmol), K2CO3 (0.199
g, 1.44 mmol) and n-Bu4NI (0.035 g, 0.096 mmol) in
DMF (4.0 mL) and the reaction mixture was stirred
overnight at room temperature. DMF was removed and
the residue was partitioned between water (3.0 mL) and
EtOAc (20 mL). The organic layer was separated and
the aqueous phase was extracted with additional EtOAc
(2ꢂ, 20 mL). The combined EtOAc extract was dried,
filtered and concentrated to a colorless oil. The oil was
purified via chromatography (Chromatotron, 2 mm
rotor, hexane/CH2Cl2/EtOAc, 3/3/1) to yield 0.396 g of
the desired product as a yellow gum: MS m/z 484 (M+
+H). Hydrogen was bubbled with stirring to a suspension
of the above methyl ester (0.39 g, 0.81 mmol) containing
10% Pd/C (wet, degussa,0.05 g) in MeOH (10 mL) for
24 h. The catalyst was filtered over Celite and the filtrate
was stripped to a solid which was purified via chroma-
(0.6 mL). An aqueous solution of 30% H2O2 (4.7 mL,
ꢁ41.1 mmol) was added to the reaction mixture over 5
min at 0 ꢀC.28 The mixture was warmed to room tem-
perature and stirred for an additional 18 h. The solvent
was removed under high vacuum and the residue taken
up in H2O (30 mL) and extracted with CH2Cl2. The
combined organic layers were dried, filtered and the
solvent evaporated. Chromatography of the residue
(hexane to hexane/EtOAc 3/1 gradient elution) pro-
vided 6-hydroxy-1,4-benzodioxan (3.85 g, 80%) as a
colorless oil: MS m/z 153 (M+ + H). CH3I (2.3 mL,
37.75 mmol) was added to a mixture of 6-hydroxy-1,4-
benzodioxan (3.83 g, 25.17 mmol), K2CO3 (7.0 g, 50.34
mmol) and n-Bu4NI (186 mg, 0.50 mmol) in DMF (10
mL) and the mixture was stirred under N2 at room
temperature for 24 h. The resulting slurry was filtered
and the filter pack was rinsed with EtOAc (3 ꢂ 15 mL).
The EtOAc solution was washed with brine (15 mL),
dried and concentrated. Chromatography of the residue
(hexane to hexane/EtOAc 4/1 gradient elution) pro-
vided 6-methoxy-1,4-benzodioxan (3.25 g, 78%) as a
colorless oil: MS m/z 167 (M+ + H).
The corresponding boronic acid was prepared by the
procedure described above.
5-Methoxy-1,3-benzopyran-4-boronic acid. This was
prepared from sesamol in a similar manner.
2,3-Dihydrobenzo[b]furan-7-boronic acid and 3,4-dihy-
dro-2H-benzopyran-8-boronic acids. These were pre-
pared from the corresponding bromides28,29 using
standard procedures.
Synthesis of benzoic acid intermediates
Methyl 4-amino-2,6-dichlorobenzoate. Thionyl chloride
(40 mL) was added to a solution of 2,6-dichloro-4-
nitrobenzoic acid30 (12.8 g, 54 mmol) in anhydrous
CH2Cl2 (60 mL) and the resulting mixture was refluxed
for 19 h. The reaction mixture was allowed to cool to
room temperature and evaporated. Additional CH2Cl2
(10 mL) was added and the solution was evaporated
again. MeOH (100 mL) was added slowly to the residue
and the mixture was refluxed for 17 h. The mixture was
allowed to cool to room temperature and was then
placed in an ice-bath. The precipitated product was
collected by filtration to give a white solid (10.8 g, 80%).
Methyl 2,6-dichloro-4-nitrobenzoate (10.8 g, 43.4 mmol),
thus obtained, was slurried in EtOH (250 mL) and a
solution of Na2S2O4 (45 g, 220 mmol) in water (100 mL)
was added dropwise. The mixture was refluxed for 2 h and
stirred at room temperature overnight. It was filtered and
concentrated to a white solid which was dissolved in 1 N
HCl (250 mL) and stirred for 2 h. The mixture was neu-
tralized with 10% NaOH and extracted with EtOAc (3 ꢂ
75 mL). The combined organic layers were dried, filtered,
evaporated and the resulting residue was re-crystallized
using EtOAc and hexane to give methyl 4-amino-2,6-
dichlorobenzoate as a yellowish solid (7.48 g, 78%).
tography (Chromatotron,
1
mm rotor, CH2Cl2/
CH3OH, 10/1) to yield 0.21 g of the titled compound as
a white solid: MS m/z 348 (M+ + Na). This material
was used to prepare 116 in a manner similar to 16.
