Note: Descriptions are shown in the official language in which they were submitted.
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
TITLE OF THE INVENTION
SUBSTITUTED N-ARYLSULFONYL-PROLINE DERIVATIVES AS POTENT
CELL ADHESION INHIBITORS
SUMMARY OF THE INVENTION
The compounds of the present invention are antagonists of the VLA-4
integrin ("very late antigen-4"; CD49d/CD29; or o~q.(31), the oc~.(3~ integrin
(LPAM-1
and ocq.~3p), and/or the cc9~i1 integrin, and are useful in the treatment,
prevention and
suppression of diseases mediated by VLA-4-, c~q.(3~-, and/or x9(31-binding and
cell
adhesion and activation.
BACKGROUND OF THE INVENTION
The present invention relates to potent substituted N-arylsulfonylated-
proline derivatives which are useful for the inhibition and prevention of
leukocyte
adhesion and leukocyte adhesion-mediated pathologies. This invention also
relates to
compositions containing such compounds and methods of treatment using such
compounds.
Many physiological processes require that cells come into close contact
with other cells andlor extracellular matrix. Such adhesion events may be
required for
cell activation, migration, proliferation and differentiation. Cell-cell and
cell-matrix
interactions are mediated through several families of cell adhesion molecules
(CAMs)
including the selectins, integrins, cadherins and immunoglobulins. CAMs play
an
essential role in both normal and pathophysiological processes. Therefore, the
targeting of specific and relevant CAMs in certain disease conditions without
interfering with normal cellular functions is essential for an effective and
safe
therapeutic agent that inhibits cell-cell and cell-matrix interactions.
The integrin superfamily is made up of structurally and functionally
related glycoproteins consisting of a and b heterodimeric, transmembrane
receptor
molecules found in various combinations on nearly every mammalian cell type.
VLA-4 ("very late antigen-4"; CD49d/CD29; or cxq.(31) is an integrin expressed
on all
leukocytes, except platelets and mature neutrophils, including dendritic cells
and
macrophage-like cells and is a key mediator of the cell-cell and cell-matrix
interactions of these cell types. The ligands for VLA-4 include vascular cell
adhesion
molecule-1 (VCAM-1) and the CS-1 domain of fibronectin (FN). VCAM-1 is a
member of the Ig superfamily and is expressed i~ vivo on endothelial cells at
sites of
-1-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
inflammation. VCAM-1 is produced by vascular endothelial cells in response to
pro-
inflammatory cytokines The CS-1 domain is a 25 amino acid sequence that arises
by
alternative splicing within a region of fibronectin. A role for VLA-4/CS-1
interactions in inflammatory conditions has been proposed (see M. J. Elices,
"The
integrin ocq.(31 (VLA-4) as a therapeutic target" in Cell Adhesion and Human
Disease,
Ciba Found. Symp., John Wiley & Sons, NY, 1995, p. 79).
ocq.(37 (also referred to as LPAM-1 and cxn.~3p) is an integrin expressed
on leukocytes and is a key mediator of leukocyte trafficking and homing in the
gastrointestinal tract. The ligands for aq.(37 include mucosal addressing cell
adhesion
molecule-1 (MadCAM-1) and, upon activation of ocq.(37~ VCAM-1 and fibronectin
(Fn). MadCAM-1 is a member of the Ig superfamily and is expressed in vivo on
endothelial cells of gut-associated mucosal tissues of the small and large
intestine
("Peyer's Patches") and lactating mammary glands. MadCAM-1 can be induced in
vitro by proinflammatory stimuli. MadCAM-1 is selectively expressed at sites
of
lymphocyte extravasation and specifically binds to the integrin, ocq.(37.
The oc9(31 integrin is found on airway smooth muscle cells, non-
intestinal epithelial cells, and neutrophils, and, less so, on hepatocytes and
basal
keratinocytes. Neutrophils, in particular, are intimately involved in acute
inflammatory responses. Attenuation of neutrophil involvement and/or
activation
would have the effect of lessening the inflammation. Thus, inhibition of
cc9(31
binding to its respective ligands would be expected to have a positive effect
in the
treatment of acute inflammatory conditions.
Neutralizing anti-a,4 antibodies or blocking peptides that inhibit the
interaction between VLA-4 and/or ocq.(37 and their ligands have been shown
efficacious both prophylactically and therapeutically in several animal models
of
disease, including i) experimental allergic encephalomyelitis, a model of
neuronal
demyelination resembling multiple sclerosis; ii) bronchial hyperresponsiveness
in
sheep and guinea pigs as models for the various phases of asthma; iii)
adjuvant-
induced arthritis in rats as a model of inflammatory arthritis; iv) adoptive
autoimmune
diabetes in the NOD mouse; v) cardiac allograft survival in mice as a model of
organ
transplantation; vi) spontaneous chronic colitis in cotton-top tamarins which
resembles human ulcerative colitis, a form of inflammatory bowel disease; vii)
contact hypersensitivity models as a model for skin allergic reactions; viii)
acute
nephrotoxic nephritis; ix) tumor metastasis; x) experimental autoimmune
thyroiditis;
-2-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
xi) ischemic tissue damage following arterial occlusion in rats; and xii)
inhibition of
TH2 T-cell cytokine production including IL-4 and IL-5 by VLA-4 antibodies
which
would attenuate allergic responses (J.Clinical Investigation 100, 3083 (1997).
The
primary mechanism of action of such antibodies appears to be the inhibition of
lymphocyte and monocyte interactions with CAMS associated with components of
the
extracellular matrix, thereby limiting leukocyte migration to extravascular
sites of
injury or inflammation and/or limiting the priming and/or activation of
leukocytes.
Animal models of these diseases may also be used to demonstrate efficacy of
small
molecule VLA-4 antagonists
There is additional evidence supporting a possible role for VLA-4
interactions :in other diseases, including rheumatoid arthritis; various
melanomas,
carcinomas, and sarcomas, including multiple myeloma; inflammatory lung
disorders;
acute respiratory distress syndrome CARDS); pulmonary fibrosis;
atherosclerotic
plaque formation; restenosis; uveitis; and circulatory shock (for examples,
see A. A.
Postigo et al., "The ec4(31/VCAM-1 adhesion pathway in physiology and
disease.",
Res. Immunol., 144, 723 (1994) and J.-X. Gao and A. C. Issekutz, "Expression
of
VCAM-1 and VLA-4 dependent T-lymphocyte adhesion to dermal fibroblasts
stimulated with proinflammatory cytokines." Immunol. 89, 375 (1996)).
At present, there is a humanized monoclonal antibody (Antegren",
Athena Neurosciences/Elan ) against VLA-4 in clinical development for the
treatment
of multiple sclerosis and Crohn's disease and a humanized monoclonal antibody
(ACT-1°/LDP-02 LeukoSite) against aq.(37 in clinical development for
the treatment .
of inflammatory bowel disease. There are also several VLA-4 antagonists in
early
clinical trials for treatment of asthma and arthritis. There still remains a
need for
potent law molecular weight inhibitors of VLA-4-, ocq.(37- and/or ec9(31
dependent cell
adhesion that have pharmacokinetic and pharmacodynamic properties suitable for
use
as human pharmaceuticals.
PCT Application No. W098/53818 discloses compounds having
activity as inhibitors of binding between VCAM-1 and cells expressing VLA-4,
and
having the formula:
R3 R4 I 5.
R1% O~.N N / \X
R2 O R6 R~
-3-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
PCT Application No. W098/53814 discloses compounds having
activity as inhibitors of binding between VCAM-1 and cells expressing VLA-4,
and
having the formula:
R7
6 B-~-~ 8 R3
R A ~R Nv X
N R~ 4
R1~Y p R R
PCT Application No. W098/53814 discloses compounds having .
activity as inhibitors of binding between VCAM-1 and cells expressing VLA-4,
and
having the formula:
Rs R4 R5
i
RwY~N~N~X~
R2 O R6 R
PCT Application Nos. W099106390, W099/06431, WO99/06432,
W099/06433, W099/06434, WO99106435, W099/06436, and W099/06437 disclose
compounds having activity as inhibitors of binding between VCAM-1 and cells
expressing VLA-4, and having the formula:
H,R4 R5
R1,S02~N~Q~OH
R2 R3 I IO
-4-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compounds of Formula I:
R4b R3a 3b
R
R4a R2 H
~ )n,N~N CO2R1
j II O X
Ari~s02 O Ar2 R5
l ~~
~A
I
or a pharmaceutically acceptable salt thereof wherein:
A is 1) N,
2) N+_O_~
X and Y are independently
selected from
1) halogen,
2) C 1 _3 alkyl,
3) C1_3alkoxy;
R1 is 1) hydrogen,
2) C1_l0alkyl,
3) aryl-C1-l0alkyl;
R2 is 1) hydrogen or
2) C1-l0alkyl;
one of R3a and
R3b is selected
from hydrogen,
C1-l0alkyl, C2_l0alkenyl,
C3-lOcycloalkyl, C02Rd, aryl and heteroaryl, and the
- other is chosen from
1) hydrogen,
2) C1-l0alkyl,
3) C2-l0alkenyl,
4) C2_ l0alkynyl,
5) C3-lOcYcloalkyl,
6) -ORd,
7) -CO2Rd,
8) -C(O)NRdRe,
9) -NRdRe>
10) -NRdS(O)mRe,
-5-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
11) -NRdC(O)Re,
12) -NRdC(O)ORe,
13) -NRdC(O)NRdRe,
14) aryl, and
15) heteroaryl,
wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four
substituents independently selected from Ra, and aryl and heteroaryl are
optionally
substituted with one to four substituents independently selected from Rb;
one of R4a and R4b is hydrogen, C1-l0alkyl, C~_lpalkenyl, C3_l0cycloalkyl,
CO~Rd, aryl and heteroaryl, and the other is chosen from
1) hydrogen,
2) C1_l0alkyl,
3) C2_l0alkenyl,
4) C2-l0alkynyl,
5) C3-lOcycloalkyl,
6) -ORd,
-C02Rd~
8) -C(O)NRdRe,
9) -~dRe
10) -NRdS(O)mRe,
11) -NRdC(O)Re,
12) -NRdC(O)ORe,
13) -NRdC(O)NRdRe,
14) -CN,
15) aryl, and
16) heteroaryl,
wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four
substituents independently selected from Ra, and aryl and heteroaryl are
optionally
substituted with one to four substituents independently selected from Rb; or
R4a and R4b together is oxo;
RS is 1) hydrogen;
2) OH;
3) OCH3; or
4) NHZ ;
-6-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Ra is 1) -ORd,
2) -NRdS(O)mRe,
3) -N02,
4) halogen
5) -S(O)mRd,
6) -SRd,
-s(O)20Rd~
-S(O)m~dRe~
9) -NRdRe~
l.0) -O(CRfRg)nNRdRe,
11) -C(O)Rd,
12) -C02Rd,
13) -C02(CRfRg)nCONRdRe,
14) -OC(O)Rd,
15) -CN,
16) -C(O)NRdRe,
1~) -~dC(O)Re~
18) -OC(O)NRdRe,
19) -NRdC(O)ORe,
20) -NRdC(O)NRdRe,
21) -CRd(N-ORe),
22) CF3,
23) -OCF3,
24) C3-gcycloalkyl, or
25) heterocyclyl;
wherein cycloalkyl
and heterocyclyl
are optionally
substituted
with one to
four groups
independently ed from Rc;
select
Rb is 1) a group selected from Ra,
2) C1-10 alkyl,
3) C2_lp alkenyl,
4) C2-10 alkynyl,
5) Arl,
6) C1_l0alkyl-Arl,
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
wherein alkyl, alkenyl, alkynyl, and Arl are optionally substituted with one
to four
substituents selected from a group independently selected from Rc;
Rc is 1) halogen,
2) amino,
3) carboxy,
4) C 1 _q.alkyl,
5) C1_q.alkoxy,
6) aryl,
7) aryl C 1 _q.alkyl,
8) hydroxy,
. 9) CF3,
10) OC(O)C1_q.alkyl,
11) OC(O)NRfRg, or
12) aryloxy;
Rd and Re are independently selected from hydrogen, C1-l0alkyl,
C2_lpalkenyl, C2_l0alkynyl, Cy and Cy C1_l0alkyl, wherein alkyl, alkenyl,
alkynyl
and Cy are optionally substituted with one to four substituents independently
selected
from Rc; or
Rd and Re together with the atoms) to which they are attached form a
heterocyclic
ring of 4 to 7 members containing 0-2 additional heteroatoms independently
selected
from oxygen, sulfur and N-Rh;
Rf and Rg are independently selected from hydrogen, C 1 _ l0allcyl, Cy and
Cy-C1_l0alkyl; or
Rf and Rg together with the carbon to which they are attached form a ring of 5
to 7
members containing 0-2 heteroatoms independently selected from oxygen, sulfur
and
nitrogen;
Rh is selected from Rf and -C(O)Rf;
Cy is selected from cycloalkyl, heterocyclyl, aryl, and heteroaryl;
Arl is selected from phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl and
triazinyl
each optionally substituted with one or two groups independently selected from
Rc;
Ar2 is 1,4-phenylene or 2,5-pyridylene;
m is 1 or 2;
n is 0, 1 or 2.