Synthesis of boronic acids: prototypic procedures
2,6 - Dimethoxybenzeneboronic acid. 1,3 - Dimethoxy-
benzene (4 g, 0.029 mol) was dissolved in freshly distilled
THF (10 mL). This solution was cooled to À78 ꢀC and n-
BuLi (24 mL, 1.6 M solution in hexanes) was added drop-
wise to the cold solution. The reaction mixture was stirred
at À78 ꢀC for 1 h, then warmed to room temperature and
stirred for an additional 1 h. The resulting mixture was
cooled again to À78 ꢀC and (MeO)3B(6.7 mL, 0.06 mol)
was added. The resulting mixture was allowed to warm to
room temperature and stirred overnight. Water (10 mL)
was added and the solution was stirred for 0.5 h, then
acidified to pH 4 with acetic acid and extracted with EtOAc
(50 mL ꢂ 3). The combined ether layers were dried, filtered
and evaporated to give 2,6-dimethoxybenzeneboronic acid
as a gum that was used without further purification.
6-Methoxy-1,4-benzodioxan-5-boronic acid. 1,4-Benzo-
dioxan-6-carboxaldehyde (5.20 g, 31.65 mmol), was
dissolved in MeOH (60 mL) containing concd H2SO4
2,6-Dichloro-4-fluorobenzoic acid. Methyl 4-amino-2, 6-
dichlorobenzoate (0.5 g, 2.27 mmol) was suspended in

2064
I. Sircar et al. / Bioorg. Med. Chem. 10 (2002) 2051–2066
15% HCl (10 mL). The suspension was stirred for 30
min and cooled to 0–5 ꢀC. Diazotization was done by
portionwise addition of NaNO2 (188 mg, 2.73 mmol).
After each addition, the flask was stoppered and stirred
for ꢁ5 min. The mixture was then stirred for 30 min at
that temperature and pre-cooled HBF4 (0.46 mL, 2.5
mmol) was added rapidly. Stirring was continued for 30
min. The tetrafluoroborate salt was filtered and
washed successively with cold water (10 mL), MeOH
(10 mL) and ether (10 mL). The solid was then dried
over concd H2SO4 in a vacuum desiccator for a few
days. The dry tetrafluoroborate salt was heated with
a Bunsen burner until it started to decompose. It was
then heated as necessary until the entire solid was mel-
ted. The fluoroboric acid fumes were collected over
water via a distilling apparatus. The product was
recovered with Et2O from the reaction flask, as well as
the distilling apparatus and the receiving flask. The
ether was evaporated and the crude product was pur-
ified by preparative TLC. The compound was loaded
with CH2Cl2 and eluted using hexane/EtOAc (50/1 to
20/1) to give methyl 2,6-dichloro-4-fluorobenzoate
(241 mg, 48%). TMSI (0.2 mL, 1.4 mmol) was added
to a solution of the above ester (157 mg, 0.7 mmol)
in CCl4 (5 mL). The mixture was then stirred under
N2 at 50 ꢀC for 2 days. Water (5 mL) was added and the
mixture was stirred for 1 h. HCl (1 N, 25 mL) was then
added and the mixture was extracted with EtOAc (3 ꢂ 30
mL). The combined organic layers were dried, filtered
and evaporated. The residue was purified on a short silica
plug using a CHCl3/MeOH gradient to give the desired
2,6-dichloro-4-fluorobenzoic acid (85 mg, 58%).
100 ꢀC. The flask was temporarily taken off of the oil
bath when nitrogen evolution was too strong. The mix-
ture was stirred at 100 ꢀC for an additional 30 min.,
diluted with water (100 mL) and extracted with EtOAc
(3 ꢂ 50 mL). The combined extracts were dried, filtered
and evaporated. The residue was purified by flash chro-
matography (hexane/EtOAc, 50/1) to give the desired
bromo ester as a pale yellow oil (1.07 g, 83%). LiOH
(1 M, 7.47 mL) was added to a solution of the above
ester (1.06 g, 3.73 mmol) in THF/MeOH (50 mL, 6/1),
and the reaction mixture was refluxed for 1 day. The
mixture was evaporated, taken up with water (50 mL)
and acidified with 1 N HCl. The aqueous solution was
extracted with EtOAc (3 ꢂ 50 mL), dried, filtered and
evaporated to give 4-bromo-2,6-dichlorobenzoic acid
(0.94 g, 99%) as a pale-yellow solid. The above acid
(0.93 g, 3.45 mmol) was dissolved in CH2Cl2 (20 mL),
thionyl chloride (2.51 mL, 34.5 mmol) was added and
the mixture was refluxed for 5 h. The mixture was then
evaporated, co-evaporated with CH2Cl2 (2 ꢂ 10 mL),
dried under vacuum and used for the coupling reaction.
2-Chloro-4-N-methylaminobenzoic acid. A solution of
tert-Boc-anhydride (1.39 g, 6.41 mmol) in dioxane (15
mL) was added with stirring to a solution of 4-amino-2-
chloro-benzoic acid (1.0 g, 5.83 mmol) in NaOH (1 N,
12.82 mL, 12.8 mmol) at 0 ꢀC. The reaction mixture was
warmed up to room temperature and stirred overnight.