_g_
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
In one embodiment of formula I Arl is pyridyl optionally substituted
with C1-3alkyl, or phenyl optionally substituted with one to two groups
independently
selected from halogen, Cl_3alkyl, phenyl, trifluoromethyl, and
trifluoromethoxy. In
one subset of this embodiment Arl is 3-substituted phenyl optionally having a
second
substituent on the 4- or 5-position wherein the substituents are independently
selected
from chloro, fluoro, bromo, methyl, phenyl, trifluoromethyl and
trifluoromethoxy. In
another subset Arl is 3-chlorophenyl or 3,5-dichlorophenyl. Examples of Arl
include
phenyl, 3-bromophenyl, 3-chlorophenyl, 3-fluorophenyl, 3-biphenyl, 3-tufluoro-
methylphenyl, 3-trifluoromethoxyphenyl, 4-chlorophenyl, 4-methylphenyl, 3,5-
dichlorophenyl, 3,4-dichlorophenyl, 3,5-bis(trifluoromethyl)phenyl, 3,5-
dimethyl-
phenyl, 5-methyl-3-pyridyl.
In another embodiment of formula I, Ar2 is 1,4-phenylene. Examples
of Ar2 include 1,4-phenylene and 2,5-pyridylene as shown below.
\ \
Arz = I / or
~s~' N s~'
In another embodiment of formula I one of X and Y is halogen and the
other is selected from halogen, C1_3alkyl and C1_3alkoxy. In one subset of
this
embodiment one of X and Y is chloro and the other is chloro or methoxy. In
another
subset X and Y are each chloro.
In another embodiment of formula I, R3a and R3b are each hydrogen,
and one of R4a and R4b is hydrogen or C1-l0alkyl, and the other is selected
from
hydrogen, C3_lOcY~loalkyl, pyridyl, NRdRe, ORd, CN, C02Rd and phenyl
optionally
substituted with C02H. In one subset of this embodiment, one of R4a and R4b is
hydrogen and the other is selected from hydrogen, phenyl, C3_bcycloalkyl,
pyridyl,
CN, ORd and CO2Rd. In another subset, one of R4a and R4b is hydrogen and the
other is NRdRe. Examples of R4~4b include hydrogen, methyl phenyl, cyclohexyl,
amino, isopropylamino, dimethylamino, 1-azetidinyl, 1-pyrrolidinyl,
cyclopropyl-
amino, hydroxy, cyano, t-butyloxy, 4-carboxyphenyl, t-butoxycarbonyl, and 4-
pyridyl.
In another embodiment of formula I, R4a and R4b are each hydrogen,
and one of R3a and R3b is selected from hydrogen, C1_l0alkyl, phenyl and
C2_lpalkenyl, and the other is selected from hydrogen, C1-l0alkyl optionally
substituted with OH, C2_l0alkenyl, C3_lOcycloalkyl, phenyl optionally
substituted
with OH or C02H, C02Rd, ORd, NRdRe, and NRdC(O)2Rd. In one subset one of
-9-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
R3a and R3b is hydrogen and the other is selected from hydrogen, phenyl
optionally
substituted with OH or C02H, C1_6alkyl optionally substituted with OH,
C3_6cycloalkyl, C02R, ORd, NRdRe and NRC(O)2R. In another subset one of R3a
and R3b is C1_6alkyl and the other is selected from C1_(alkyl and ORd. In
another
subset R3a and R3b are each C1_6alkyl or C2_6alkenyl. Examples of R3a/3b
include
hydrogen, methyl, phenyl, hydroxy, cyclohexyl, carboxy, hydroxymethyl,
methoxy, 4-
hydroxyphenyl, 4-carboxyphenyl, dimethylamino, allyl, and
allyloxycarbonylamino.
One embodiment of formula I provides compounds of formula Ia:
Rqb R3a R3b
R4a R2 H
~NuC02H
N
CI \ Sp2 O
O CI
CI H I A
CI
Ia
wherein A is N or N+O-;
R2 is H or methyl;
one of R3a and R3b is selected from H, C1_6alkyl, C2_6alkenyl and phenyl, and
the
other is selected from H, phenyl optionally substituted with OH or C02H,
C1_6alkyl
optionally substituted with OH, C2-(alkenyl, C3_6cycloalkyl, C02Rd, ORd, NRdRe
and NRdC(O)2Rd;
one of R4a and R4b is selected from H and C1_6alkyl, and the other is selected
from
H, phenyl, C3_6eycloalkyl, pyridyl, CN, ORd, NRdRe and C02Rd; or
R4a and R4b together is oxo; or
a pharmaceutically acceptable salt thereof.
In one subset of formula Ia, R3a and R3b are each hydrogen; one of
R4a and R4b is hydrogen and the other is selected from phenyl, C3_6cycloalkyl,
hydroxy, C 1 _5alkoxy, C02H, pyridyl, cyano, and NRdRe. In one embodiment R4a
or
R4b is phenyl; in another embodiment R4a or R4b is NRdRe wherein Rd and Re are
independently selected from hydrogen and C1_l0alkyl; in another embodiment R4a
or
R4b is NRdRe wherein Rd and Re together with the atom to which they are
attached
form a heterocyclic ring of 4 to 7 members containing 0 additional heteroatom.
- 10-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
In another subset of formula Ia, R4a and R4b are each hydrogen; one
of R3a and R3b is hydrogen, and the other is selected from phenyl optionally
substituted with OH or CO~H, C1_6alkyl optionally substituted with OH,
C~_6alkenyl, C3_6cycloalkyl, C02Rd, ORd, NRdRe and NRdC(O)2Rd.
In another subset of formula Ia one of R3a and R3b is hydrogen, and
the other is selected from phenyl optionally substituted with OH or C02H,
C1_6alkyl
optionally substituted with OH, C2_galkenyl, C3_6cycloalkyl, CO2Rd, ORd, NRdRe
and NRdC(O)2Rd; and one of R4a and R4b is hydrogen and the other is selected
from
phenyl, C3_6cycloalkyl, hydroxy, C1_5alkoxy, CO~H, pyridyl, cyano, and NRdRe.
Representative compounds of formula I are as follows:
3a
Rib R sb
R
R4a
3 R2 H
N~C02H
1N
O=S=O O ~ O CI
5
of ~ c2 ~ N ~ R
R U R H
CI i Ny
~~~n
R3a/g3b/R4a/R4b* R2 Rcl/Rc2 n/RS
CH3 3,5-diCl 0/H
H 3,5-diCl 0/H
H 3,5-diCl 1/H
3-OH/3-CH3 H 3,5-diCl 0/H
4(S)-Ph H 3,5-diCl 0/H
3(R)-Ph H 3,5-diCl 0/H
4(S)-Ph CH3 3,5-diCl 0/H
3(S)-Ph CH3 3,5-diCl 0/H
3(R)-cHex H 3,5-diCl 0/H
4(S)-cHex H 3,5-diCl 0/H
3(S)-cHex H 3,5-diCl 0/H
traps-3-C02H CH3 3,5-diCl 0/H
traps-3-Ph H 3,5-diCl 0/H
-11-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
R3a/R3b/R4a/R.4b* R2 Rc1/Rc2 n/R5
trans-3-CH20H CH3 3,5-diCl 0/H
3(R)-OCH3 H 3,5-diCl 0/H
4(R)-NH-iPr CH3 3-CI 0/H
4(R)-N(CH3)2 CH3 3-CI 0/H
4(R)-1-azetidin 1 CH3 3-CI O/H
4(R)-1- yrrolidin CH3 3-CI O/H
1
4(R)-NH2 CH3 3,5-diCl 0/H
4(R)-NH-cPro CH3 3,5-diCl O/H
4(R)-1-azetidinyl CH3 3,5-diCl 0/H
4(R)-OH CH3 3,5-diCl 0/H
3(S)-CH3 H 3,5-diCl 0/H
3(S)-OH H 3,5-diCl 0/H
4(R)-CN H 3,5-diCl 0/H
4(R)-O-C(CH3)3 H 3,5-diCl 0/H
3-(4-OH-Ph) H 3,5-diCl 0/H
3-CH3/3-CH3 H 3,5-diCl 0/H
CH3 3,5-diCl 1/H
4(S)-Ph H H/H 0/H
4(S)-Ph H 3-OCF3 O/H
4(S)-Ph H 4-CI OlH
4(S)-Ph H 3-Br 0/H
4(S)-Ph H 3,4-diCl 0/H
4(S)-Ph H 3,5-(CF3)2 0/H
4(S)-Ph H 4-CH3 0/H
4(S)-Ph H 3,5-(CH3)2 0/H
4(S)-Ph H 3-F 0/H
4(S)-Ph H 3-CI 0/H
4(S)-Ph H 3-CF3 0/H
4(S)-Ph H 3-Ph 0/H
CH3 3,5-diCl 0/0H
CH3 3,5-diCl 0/OCH3
CH3 3,5-diCl 0/NH2
-12-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
g3a/g3b~g4a/g4b* g2 gcl/gc2 n/g5
3,3-(CH3)~ H 3-Cl O/H
3,3-(CH3)2 H 3-CI 1/H
4(R)-1-azetidinyl H 3,5-diCl 0/H
3(R)-(4-C02H-Ph) H 3,5-diCl 0/H
3(S)-(4-CO2H-Ph) H 3,5-diCl 0/H
4(R)-(4-C02H-Ph) H 3,5-diCl 0/H
4(R)-C02C(CH3)3 H 3,5-diCl OlH
4(R)-Ph H 3,5-diCl 0/H
4(R)-4- yrid 1 H 3,5-diCl 0/H
3(R)-NHC02CH~CH=CH CH3 3,5-diCl O/H
3(R)-N(CH3)2 CH3 3,5-diCl 0/H
3-Ph/3-CO~H CH3 3,5-diCl 0/H
4-CH3/4-C02H H 3,5-diCl 0/H
4(R)-cHex H 3,5-diCl 0/H
3(S)-NHC02CH2CH=CH CH3 3,5-diCl H
4-oxo-3,3-(CH2CH=CH)2H 3,5-diCl H
~' when no value is given, the variables) is hydrogen.
H
N C02H
N
O=S=00 \ O CI
/ ~ H
CI ~ CI CI ~ N
H
N C02H
N
O=S=O O \ O OCH3
/ ~ /
H
CI CI CI ~ N
- 13-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
H
N C02H
N
O=S=O O
O CI
I
I \
CI \ CI N H I i N
CI
H
N C02H
O=S=O O
O CI
H3C \ CI ~ N
H
N C02H
N
O=S=O O
O CI
I
I
CI ~ CI N H I i N
CI
In another aspect the present invention provides a method for the
prevention or treatment of diseases, disorders, conditions or symptoms
mediated by
cell adhesion in a mammal which comprises administering to said mammal an
effective amount of a compound of formula I.
In one embodiment said disease or disorder is selected from asthma,
allergic rhinitis, multiple sclerosis, atherosclerosis, inflammatory bowel
disease,
rheumatoid arthritis, and organ transplantation.
- 14-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
In another aspect the present invention provides a method for
preventing the action of VLA-4 in a mammal which comprises administering fo
said
mammal a therapeutically effective amount of a compound of formula I.
Another aspect of the present invention provides a pharmaceutical
composition which comprises a compound of formula I and a pharmaceutically
acceptable carrier.
"Alkyl", as well as other groups having the prefix "alk", such as
alkoxy, alkanoyl, means carbon chains which may be linear or branched or
combinations thereof. Examples of alkyl groups include methyl, ethyl, propyl,
isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl,
and the like.
"Alkenyl" means carbon chains which contain at least one carbon-
carbon double bond, and which may be linear or branched or combinations
thereof.
Examples of alkenyl include vinyl, allyl, isopropenyl, pentenyl, hexenyl,
heptenyl, 1-
propenyl, 2-butenyl, 2.-methyl-2-butenyl, and the lilce.
"Alkynyl" means carbon chains which contain at least one carbon-
carbon triple bond, and which may be linear or branched or combinations
thereof.
Examples of alkynyl include ethynyl, propargyl, 3-methyl-1-pentynyl, 2-
heptynyl and
the like.
"Cycloalkyl" means mono- or bicyclic saturated carbocyclic rings, each
of which having from 3 to 10 carbon atoms. The term also includes monocyclic
rings
fused to an aryl group in which the point of attachment is on the non-aromatic
portion.
Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl,
cycloheptyl, tetrahydronaphthyl, decahydronaphthyl, indanyl, and the like.
"Aryl" means mono- or bicyclic aromatic rings containing only carbon
atoms. The term also includes aryl group fused to a monocyclic cycloalkyl or
monocyclic heterocyclyl group in which the point of attachment is on the
aromatic
portion. Examples of aryl include phenyl, naphthyl, indanyl, indenyl,
tetrahydronaphthyl, 2,3-dihydrobenzofuranyl, dihydrobenzopyranyl, 1,4-
benzodioxanyl, and the like.
"Heteroaryl" means a mono- or bicyclic aromatic ring containing at
least one heteroatom selected from N, O and S, with each ring containing 5 to
6
atoms. Examples of heteroaryl include pyrrolyl, isoxazolyl, isothiazolyl,
pyrazolyl,
pyridyl, oxazolyl, oxadiazolyl, thiadiazolyl, thiazolyl, imidazolyl,
triazolyl, tetrazolyl,
furanyl, triazinyl, thienyl, pyrimidyl, pyridazinyl, pyrazinyl, benzoxazolyl,
-15-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
benzothiazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, furo(2,3-
b)pyridyl,
quinolyl, indolyl, isoquinolyl, and the like.
"Heterocyclyl" means mono- or bicyclic saturated rings containing at
least one heteroatom selected from N, S and O, each of said ring having from 3
to 10
atoms in which the point of attachment may be carbon or nitrogen. The term
also
includes monocyclic heterocycle fused to an aryl or heteroaryl group in which
the
point of attachment is on the non-aromatic portion. Examples of "heterocyclyl"
include pyrrolidinyl, piperidinyl, piperazinyl, imidazolidinyl, 2,3-
dihydrofuro(2,3-
b)pyridyl, benzoxazinyl, tetrahydrohydroquinolinyl, tetrahydroisoquinolinyl,
dihydroindolyl, and the like. The term also includes partially unsaturated
monocyclic
rings that are not aromatic, such as 2- or 4-pyridones attached through the
nitrogen or
N-substituted-(1H,3H)-pyrimidine-2,4-diones (N-substituted uracils).
"Halogen" includes fluorine, chlorine, bromine and iodine.
Optical Isomers - Diastereomers - Geometric Isomers - Tautomers
Compounds of Formula I contain one or more asymmetric centers and
can thus occur as racemates and racemic mixtures, single enantiomers,
diastereomeric
mixtures and individual diastereomers. The present invention is meant to
comprehend all such isomeric forms of the compounds of Formula I.
Some of the compounds described herein contain olefinic double
bonds, and unless specified otherwise, are meant to include both E and Z
geometric
isomers.
Some of the compounds described herein may exist with different
points of attachment of hydrogen, referred to as tautomers. Such an example
may be a
ketone and its enol form known as keto-enol tautomers. The individual
tautomers as
well as mixture thereof are encompassed with compounds of Formula I.
Compounds of the Formula I may be separated into diastereoisomeric
pairs of enantiomers by, for example, fractional crystallization from a
suitable solvent,
for example MeOH or ethyl acetate or a mixture thereof. The pair of
enantiomers
thus obtained may be separated into individual stereoisomers by conventional
means,
for example by the use of an optically active amine as a resolving agent or on
a chiral
HPLC column.
-16-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Alternatively, any enantiomer of a compound of the general Formula I
or Ia may be obtained by stereospecific synthesis using optically pure
starting
materials or reagents of known configuration.
Salts
The term "pharmaceutically acceptable salts" refers to salts prepared
from pharmaceutically acceptable non-toxic bases or acids including inorganic
or
organic bases and inorganic or organic acids. Salts derived from inorganic
bases
include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium,
magnesium,
manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly
preferred are the ammonium, calcium, magnesium, potassium, and sodium salts.
Salts derived from pharmaceutically acceptable organic non-toxic bases include
salts
of primary, secondary, and tertiary amines, substituted amines including
naturally
occurring substituted amines, cyclic amines, and basic ion exchange resins,
such as
arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine,
diethylamine, 2-
. diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine,
N-
ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,
hydrabamine,
isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine,
polyamine resins, procaine, purines, theobromine, triethylamine,
trimethylamine,
tripropylamine, tromethamine, and the like.
When the compound of the present invention is basic, salts may be
prepared from pharmaceutically acceptable non-toxic acids, including inorganic
and
organic acids. Such acids include acetic, benzenesulfonic, benzoic,
camphorsulfonic,
citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic,
hydrochloric,
isethionic, lactic, malefic, malic, mandelic, methanesulfonic, mucic, nitric,
pamoic,
pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid,
and the
like. Particularly preferred are citric, hydrobromic, hydrochloric, malefic,
phosphoric,
sulfuric, and tartaric acids.
It will be understood that, as used herein, references to the compounds
of Formula I are meant to also include the pharmaceutically acceptable salts.
Utilities
The ability of the compounds of Formula I to antagonize the actions of
VLA-4 and/or a4(37 integrin malees them useful for preventing or reversing the
-17-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
symptoms, disorders or diseases induced by the binding of VLA-4 and or a4(37
to
their various respective ligands. Thus, these antagonists will inhibit cell
adhesion
processes including cell activation, migration, proliferation and
differentiation.
Accordingly, another aspect of the present invention provides a method for the
treatment (including prevention, alleviation, amelioration or suppression) of
diseases
or disorders or symptoms mediated by VLA-4 and/or a4~i7 binding and cell
adhesion
and activation, which comprises administering to a mammal an effective amount
of a
compound of Formula I. Such diseases, disorders, conditions or symptoms are
for
example (1) multiple sclerosis, (2) asthma, (3) allergic rhinitis, (4)
allergic
conjunctivitis, (5) inflammatory lung diseases, (6) rheumatoid arthritis, (7)
septic
arthritis, (8) type I diabetes, (9) organ transplantation rejection, (10)
restenosis, (11)
autologous bone marrow transplantation, (12) inflammatory sequelae of viral
infections, (13) myocarditis, (14) inflammatory bowel disease including
ulcerative
colitis and Crohn's disease, (15) certain types of toxic and immune-based
nephritis,
(16) contact dermal hypersensitivity, (17) psoriasis, (18) tumor metastasis,
(19)
atherosclerosis, and (20) hepatitis.
The utilities of the present compounds in these diseases or disorders
may be demonstrated in animal disease models that have been reported in the
literature. The following are examples of such animal disease models:
i) experimental allergic encephalomyelitis, a model of neuronal
demyelination resembling multiple sclerosis (for example, see T. Yednock et
al.,
"Prevention of experimental autoimmune encephalomyelitis by antibodies against
~4~1 integrin." Nature, 356, 63 (1993) and E. Keszthelyi et al., "Evidence for
a
prolonged role of a4 integrin throughout active experimental allergic
encephalomyelitis." Neurolo~y, 47, 1053 (1996));
ii) bronchial hyperresponsiveness in sheep and guinea pigs as models
for the various phases of asthma (for example, see W. M. Abraham et al., "ocq.-
Integrins mediate antigen-induced late bronchial responses and prolonged
airway
hyperresponsiveness in sheep." J. Clin. Invest. 93, 776 (1993) and A. A. Y.
Milne and
P. P. Piper, "Role of VLA-4 integrin in leucocyte recruitment and bronchial
hyperresponsiveness in the gunea-pig." Eur. J. Pharmacol., 282, 243 (1995));
iii) adjuvant-induced arthritis in rats as a model of inflammatory
arthritis (see C. Barbadillo et al., "Anti-VLA-4 mAb prevents adjuvant
arthritis in
Lewis rats." Arthr. Rheuma. (Suppl.), 36 95 (1993) and D. Seiffge, "Protective
effects
-18-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
of monoclonal antibody to VLA-4 on leukocyte adhesion and course of disease in
adjuvant arthritis in rats." J. Rheumatol., 23, 12 (1996));
iv) adoptive autoimmune diabetes in the NOD mouse (see J. L. Baron
et al., "The pathogenesis of adoptive murine autoimmune diabetes requires an
interaction between a4-integrins and vascular cell adhesion molecule-1.", J.