Dioxane was distilled, water was added to the residue
and the solution was extracted with ether. The aqueous
solution was acidified and the precipitate was filtered,
washed with 1 N HCl, water and dried under vacuum to
yield 2.7 g of the desired N-Boc acid: MS m/z 294 (M+
+ Na). NaOMe (0.22 g, 4.1 mmol) was added, under
N2, to a solution of the above acid (0.52 g, 1.85 mmol)
in DMF (7 mL). The mixture was cooled in ice and
MeI (0.5 L, 7.5 mmol) was added. After stirring for 6 h,
DMF was removed and the residue was partitioned
between EtOAc and water. The EtOAc layer was sepa-
rated and the aqueous layer was extracted with addi-
tional EtOAC. The combined EtOAc extract was
washed with water, dried, filtered, and evaporated.
Flash chromatography of the residue (hexane/EtOAc, 1/
1) provided 0.27 g of 2-chloro-4-N-(boc)methylamino-
benzoic acid methyl ester: MS m/z 322 (M+ + Na).
TFA (3 mL) was added to a solution of the above Boc-
ester (0.21 g) in CH2Cl2 (10 mL) and the solution was
stirred for 2 h. Workup similar to that described for 16
gave 0.13 g of 2-chloro-4-N-methylaminobenzoic acid
methyl ester: MS m/z 200 (M+ + H). The methyl ester was
hydrolyzed to give 81 mg of the titled acid that was used as
is for the coupling reaction: MS m/z 185 (M+ + H).
2,6-Dichloro-4-hydroxybenzoic acid. Methyl 4-amino-
2,6-dichlorobenzoate (0.5 g, 2.27 mmol) was diazotized
with NaNO2 (188 mg, 2.73 mmol) as described above.
The reaction mixture was stirred for 30 min at 0–5 ꢀC
and added dropwise to boiling water (50 mL). The
resulting solution was refluxed for 2 h, allowed to cool
to room temperature and extracted with EtOAc (3 ꢂ 50
mL). The extract was dried, filtered and evaporated.
The crude product was then purified twice by pre-
parative TLC using CH2Cl2 as the solvent to give the
desired ester as a dark orange oil which solidified on
standing (275 mg, 55%). NaOH (1 N, 3.6 mL) was
added to a solution of the above ester (265 mg, 1.2
mmol) in THF/MeOH (25 mL, 6/1) and the mixture was
refluxed for 1 day. Additional NaOH (1 N, 3.6 mL) was
added and the mixture was refluxed for an additional 24 h.
The mixture was evaporated and the residue was taken up
with water (50 mL). The aqueous solution was acidified
with 1 N HCl and extracted with EtOAc/MeOH (95/5, 3
ꢂ 50 mL). The extract was dried, filtered and evapo-
rated to give the desired acid as a brownish solid (248
mg). This material was used for the coupling reaction.
2-Chloro-4-(N-methylsulfonyl)methylaminobenzoic acid.
MeSO2Cl (0.4 mL, 5 mmol) was added under N2 to a
solution of 2-chloro-4-N-methylamino benzoic acid
methyl ester (0.25 g, 1.25 mmol) in CH2Cl2 (20 mL)
containing pyridine (0.4 mL, 5 mmol). The mixture was
refluxed for 4 h. Additional CH2Cl2 (40 mL) was added
and the solution was washed successively with 1 N HCl
(3 ꢂ 20 mL) and water. It was dried, filtered and eva-
porated. The residue was purified via chromatography
(hexane/EtOAc, 3/1 to 1/1) to give 0.26 g of the desired
4-Bromo-2,6-dichlorobenzoyl chloride. Methyl 4-amino-
2,6-dichlorobenzoate (1.00 g, 4.54 mmol) was suspended
in 40% aq HBr and the mixture was cooled to 0–5 ꢀC.
NaNO2 (376 mg, 5.45 mmol) was added in small por-
tions. After each addition, the flask was stoppered and
stirred for ꢁ5 min. Copper powder (100 mg, 1.6 mmol)
was added and the mixture was slowly warmed to

I. Sircar et al. / Bioorg. Med. Chem. 10 (2002) 2051–2066
2065
methyl ester: MS m/z 278 (M+ + H), 300 (M+ + Na).
References and Notes
The methyl ester was hydrolyzed to give 85 mg of the
titled acid that was used as is for the coupling reaction :
MS m/z 264 (M+ + H).
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internal standard. For the Jurkat cell adhesion assay,
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and adhesion buffers. Standard error of the mean for
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Acknowledgements
The authors thank Dr. Kenneth Locke for critical
review of the manuscript. We also express our gratitude
to Tanabe Seiyaku Co. Ltd., Japan for their support in
this project.
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