Clin.
Invest., 93, 1700 (1994), A. Jakubowski et al., "Vascular cell adhesion
molecule-Ig
fusion protein selectively targets activated cc4-integrin receptors in vivo:
Inhibition of
autoimmune diabetes in an adoptive transfer model in nonobese diabetic mice."
J.
Immunol., 155, 938 (1995), and X. D. Yang et al., "Involvement of beta 7
integrin and
mucosal addressin cell adhesion molecule-1 (MadCAM-1) in the development of
diabetes in nonobese diabetic mice", Diabetes, 46, 1542 (1997));
v) cardiac allograft survival in mice as a model of organ transplantation
(see M. Isobe et al., "Effect of anti-VCAM-1 and anti-VLA-4 monoclonal
antibodies
on cardiac allograft survival.and response to soluble antigens in mice.",
Tranplant.
Proc., 26, 867 (1994) and S. Molossi et al., "Blockade of very late antigen-4
integrin
binding to fibronectin with connecting segment-1 peptide reduces accelerated
coronary arteripathy in rabbit cardiac allografts." J. Clin Invest., 95, 2601
(1995));
vi) spontaneous chronic colitis in cotton-top tamarins which resembles
human ulcerative colitis, a form of inflammatory bowel disease (see D. K.
Podolsky et
al., "Attenuation of colitis in the Cotton-top tamarin by anti-oc4 integrin
monoclonal
antibody.", J. Clin. Invest., 92, 372 (1993));
vii) contact hypersensitivity models as a model for skin allergic
reactions (see T. A. Ferguson and T. S. Kupper, "Antigen-independent processes
in
antigen-specific immunity.", J. Immunol., 150, 1172 (1993) and P. L. Chisholm
et al.,
"Monoclonal antibodies to the integrin a-4 subunit inhibit the murine contact
hypersensitivity response." Eur. J. Immunol., 23, 682 (1993));
viii) acute nephrotoxic nephritis (see M. S. Mulligan et al.,
"Requirements for leukocyte adhesion molecules in nephrotoxic nephritis.", J.
Clin.
Invest., 91, 577 (1993));
ix) tumor metastasis (for examples, see M. Edward, "Integrins and
other adhesion molecules involved in melanocytic tumor progression.", Curr.
Opin.
Oncol., 7, 185 (1995));
-19-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
x) experimental autoimmune thyroiditis (see R. W. McMurray et al.,
"The role of a4 integrin and intercellular adhesion molecule-1 (ICAM-1) in
murine
experimental autoimmune thyroiditis." Autoimmunity, 23, 9 (1996);
xi) ischemic tissue damage following arterial occlusion in rats (see F.
Squadrito et al., "Leukocyte integrin very late antigen-4/vascular cell
adhesion
molecule-1 adhesion pathway in splanchnic artery occlusion shock." Eur. J.
Pharmacol., 318, 153 (1996; and
xii) inhibition of TH2 T-cell cytokine production including IL-4 and
IL-5 by VLA-4 antibodies which would attenuate allergic responses (J.Clinical
Investigation 100, 3083 (1997).
Dose Ranges
The magnitude of prophylactic or therapeutic dose of a compound of
Formula I will, of course, vary with the nature of the severity of the
condition to be
treated and with the particular compound of Formula I and its route of
administration.
It will also vary according to the age, weight and response of the individual
patient.
In general, the daily dose range lie within the range of from about 0.001 mg
to about
100 mg per kg body weight of a mammal, preferably 0.01 mg to about 50 mg per
kg,
and most preferably 0.1 to 10 mg per kg, in single or divided doses. On the
other
hand, it may be necessary to use dosages outside these limits in some cases.
For use where a composition for intravenous administration is
employed, a suitable dosage range is from about 0.001 mg to about 25 mg
(preferably
from 0.01 mg to about 1 mg) of a compound of Formula I per kg of body weight
per
day and for cytoprotective use from about 0.1 mg to about 100 mg (preferably
from
about 1 mg to about 100 mg and more preferably from about l mg to about 10 mg)
of
a compound of Formula I per kg of body weight per day.
In the case where an oral composition is employed, a suitable dosage
range is, e.g. from about 0.01 mg to about 100 mg of a compound of Formula I
per kg
of body weight per day, preferably from about 0.1 mg to about 10 mg per kg and
for
cytoprotective use from 0.1 mg to about 100 rng (preferably from about 1 mg to
about
100 mg and more preferably from about 10 mg to about 100 mg) of a compound of
Formula I per kg of body weight per day.
-20-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
For the treatment of diseases of the eye, ophthalmic preparations for
ocular administration comprising 0.001-1% by weight solutions or suspensions
of the
compounds of Formula I in an acceptable ophthalmic formulation maybe used.
Pharmaceutical Compositions
Another aspect of the present invention provides pharmaceutical
compositions which comprises a compound of Formula I and a pharmaceutically
acceptable carrier. The term "composition", as in pharmaceutical composition,
is
intended to encompass a product comprising the active ingredient(s), and the
inert
ingredients) (pharmaceutically acceptable excipients) that make up the
carrier, as
well as any product which results, directly or indirectly, from combination,
complexation or aggregation of any two or more of the ingredients, or from
dissociation of one or more of the ingredients, or from other types of
reactions or
interactions of one or more of the ingredients. Accordingly, the
pharmaceutical
compositions of the present invention encompass any composition made by
admixing
a compound of Formula I, additional active ingredient(s), and pha~.-
maceutically
acceptable excipients.
Any suitable route of administration may be employed for providing a
mammal, especially a human with an effective dosage of a compound of the
present
invention. For example, oral, rectal, topical, parenteral, ocular, pulmonary,
nasal, and
the like may be employed. Dosage forms include tablets, troches, dispersions,
suspensions, solutions, capsules, creams, ointments, aerosols, and the like.
The pharmaceutical compositions of the present invention comprise a
compound of Formula I as an active ingredient or a pharmaceutically acceptable
salt
thereof, and may also contain a pharmaceutically acceptable carrier and
optionally
other therapeutic ingredients. The term "pharmaceutically acceptable salts"
refers to
salts prepared from pharmaceutically acceptable non-toxic bases or acids
including
inorganic bases or acids and organic bases or acids.
The compositions include compositions suitable for oral, rectal,
topical, parenteral (including subcutaneous, intramuscular, and intravenous),
ocular
(ophthalmic), pulmonary (aerosol inhalation), or nasal administration,
although the
most suitable route in any given case will depend on the nature and severity
of the
conditions being treated and on the nature of the active ingredient. They may
be
-21-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
conveniently presented in unit dosage form and prepared by any of the methods
well-
known in the art of pharmacy.
For administration by inhalation, the compounds of the present
invention are conveniently delivered in the form of an aerosol spray
presentation from
pressurized paclcs or nebulizers. The compounds may also be delivered as
powders
which may be formulated and the powder composition may be inhaled with the aid
of
an insufflation powder inhaler device. The preferred delivery systems for
inhalation
are metered dose inhalation (MDI) aerosol, which may be formulated as a
suspension
or solution of a compound of Formula I in suitable propellants, such as
fluorocarbons
or hydrocarbons and dry powder inhalation (DPI) aerosol, which may be
formulated
as a dry powder of a compound of Formula I with or without additional
excipients.
Suitable topical formulations of a compound of formula I include
transdermal devices, aerosols, creams, ointments, lotions, dusting powders,
and the
like.
In practical use, the compounds of Formula I can be combined as the
active ingredient in intimate admixture with a pharmaceutical carrier
according to
conventional pharmaceutical compounding techniques. The carrier may take a
wide
variety of forms depending on the form of preparation desired for
administration, e.g.,
oral or parenteral (including intravenous). In preparing the compositions for
oral
dosage form, any of the usual pharmaceutical media may be employed, such as,
for
example, water, glycols, oils, alcohols, flavoring agents, preservatives,
coloring agents
and the like in the case of oral liquid preparations, such as, for example,
suspensions,
elixirs and solutions; or carriers such as starches, sugars, microcrystalline
cellulose,
diluents, granulating agents, lubricants, binders, disintegrating agents and
the like in
the case of oral solid preparations such as, for example, powders, capsules
and tablets,
with the solid oral preparations being preferred over the liquid preparations.
Because
of their ease of administration, tablets and capsules represent the most
advantageous
oral dosage unit form in which case solid pharmaceutical carriers are
obviously
employed. If desired, tablets may be coated by standard aqueous or nonaqueous
techniques.
In addition to the common dosage forms set out above, the compounds
of Formula I may also be administered by controlled release means and/or
delivery
devices such as those described in U.S. Patent Nos. 3,845,770; 3,916,899;
3,536,809;
3,598,123; 3,630,200 and 4,008,719.
-22-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Pharmaceutical compositions of the present invention suitable for oral
administration may be presented as discrete units such as capsules, cachets or
tablets
each containing a predetermined amount of the active ingredient, as a powder
or
granules or as a solution or a suspension in an aqueous liquid, a non-aqueous
liquid,
an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions
may be
prepared by any of the methods of pharmacy but all methods include the step of
bringing into association the active ingredient with the carrier which
constitutes one
or more necessary ingredients. In general, the compositions are prepared by
uniformly and intimately admixing the active ingredient with liquid carriers
or finely
divided solid carriers or both, and then, if necessary, shaping the product
into the
desired presentation. For example, a tablet may be prepared by compression or
molding, optionally with one or more accessory ingredients. Compressed tablets
may
be prepared by compressing in a suitable machine, the active ingredient in a
free-
flowing form such as powder or granules, optionally mixed with a binder,
lubricant,
inert diluent, surface active or dispersing agent. Molded tablets may be made
by
molding in a suitable machine, a mixture of the powdered compound moistened
with
an inert liquid diluent. Desirably, each tablet contains from about 1 mg to
about 500
mg of the active ingredient and each cachet or capsule contains from about 1
to about
500 mg of the active ingredient.
The following are examples of representative pharmaceutical dosage
forms for the compounds of Formula I:
Iniectable Suspension LM.) mg/~
Compound of Formula I 10
Methylcellulose 5.0
Tween 80 0.5
Benzyl alcohol 9.0
Benzalkonium chloride 1.0
Water for injection to a total volume of 1 mL
-23-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Tablet m~/tablet
Compound of Formula25
I
Microcrystalline 415
Cellulose
Povidone 14.0
Pregelatinized Starch 43.5
Magnesium Stearate 2.5
500
Capsule mg/cacapsule
Compound of Formula I 25
Lactose Powder 573.5
Magnesium Stearate 1.5
600
Aerosol Per canister
Compound of Formula I 24 rng
Lecithin, NF Liq. Conc. 1.2 mg
Trichlorofluoromethane, NF 4.025 g
Dichlorodifluoromethane, NF 12.15 g
Combination Therapy
Compounds of Formula I may be used in combination with other drugs
that are used in the treatment/prevention/suppression or amelioration of the
diseases
or conditions for which compounds of Formula I are useful. Such other drugs
may be
administered, by a route and in an amount commonly used therefor,
contemporaneously or sequentially with a compound of Formula I. When a
compound of Formula I is used contemporaneously with one or more other drugs,
a
pharmaceutical composition containing such other drugs in addition to the
compound
of Formula I is preferred. Accordingly, the pharmaceutical compositions of the
present invention include those that also contain one or more other active
ingredients,
in addition to a compound of Formula I. Examples of other active ingredients
that
may be combined with a compound of Formula I, either administered separately
or in
the same pharmaceutical compositions, include, but are not limited to:
-24-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
(a) other VLA-4 antagonists such as those described in US 5,510,332,
W097/03094,
W097/02289, W096/40781, W096/22966, W096/20216, W096/01644,
W096/06108, W095/15973 and W096/31206; (b) steroids such as beclomethasone,
methylprednisolone, betamethasone, prednisone, dexamethasone, and
hydrocortisone;
(c) immunosuppressants such as cyclosporin, tacrolimus, rapamycin and other FK-
506
type immunosuppressants; (d) antihistamines (Hl-histamine antagonists) such as
bromopheniramine, chlorpheniramine, dexchlorpheniramine, triprolidine,
clemastine,
diphenhydramine, diphenylpyraline, tripelennamine, hydroxyzine, methdilazine,
promethazine, trimeprazine, azatadine, cyproheptadine, antazoline, pheniramine
pyrilamine, astemizole, terfenadine, loratadine, cetirizine, fexofenadine,
descarboethoxyloratadine, and the like; (e) non-steroidal anti-asthmatics such
as b2-
agonists (terbutaline, metaproterenol, fenoterol, isoetharine, albuterol,
bitolterol,
salmeterol and pirbuterol), theophylline, cromolyn sodium, atropine,
ipratropium
bromide, leulcotriene antagonists (zafirlukast, montelulcast, pranlukast,
iralukast,
pobilukast, SKB-106,203), leukotriene biosynthesis inhibitors (zileuton, BAY-
1005);
(f) non-steroidal antiinflarnmatory agents (NSAIDs) such as propionic acid
derivatives (alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen,
fenoprofen, fluprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen,
miroprofen,
naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid, and
tioxaprofen), acetic acid derivatives (indomethacin, acemetacin, alclofenac,
clidanac,
diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac,
isoxepac,
oxpinac, sulindac, tiopinac, tolmetin, zidometacin, and zomepirac), fenamic
acid
derivatives (flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid
and
tolfenamic acid), biphenylcarboxylic acid derivatives (diflunisal and
flufenisal),
oxicams (isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (acetyl
salicylic
acid, sulfasalazine) and the pyrazolones (apazone, bezpiperylon, feprazone,
mofebutazone, oxyphenbutazone, phenylbutazone); (g) cyclooxygenase-2 (COX-2)
inhibitors such as celecoxib, rofecoxib, and parecoxib; (h) inhibitors of
phosphodiesterase type IV (PDE-IV); (i) antagonists of the chemokine
receptors,
especially CCR-1, CCR-2, and CCR-3; (j) cholesterol lowering agents such as
HMG-
CoA reductase inhibitors (lovastatin, simvastatin, pravastatin, fluvastatin,
atorvastatin,
and other statins), sequestrants (cholestyramine and colestipol), nicotinic
acid,
fenofibric acid derivatives (gemfibrozil, clofibrat, fenofibrate and
benzafibrate), and
probucol; (k) anti-diabetic agents such as insulin, sulfonylureas, biguanides
-25-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
(metformin), a-glucosidase inhibitors (acarbose) and glitazones (troglitazone,
pioglitazone, englitazone, MCC-555, BRI~1.9653 and the like); (1) preparations
of
interferon beta (interferon beta-la, interferon beta-lb); (m) anticholinergic
agents
such as muscarinic antagonists (ipratropium nad tiatropium); (n) other
compounds
such as 5-aminosalicylic acid and prodrugs thereof, antimetabolites such as
azathioprine and 6-mercaptopurine, and cytotoxic cancer chemotherapeutic
agents.
The weight ratio of the compound of the Formula I to the second active
ingredient may be varied and will depend upon the effective dose of each
ingredient.
Generally, an effective dose of each will be used. Thus, for example, when a
compound of the Formula I is combined with an NSAID the weight ratio of the
compound of the Formula I to the NSAID will generally range from about 1000:1
to
about 1:1000, preferably about 200:1 to about 1:200. Combinations of a
compound of
the Formula I and other active ingredients will generally also be within the
aforementioned range, but in each case, an effective dose of each active
ingredient
should be used.
Abbreviations used in the following Schemes and Examples:
4-DMAP: 4-dimethylaminopyridine
Ac20: acetic anhydride
AcCN: acetonitrile
Ag20: silver(I) oxide
AIBN: 2,2'-azobisisobutyronitrile
BF3-Et20: borontrifluoride etherate
BH3-DMS: borane dimethylsulfide complex
Bn: benzyl
BOC: tent-butoxycarbonyl
BOC-ON 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile
BOP: benzotriazol-1-yloxy-tris (dimethylamino)-phosphonium
hexafluorophosphate
brine: saturated sodium chloride solution
CBZ: benzyloxycarbonyl
Cy3P: tricyclohexylphosphine
DBU: 1,8-diazobicyclo[5.4.0]undec-7-ene
DCC: dicyclohexylcarbodiimide
DIBAL-H: diisobutylaluminum hydride
-26-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
DIPEA: N,N-diisopropylethylamine
DME: 1,2-dimethoxyethane
DMF: dimethylformamide
DMPU: 1,3-dimethyl-3,4,5,6-tetrahydro-2(lIl)-pyrimidinone
DMSO: dimethylsulfoxide
EDC: 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride
Et: ethyl
Et20: diethyl ether
EtOAc: ethyl acetate
EtOH: ethanol
FMOC: - 9-fluorenylmethoxylcarbonyl
g or gm: gram
h or hr: hours
HATU: O-(7-azabenzotriazol-1-yl)-1,1, 3, 3-tetramethyluronium
hexafluorophosphate
HBTU: O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
HOAc: acetic acid
HOAt: 1-hydroxy-7-azabenzotriazole
HOBt: 1-hydroxybenzotriazole
HPLC: high pressure liquid chromatography
in vacaco:rotoevaporation
KOAc: potassium acetate
LDA: lithium diisopropylamide
LiHMDS: lithium hexamethyldisilylamide
mCPBA: meta-chloroperbenzoic acid
Me: methyl
MeI: methyl iodide
MeOH: methanol
mg: milligram
MHz: megahertz
min: minutes
mL: milliliter
mmol: millimole
MPLC: medium pressure liquid chromatography
_27_
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
MS or ms: mass spectrum
MsCI: methanesulfonyl chloride
NBS: N-bromosuccinimide
NMO: 4-methyl-morpholine-N-oxide
Pd2dba3: tris(dibenzylideneacetone) dipalladium(0)
Ph: phenyl
Ph3P: triphenylphosphine
pTSA: para-toluenesulfonic acid
PyBOP: (benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate
rt: room temperature
TBAF: ~ tetrabutylammonium fluoride
TBSCI: tert-butyldimethylsilyl chloride
t-Bu3P: tri-tert-butylphosphine
TEA: triethylamine
TFA: trifluoroacetic acid
THF: THF
TLC: thin layer chromatography
TMSCHN2: trimethylsiliyldiazomethane
TMSCI: trimethylsilyl chloride
TMSI: trimethylsilyl iodide
TPAP: tetrapropylammonium perruthenate
TsCI: para-toluene sulfonyl
chloride
Compounds of the present invention may be prepared by procedures
illustrated in the accompanying schemes. In the first method (Scheme 1), a
substituted pyridyl-4-carboxylic acid derivative A is treated with thionyl
chloride to
make the carboxylic acid chloride derivative which is subsequently reacted
with a 4-
amino-(L)-phenylalanine derivative to yield the amide B. The N-BOC-protecting
group in B is removed with strong acid (TFA or HCl) to afford the free amine
_C. An
appropriately substitued 2-azetidinyl-, 2.-pyrrolidinyl-, or 2-piperidinyl-
carboxylate D
is sulfonylated with a substitued arylsulfonyl chloride in the presence of
base (DIPEA
or Na~,C03) to yield sulfonamide E which, if containing an ester protecting
group, is
treated with hydroxide to afford the free acid. Amine C and acid E are reacted
together in the presence of an appropriate coupling agent (eg., PyBOP,
HBTU/HOAt,
-2~-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
premake the acid chloride of E, etc.) to afford amide F. The ester in F can be
hydrolyzed with hydroxide (if RS is n-alkyl) or TFA or HCl (if RS is tert-
butyl).
-29-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Scheme 1
O X ~ N CO2R5
HO I ~ R6 1. SOCI2 _SOCI2 _ BOC O X TFA
Y ~ A 2BOCN C02R5 Ar2N ~ R6
ArNH B Y ~ A
2
H2N CO2R5
O X
Ar2N ~ R6
C ~ iA
- Y
Rq b R3a R3b R4b R3a R3b
R4a R2 ArISO2C1 R4a R2
( ) ' ~ DIPEA or Na2C03 ( )n, ~CO H
n N C02R N 2
D H p,r1~s02
E
coupling
agent N C02R5
C + E
i O X
Ar1~S02 O Ar2N ~ Rs
~A
Y
R4b R3a R3b
OH-or H+ R4a
R2 H
( )n. ~N C02H
O X
Ar1~S02 O Ar2N ~ R6
G l j
Y ,A
R4b Rsa R3b
R4a R2
( )n.N
-30-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Compounds of the present invention may be prepared by procedures detailed in
the
following examples. The examples are provided to illustrative the present
invention
and are not to be construed as limiting its scope in any manner.
REFERENCE EXAMPLE 1
3,5-Dichloroisonicotinic acid.
To a solution of 3,5-dichloropyridine (10.00 g, 67.57 mmol) in 70 mL
of THF was added 35.4 mL of a 2.0 M solution of LDA in THF at -78°C.
The
reaction was stirred for 1 h, then C02 gas was bubbled through the solution
for 20
mins. The reaction was allowed to warm to rt over 1 h then quenched with 1N
NaOH
(100 mL) and washed with Et20 (50 mL). The aqueous layer was acidified with
cone
HCl which caused a precipitate to form. The precipitate was collected by
filtration
and recrystallized from EtOH to give the title compound as a pale yellow solid
(7.1 g,
36.97 mmol, 55%).
1H NMR (500 MHz , DMSO-d6): ~ 8.73 (s, 2H).
REFERENCE EXAMPLE 2
4-((3,5-dichloroisonicotino~)amino)-(L)-phenylalanine, meth l~hydrochloride.
Step A N-(BOC)-4-((3',5'-dichloroisonicotinoyl)amino)-(L)-phenylalanine
methyl ester.
A slurry of 3,5-dichloroisonicotinic acid (3.1 g, 16.11 mmol) in 10 mL
of CH2C12 was treated with DMF (50 ~,L) and thionyl chloride (1.23 mL, 16.91
mmol) and heated to reflux for 5 h. The reaction was concentrated to give a
yellow
oil. This oil was dissolved in 5 mL of CH2Cl2 and added to N-(BOC)-4-amino-(L)-
phenylalanine, methyl ester (4.00g, 14.39 mmol) and 4-methylmorpholine (2.7
mL,
24.21 mmol) in 25 mL of CH2Cl2 at 0°C. After stirring for 2 h at this
temperature,
the reaction was quenched with water (50 mL) and extracted into CH2C12 (3 x
100
mL). The combined organics were combined, dried over anhydrous MgS04 and
concentrated if2 vacuo to give a yellow solid. Trituration with CH2C12 gave
5.5 g of a
while solid
1H NMR (500 MHz , CDCl3): 8 8.63 (s, 2H); 7.58 (d, J=8.2 Hz, 2H); 7.23 (d,
J=8.2
Hz, 2H); 6.91 (d, J=8.4 Hz, 1H); 4.39 (m, 1H); 3.70 (s, 3H); 3.11 (m, 1H);
2.91 (m,
1H); 2.00 (s, 9H);
-31-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
MS mle 468.20 (M+).
Step B 4-((3',5'-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester
hydrochloride
N-(BOC)-4-((3',5'-dichloroisonicotinoyl)amino)-(L)-phenylalanine,
methyl ester (2.50 g, 5.34 mmol) was dissolved in EtOAc (40 mL) and treated
with
HCl (gas). Concentration irz vacuo gave the title compound as a yellow solid
(2.05 g,'
4.59 mmol, 86°l0).
1H NMR (500 MHz , CD3OD): ~ 8.69 (s, 2H); 7.68 (d, J= 8.5 Hz, 2H); 7.31 (m, J=
8.5 Hz, 2H); 4.35 (t, J=6.9Hz, 1H); 3.83 (s, 3H); 3.29 (m, 1H); 3.21 (m, 1H);
MS mle
368.13 (M+):
REFERENCE EXAMPLE 3
N-(3,5-Dichlorobenzenesulfonyl)-(L)-proline
Step A N-(3,5-Dichlorobenzenesulfon, l~proline, methyl ester
To a mixture of (L)-proline, methyl ester hydrochloride (838 mg, 5.06
mmol) in CH2Cl2 (25 mL) at 0°C were added DIPEA (2.64 mL, 15.2 mmol)
and a
solution of 3,5-dichlorobenzenesulfonyl chloride (1.49 g, 6.07 mmol) in CH2C12
(5
mL). The cooling bath was removed, and the mixture was stirred overnight at
rt. It
was then diluted with CH2C12, washed with 1N hydrochloric acid, saturated
NaHCO3, saturated brine solution, dried over anhydrous Na2S04, and
rotoevaporated. The product was purified by flash column chromatography on
silica
gel eluted with 10°7o acetone in hexanes to yield N-(3,5-
dichlorobenzenesulfonyl)-(L)-
proline, methyl ester; yield 1.49 g.
Step B N-(3,5-Dichlorobenzenesulfon, l~ rp cline
N-(3,5-dichlorobenzenesulfonyl)-(L)-proline, methyl ester from Step A
was dissolved in ethanol (50 mL) and treated with 0.2N sodium hydroxide (26.6
mL)
for 1.5 h at rt. The mixture was acidified with glacial HOAc, concentrated by
roroevaporation, and the residue dissolved in CH2Cl2, washed with water,
saturated
brine solution, dried over anhydrous Na2S04, and evaporated to give the title
compound; yield 1.4 g.
-32-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
400 MHz 1H NMR (CD30D): 8 1.80-2.15 (m, 4H); 3.35-4.45 (m, 2H); 4.30 (dd, 1H);
7.76 (m, 1H); 7.83 (m, 2H).
REFERENCE EXAMPLE 4
N-(3 5-Dichlorobenzenesulfonxl)-2-meth 1-y (L)-proline
Step A 2-Meth 1-~proline, methyl ester, hydrochloride.
To a solution of anhydrous MeOH (65 mL) at 0°C was added thionyl
chloride (9.03 mL, 124 mmol) slowly over a 5 min period. 2-Methyl-(L)-proline
(Bachem AG, Cat. No. F-3440, Bubendorf, Switzerland) (4.0 gm, 31 mmol) was
added in one 'portion. The reaction was stirred at 0°C for 5 min and
then warmed to rt.
The reaction was then heated to 70°C overnight. The reaction was cooled
and the
solvent removed to yield 2-methyl-(L)-proline, methyl ester hydrochloride as a
white
solid (5.72 gm).
Step B N-(3 5-Dichlorobenzenesulfonyl)-2-meth 1-y (L)-proline, methyl ester.
2-Methyl-(L)-proline, methyl ester hydrochloride (95 gm, 52.9 mmol)
was dissolved in a mixture (1:l) of dry THF and CH2Cl2 (250 mL). A solution of
3,5-dichlorobenzenesulfonyl chloride (13.0 gm, 52.9 rnmol) in dry CH2Cl2 (20
mL)
was added. The reaction was cooled in an ice bath and diisopropylethylamine
(20.5
gm, 158.6 mmol) was added. The ice bath was removed and the reaction mixture
was
stirred overnight at rt. The reaction was concentrated and dried to a tan
solid (18.68
gm). This solid was purified on a Biotage 40M chromatography system eluted
with
10% EtOAc in hexanes to yield N-(3,5-dichlorobenzenesulfonyl)-2-methyl-(L)-
proline, methyl ester as a pure white solid (16.8 gm, 85% yield).
Mass spectrum (m/e) 352 (M+1).
EXAMPLE 1
N-(N-(3 5-dichlorobenzenesulfonyl)-2-methyl-(L)-prolyl)-4-((3',5'-dichloro-
isonicotinoyl)amino)-(L)-phenylalanine, meth liter.
To a suspension of 4-((3,5-dichloroisonicotinoyl)amino)-(L)-
phenylalanine, methyl ester hydrochloride (Reference Example 3; 1.00 g, 2.23
mmol),
PyBOP (1.16 g, 2.23 mmol) and N-((3,5-dichlorobenzene)sulfonyl)-2-methyl-(L)-
proline (Reference Example 4; 0.70 g, 2.13 mmol) in CH2C12 (10 mL) was added
-33-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
DIPEA (1.0 mL, 5.6 mmol). The reaction was stirred at rt for 20 h then
concentrated
in vacuo. Flash column chromatography on silica gel eluted with hexane/ethyl
acetate
(1:1) gave 1.41 g (2.04 mmol, 96%) of a white solid.
1H NMR (500 MHz , CDC13): 8 8.58 (s, 2H), 8.36 (s, 1H), 7.80 (d, J=l.8Hz, 2H),
7.62 (d, J=8.5Hz, 2H), 7.25 (d, J=8.5Hz, 2H), 7.14 (d, J=7.6Hz, 1H), 4.88 (m,
1H),
3.85 (s, 3H), 3.60 (m, 1H), 3.37 (m, 1H), 3.32 (dd, J=5.5, l4Hz, 1H), 3.16
(dd, J=6.6,
l4Hz, 1H), 2.35 (m, 1H), 1.86 (m, 1H), 1.72 (m, 2H), 1.64 (s, 3H); 13C NMR
(125
MHz , CDCl3): b 173.3, 171.4, 160.0, 147.7, 142.6, 142.0, 135.9, 135.3, 133.3,
132.8,
130.0, 129.0, 125.9, 120.6, 70.3, 53.6, 52.5, 50.0, 40.7, 37.3, 22.9, 22.5;
MS m/e 687.2 (M+)
EXAMPLE 2
N-(N-(3,5-Dichlorobenzenesulfonyl)-2-meth 1-~prolyl)-4-((3',5'-dichloro-
isonicotin ~l)amino)-(L)-phenylalanine
To a solution of N-(N-(3,5-dichlorobenzenesulfonyl)-2-methyl-(L)-
prolyl)-4-((3',5'-dichloroisonicotinoyl)amino)-(L)-phenylalanine, methyl ester
(Example 1) in MeOH (5 mL) was added 3 mL of a 1M solution of NaOH. The
reaction was stirred at rt for 1 h then diluted with 25 mL of water and washed
with
CH2C12 (25 mL) which was discarded. The aqueous layer was acidified with conc.
HCl to pH=1 and extracted with CH2C12 (3 x 50 mL). The combined organics were
dried over anhydrous MgSO4 and concentrated in vacuo to give the title
compound as
a white solid (1.10 g, 1.63 mmol, 80%).
1H NMR (500 MHz , CD30D): S 8.63 (s, 2H), 7.79 (d, J=2Hz, 2H), 7.73 (t,
J=l.9Hz,
1H), 7.59 (d, J=8.4Hz, 2H), ?.52 (d, J=7.6Hz, 1H), 7.28 (d, J=8.5Hz, 2H), 4.71
(m,
1H), 3.41 (m, 1H), 3.30 (m, 2H), 3.10 (dd, J=8.2, l4Hz, 1H), 2.14 (m, 1H),
1.81 (m,
2H), 1.70 (m, 1H), 1.60 (s, 3H); 13C NMR (125 MHz , CD3OD): 8173.3, 171.4,
160.0, 147.7, 142.6, 142.0, 135.9, 135.3, 133.3, 132.8, 130.0, 129.0, 125.9,
120.6,
70.3, 53.6, 52.5, 50.0, 40.7, 37.3, 22.9, 22.5;
MS mle 673.2 (M+).
EXAMPLE 3
N-(N-(3 5-dichlorobenzenesulfon 1~-(L)-prol 13',5'-dichloroisonicotino
amino)-(L)-phenyl-alanine
-34-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
N-(N-(3,5-Dichlorobenzenesulfonyl)-(L)-prolyl)-4-((3',5'-dichloro-
isonicotinoyl)amino)-(L)-phenylalanine was prepared according to the procedure
described in Example 2, substituting (N-(3,5-dichlorobenzenesulfonyl)-(L)-
proline,
methyl ester (Reference Example 3) for (N-(3,5-dichlorobenzenesulfonyl)-2-
methyl-
(L)-proline, methyl ester.
1H NMR (500 MHz, CD30D): 7.20 (s, 1H); 6.76 (s, 1H); 6.36 (m, 2H); 6.18 (m,
2H);
5.90 (m, 2H); 3.30 (m, 1H); 2.04 (m, 1H); 1.86 (m, 2H); 1.66 (m, 1H); 0.46 (m,
4H);
MS mle 676.1 (M+).
EXAMPLE 4
N-(N-(3,5-Dichlorobenzenesulfon~)-azetidine-2(S)-carbonyl)-4-((3' 5'-dichloro-
isonicotinoyl)amino)-(L)-phenylalanine
Step A N-(3,5-Dichlorobenzenesulfonyl)-azetidine-2(S)-carboxylic acid.
N-(3,5-Dichlorobenzenesulfonyl)-azetidine-2(S)-carboxylic acid was
prepared according to the procedure described in Reference Example 3,
substituting
azetidine-2(S)-carboxylic acid, methyl ester for (L)-proline, methyl ester.
500 MHz 1H NMR (CDCl3): 7.80 (s, 2H); 7.60 (s, 1H); 4.80 (m, 1H); 4.00 (m,
1H);
3.80 (m, 1H); 2.50 (m, 2H)
Step B N-(N-(3,5-Dichlorobenzenesulfonyl)-azetidine-2(S)-carbon 1y )~4-
((3' ,5'-dichloroisonicotinoyl)amino~LL)-phenylalanine.
N-(N-(3,5-Dichlorobenzenesulfonyl)-azetidine-2(S)-carbonyl)-4-
((3',5'-dichloroisonicotinoyl)amino)-(L)-phenylalanine was prepared according
to the
procedure described in Example 2, substituting (N-(3,5-
dichlorobenzenesulfonyl)-
azetidine-2(S)-carboxylic acid, methyl ester for (N-(3,5-
dichlorobenzenesulfonyl)-2-
methyl-(L)-proline, methyl ester.
MS s~z/e 647.2 (M+).
EXAMPLE 5
N-(N-f(3,5-dichlorobenzene)sulfonyll-2-meth 1-~prol 1~~3'-chloro-5'-methox~
isonicotinoyl)aminol-(L)-phenylalanine
Step A N-(N-f(3,5-dichlorbbenzene)sulfonyll-2-meth 1-~prolyl-(L)-4-
nitrophen~lalanine, meth 1
-35-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
To a suspension of N-(3,5-dichlorobenzenesulfonyl)-2-methyl-(L)-
proline (1.25 g, 3.85 mmol), (L)-4-nitrophenylalanine, methyl ester
hydrochloride
(1.10 g, 4.24 mmol) and PyBOP (2.20 g, 4.21 mmol) in CH2C12 (15 mL) was added
DIPEA (1.8 mL, 9.64 mmol). After stirring for 20 h, the reaction was
concentrated
and purified by flash column chromatography on silica gel eluted with
hexane/EtOAc
(2:1) to give N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-methyl-(L)-prolyl-(L)-4-
nitrophenylalanine, methyl ester as a white foam (1.53 g, 73%).
Step B (N-f(3,5-dichlorobenzene)sulfonyll-2-methyl-(L)-prolyl-4-amino-
phenylalanine, meth. l ester
To a solution of N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-methyl-(L)-
prolyl-(L)-4-nitrophenylalanine, methyl ester (1.53 g, 2.89 mmol) in MeOH (25
mL)
was added tin(II) chloride dihydrate (6.4 g, 28.19 mmol) and the reaction was
stirred
for 6 h at 55°C. The reaction was cooled and quenched by the addition
of 1N aqueous
NaOH (50 mL) which caused a precipitate to form. This precipitate was removed
by
filtration and the resulted clear solution was washed with saturated aqueous
NaCl (50
mL), dried over anhydrous MgS04 and concentrated ira vacuo to give the title
compound as a white foam (1.23 g, 85%).
1H NMR (500 MHz , CD30D): 8 7.75 (d, J=2.lHz, 2H), 7.54 (t, J=l.9Hz, 1H), 6.92
(d, J=8.2Hz, 2H), 6.60 (d, J=8.3Hz, 2H), 4.76 (q, J=4.lHz, 1H), 3.75 (s, 3H),
3.48 (m,
1H), 3.34 (m, 1H), 3.13 (dd, J=5.5, 14.2Hz, 1H), 2.98 (dd, J=6.9, 14.2Hz, 1H),
2.30
(m, 1H), 1.77 (m, 1H), 1.65 (m, 2H), 1.60 (s, 3H); MS mle 514.3 (M+).
Step C N-(N-f(3,5-dichlorobenzene)sttlfonyll-2-meth~L)-prolyl)-4-[(3'-
chloro-5'-methoxy-isonicotinoyl)aminol-(L)-phenylalanine meth
ester
To a suspension of 3-chloro-5-methoxy-isonicotinic acid (0.020 g,
0.107 mmol), N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-methyl-(L)-prolyl)-4-amino-
(L)-phenylalanine methyl ester (0.050 g, 0.097 mmol) and PyBOP (0.061 g,
0.1169
mmol) in CH2Cl2 (0.250 mL) was added DIPEA (0.027 mL, 0.1461 mmol). The
reaction was stirred for 5 h then concentrated and purified by preparative
HPLC. The
title compound was isolated as a while solid (0.020 g); MS nZle 683.2 (M~).
-36-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Step D N-(N-f(3,5-dichlorobenzene)sulfonyll-2-methyl-(L)-prolXl)-4-f(3'-
chloro-5'-methoxy-isonicotinoyl)aminol-(L)-phenylalanine
To a solution of N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-methyl-(L)-
prolyl)-4-[(3'-chloro-5'-methoxy-isonicotinoyl)amino]-(L)-phenylalanine,
methyl
ester in MeOH (1 mL) was added 1N NaOH (0.2 mL). The reaction was stirred for
1
hr, then purified by preparative HPLC to give the title compound as a white
solid
(0.015 g); MS nz/e 669.2 (M+)
EXAMPLE 6
N-(N-(3,5-dichlorobenzenesulfonyl)-2-meth)-prolyl)-(L)-3-(6-((3',5'-dichloro-
isonicotinoyl)amino)-3-~yridyl)alanine
Step A 2-Bromo-5-bromometh~p, rid
2-Bromo-5-methylpyridine (4.01 g, 23.31 mmol), NBS (5.19 g, 29.14
mmol), and AIBN (0.19 g, 1.17 mmol) were dissolved in CC14 (46.6 mL, 23.31
mmol). The reaction mixture was heated to 75°C for 4 h. The residue was
quenched
with water and extracted from EtOAc. The organic layer was dried over
anhydrous
MgS04 and concentrated ifi vacuo. The crude mixture was purified by flash
column
chromatography on silica gel eluted with 7% EtOAc/hexane to give 2.65 g of the
title
compound. MS mle 251.9 (M+).
Step B 2-bromo-5-tert-but l~-(diphen l~ lene~lycinate meth~p ridine.
2-Bromo-5-bromomethylpyridine (2.65 g, 10.65 mmol), tert-butyl N-
(diphenylmethylene) glycinate (3.15 g, 10.65 mmol), 10% cesium hydroxide
monohydrate (17.88 g, 106.5 mmol) and O-Allyl-N-(9-anthracenylmethyl)
cinchonidiniurn bromide (0.645 g, 1.065 mmol) were dissolved in CH2C12 (31 mL,
10.65 mmol). The reaction mixture was cooled to -78°C for 4 h and then
to -50°C for
16 h. The residue was quenched with water and extracted from ether. The
organic
layer was washed with brine, dried over anhydrous MgS04, and concentrated isZ
vacuo. The resulting crude mixture was carried on to the next step. MS mle
411.2
(M+)
Step C 3-(6-bromo-3-p~ridine)alanine tert-butyl ester.
-37-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
A crude mixture containing 2-bromo-5-butyl N-(diphenylmethylene)-
glycinate methyl pyridine (10.65 mmol) was dissolved in a mixture of THF (10
mL),
distilled water (10 mL), and HOAc (5 mL). The reaction mixture was mixed at rt
for
1.5 h. The residue was quenched with 2 N HCl and water. The resulting aqueous
layer was made basic with concentrated NaOH and extracted from EtOAc. The
organic layer was dried over anhydrous MgS04 and concentrated irz vacuo to
give 1.5
g of the desired product.
MS rule 247.0 (M+). (mass spectrum shows the desired product minus the mass of
tert-butyl).
Step D ' N-(N-(3,5-dichlorobenzenesulfonyl)-2-methyl-(L)-prol~L)-3-(6-
bromo-3-p.~d~)alanine, tert-but.1
3-(6-Bromo-3-pyridine)alanine, tert-butyl ester (0.75 g, 2.5 mmol), N-
3,5-dichlorosulfonyl-2-methyl-(L)-proline (0.93 g, 2.75 mmol), and PyBOP (1.43
g,
2.75 mmol) were dissolved in CH2C12 (2.5 mL, 2.5 mmol). DIPEA (0.69 mL, 3.75
mmol) was added to the reaction mixture which was stirred at rt for 20 h. The
residue
was quenched with water and extracted from EtOAc. The resulting organic layer
was
washed with brine, dried over anhydrous MgS04 and concentrated i~z vacuo. The
crude mixture was purified by flash chromatography, eluting with 35% ethyl
acetate/hexane to give 0.78 g of the desired product.
MS y~ile 622.2 (M+).
Step E 3,5-Dichloropyridine-4-carboxylic acid chloride
3,5-Dichloropyridine-4-carboxylic acid (0.5 g, 2.6 mmol; Reference
Example 1) was dissolved in CH2C12 (1.75 mL) and DMF (50 ~,L). Thionyl
chloride
(0.21 mLa 2.86 mL) was added and the reaction mixture .was heated at
50°C for 20 h.
The residue was concentrated in vacuo. The formation of the acid chloride was
observed by TLC (50% EtOAc/hexane) and the crude product was carried on to the
next step.
Step F 3 5-Dichloropyridine-4-carbox ay mide.
3,5-Dichloropyridine-4-carboxylic acid chloride (0.54 g, 2.6 mmol)
was dissolved in CH2C12 (2.6 mL, 2.6 mmol) and NH3 (2.0 M in dioxane) (6.5 mL)
was added. The reaction mixture was cooled to 0°C for 30 min. The
residue was
-38-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
quenched with water and extracted with EtOAc. The organic layer was washed
with
brine, dried over anhydrous MgS04 and concentrated isi vacuo. The crude
mixture
was purified by preparative HPLC to give 0.135 g of the desired product.
MS axle 190.9 (M+).
Step G N-(N-(3,5-dichlorobenzenesulfonyl)-2-methyl-(L)-prol. l~~-3-(6-
((3',5'-dichloroisonicotino~)amino)-3-pyridyl)alanine, tert-butyl ester
3,5-Dichloropyridine-4-carboxamide (0.019 g, 0.097 mmol), N-(N-
(3,5-dichlorobenzenesulfonyl)-2-methyl-(L)-prolyl)-(L)-3-(6-bromo-3-
pyridyl)alanine,
tert-butyl ester (0.05 g, 0.081 mmol), cesium carbonate (0.037 g, 0.113 mmol),
tris
(dibenzylideneacetone)dipalladium (0) (0.003 g, 0.0032 mmol), and 9,9-dimethyl-
4,5-
bis(diphenylphosphino)xanthene (0.0056 g, 0.0098 mmol) were dissolved in THF
(0.5
mL). The reaction mixture was heated at 50°C for 20 h. The residue was
dissolved in
EtOAc, filtered through a pad of silica gel and concentrated ire vacuo. The
crude
mixture was purified by preparative HPLC to give 0.016 g of the desired
product.
MS r~zle 732.2 (M+)
Step H N-(N-(3,5-dichlorobenzenesulfonyl)-2-methyl-(L)-prolyl)-(L)-3~6-
((3' ,5'-dichloroisonicotinoyl)amino)-3-p~yl)alanine
N-(N-(3,5-dichlorobenzenesulfonyl)-2-methyl-(L)-prolyl)-(L)-3-(6-
((3',5'-dichloroisonicotinoyl)amino)-3-pyridyl)alanine, tert-butyl ester
(0.016 g, 0.02
mmol) was dissolved in CH2C12 (2 mL) and TFA (1 mL) was added. The reaction
mixture was stirred at rt for 2 h. The residue was concentrated in vacuo and
purified
by preparative HPLC to give 12 mg of the desired product which was
crystallized
from Et20/hexane. Enantiomerically pure material was obtained after further
purification by preparative HPLC.
500 MHz 1H NMR (CD3OD): 8.64 (s, 1H); 8.28 (s, 1H); 8.04 (m, 2H); 7.96 (m,
2H);
7.74 (m, 2H); 4.8 (s, 1H); 3.5 (m, 1H); 3.4 (m, 2H); 3.2 (m, 1H); 1.8 (m, 4H);
1.3 (s,
3H);
MS ~z/e 676.1 (M+)
EXAMPLE 7
N-(N-f (3,5-dichlorobenzene)sulfon 1~1-(L)-prolyl)-4-f (3 5-
dichloroisonicotino.
oxide)aminol-(L)-phenylalanine
-39-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Step A N-(N-f(3,5-dichlorobenzene)sulfon l~prolyl)-4-f(3,5-dichloro-
isonicotinoyl-N-oxide)aminol-(L)-phenylalanine, methyl ester.
N-(N-[(3,5-dichlorobenzene)sulfonyl]-(L)-prolyl)-4-[(3,5-dichloro-
isonicotinoyl)amino]L) phenylalanine, methyl ester (0.105 g, 0.156 mmol) and
mCPBA (0.135 g, 0.781 mmol) were dissolved in CH2Cl2. The reaction mixture was
stirred at rt. TLC did not show completion so the reaction was heated to
50°C and
was monitored by TLC. When the reaction was finished, the residue was quenched
with aqueous NaHC03 and the product was extracted with EtOAc. The organic
layer
was washed with brine. The resulting organic layer was dried over anhydrous
MgS04
and concentrated in vacuo. The crude mixture was purified by preparative HPLC
to
yield the desired product.
MS rule 691.2 (M+)
Step B N-(N-f(3,5-dichlorobenzene)sulfon l~prol,1~[~3',5'-
dichloroisonicotinoyl-N-oxide)aminol-(L)-phenylalanine.
N-(N-[(3,5-dichlorobenzene)sulfonyl]-(L)-prolyl)-4-[(3,5-dichloro-
isonicotinoyl-N-oxide)amino]-(L)-phenylalanine, methyl ester (0.156 mmol) was
dissolved in MeOH and 1N NaOH was added until the reaction turned clear (about
1
mL). The reaction mixture was stirred at rt for about 30 mins. TLC showed that
the
reaction was complete. A few drops of TFA were added to the solution and the
resulting solid was purified by preparative HPLC to give 0.050 g (47.6 %
yield) of the
title compound.
MS fnle 677.2 (M+)
EXAMPLE 8
N-(N-f (3,5-dichlorobenzene)sulfonvll-3-hvdroxv-3-methyl-(L)-brolvl)-4-f (3,5-
dichloroisonicotinoyl)aminol-(L)-phenylalanine
Step A N-f(3,5 Dichlorobenzene)sulfon~ll-glycine tart-butyl ester.
To a solution of t-butyl glycine hydrochloride (2.0g, 11.9mmol ),
DIPEA (3.1g, 23.9mmo1) in CH2Cl2 at 0°C was added 3,5-dichlorobenzene
sulfonyl
chloride (2.1g, 11.9 mmol). The reaction was allowed to warm to rt overnight
and
was concentrated i~a vacuo. The residue was then diluted with EtOAc and washed
with 1M HCl, NaHC03, brine, dried over anhydrous MgSOq. and concentrated in
-40-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
vaccco. The pale yellow solid was then triturated with hexanes. The crude
mixture
was used in the subsequent step.
Step B N-f(3,5-dichlorobenzene)sulfonyll-3-h droxy-3-methy(L)-proline tert-
but, l ester.
To a solution of the product from Step A (0.28, 0.59mmo1) in CH2Cl2
(1 mL) was added methyl vinyl lcetone (0.048, 0.59 mmol), and then DBU
(0.1978,
1.29 mmol). The reaction was allowed to stir overnight. The reaction mixture
was
diluted with EtOAc, and then washed with 1M HCI, NaHC03, and brine, dried over
anhydrous MgS04 and concentrated in vacuo to yield an orange oil. The crude
mixture was used in the subsequent step.
Major diastereomer: 500 MHz IH NMR (CDC13): 7.74(d, 2H); 7.54 (t, 1H); 4.01
(s,
1H); 3.65 (t, 1H); 3.5 (m, 1H); 2.14 (q, 1H); 1.9 (m, 1H); 1.5 (s, 9H); 1.42
(s, 3H)
Step C N-f (3,5-dichlorobenzene)sulfon 1~, droxy-3-meth.~proline.
To a solution of the product from Step B in CH2C12 (2 mL) was added
TFA (2mL). The solution was allowed to stir for 4 h, concentrated in vacuo and
azeotroped with toluene to remove the excess TFA. The solvent was removed by
rotoevaporation and the crude product used without further purification in the
subsequent step.
Step D N-(N-f(3,5-dichlorobenzene)sulfonyll-3-hydroxy-3-methyl-(L~prol.
4-f(3,5-dichloroisonicotinoyl)aminol-(L)-phenylalanine meth l
To a solution of N-[(3,5-dichlorobenzene)sulfonyl]-3-hydroxy-3
methy-(L)-proline from Step C (0.1g, 0.28 mmol), 3,5-(dichloroisonicotinoyl)
amino-
(L)-phenylalanine (0.1148, 0.28 mmol), HOAt (0.06 g, 0.42 mmol) in CH2C12 at
0°C
was added DIPEA base (0.0738, 0.56 mmol) and HATU (0.I078, 0.28 mmol). The
reaction was allowed to warm to rt overnight. The reaction was diluted with
EtOAc,
then washed with 1M HCI, NaHC03, brine, dried over anhydrous MgS04 and
concentrated in vacuo. The crude mixture was purified by flash column
chromatography on silica gel with gradual elutions of 75% hexaneslEt20 to 100%
Et20 to yield 0.1337 g of the desired product which was used in the subsequent
step.
-41 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Step E N-(N-f(3,5-dichlorobenzene)sulfon 1y 1-3-h droxy-3-meth 1-~prolyl)
4-f (3,5-dichloroisonicotinovl)aminol-(L)-phenylalanine.
To a solution of N-(N-[(3,5-dichlorobenzene)sulfonyl]-3-hydroxy-3
methyl-(L)-prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-phenylalanine,
methyl
ester (0.134 g) from Step D in THF was added 1M LiOH. The reaction was allowed
to stir at rt for 4 h. The reaction was then quenched with 2N HCl and
extracted with
EtOAc. The organic layer was dried over anhydrous MgS04 and concentrated i~z
vacuo.
MS f~zle 691.2 (M+)
EXAMPLE 9
N-(N-f (3,5-dichlorobenzene)sulfonvll-4-(S)-phenyl-(L)-nrolvl)-4-f (3,5-
dichloro
isonicotinoyl)aminol-(L)-phenylalanine
Step A 4-(S)-phenyl-(L)-proline.
To a solution of (4S,2S) N-BOC-4-phenyl-pyrrolidine-2-carboxylic
acid (1.0g, 3.4mmo1) in CH2Cl2 at 0°C, was added TFA (15 mL). The
reaction was
allowed warm to rt for 4 h. The reaction was concentrated ifz vacaao and
azeotroped
with toluene. The crude product was used in the subsequent step.
Step B N-f(3,5-dichlorobenzene)sulfonyll-4~S)-phenyl-(L)-proline.
To a solution of the product from Step A (0.66g, 3.43 mmol) in
saturated aqueous Na2C03 was added 3,5-dichlorobenzene sulfonyl chloride (1.79
g,
6.86 mmol). The reaction was allowed to stir overnight at rt. The reaction was
diluted with Et20 and water, and the Et20 layer was discarded. The aqueous
layer
was acidified with 2N HCl to a pH = 4 and extracted with EtOAc. The organic
layers
were collected washed with brine and dried over anhydrous MgS04 and
concentrated
in vaca~o to afford 1.1 g of a pale orange solid. The crude product was used
in the
subsequent step
Step C N-(N-f(3a5-dichlorobenzene)sulfonyll-4-(S~phenyl-(L)-prol.1
f(3,5-dichloroisonicotinoyl)aminol-(L)-phenylalanine methyl ester.
To a solution of N-[(3,5-dichlorobenzene)sulfonyl]-4-(S)-phenyl-(L)-
proline from Step B (0.1g, 0.25 mmol), 3,5-(dichloroisonicotinoyl) amino-(L)-
phenylalanine (0.102g, 0.25 mmol), and HOAt (0.051g, 0.375 mmol) in CH2C12 at
-42-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
0°C was added DIPEA (0.065g, 0.5 mmol) and HATU (O.OlSg, 0.275 mmol).
The
reaction was allowed to warm to nt overnight. The reaction was diluted with
EtOAc,
then washed with 1M HCI, NaHC03, and brine, dried over anhydrous MgS04 and
concentrated in vacuo. The crude mixture was purified by flash column
chromatography on silica gel with gradual elutions of 75% Hexanes/Et20 to 100%
Et20 to give 66.2 mg of the desired product. The purified solid was used in
the
subsequent step.
MS mle 751.2 (M+)
Step D N-(N-((3,5-dichlorobenzene)sulfon 1~(S)-phen 1-y (L)-prolxl)-4-
f(3,5-dichloroisonicotinoyl)aminol-(L)-phenylalanine.
To a solution of the product from Step C (0.066g, 0.088 mmol) in THF
was added 1M LiOH (1m1). The reaction was allowed to stir at rt for 4 h. Then
reaction was acidified with 2N HCl to a pH of 4 and extracted with EtOAc. The
organic layers were washed with brine, dried over anhydrous MgS04 and
concentrated in vacuo to yield N-(N-[(3,5-dichlorobenzene)sulfonyl]-4-(S)-
phenyl-
(L)-prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-phenylalanine.
MS mle 737.2 (M+)
EXAMPLE 10
N-(N-f (3,5-dichlorobenzene)sulfonyll-3-(R)-phen 1-~prolyl)-4- f (3,5-dichloro-
isonicotinoxl)aminol-(L)-phen lad lanine
Step A N-(N-f (3,5-dichlorobenzene)sulfonyll-3-(R)-phen.1-~prol.1
f (3,5-dichloroisonicotinoyl)aminol-(L)-phenylalanine.
N-(N-[(3,5-dichlorobenzene)sulfonyl]-3-(R)-phenyl-(L)-prolyl)-4-
[(3,5-dichloroisonicotinoyl)amino]-(L)-phenylalanine was prepared by the
procedures
described in Example 9 substituting 3(R)-phenylpyrrolidine-2(S)-carboxylic
acid for
4(S)-phenyl-(L)-proline in Step A.
MS m/s 737.2 (M+)
EXAMPLE 11
N-(N-f(3,5-dichlorobenzene)sulfonyll-2-methyl-4-(S)-phen 1-~prolyl)-4-f(3,5-
dichloroisonicotinoyl)aminol-(L)-phenylalanine
Step A N-BOC-4-(S)-phen 1-Y (L)-proline, meth 1 ester.
- 43 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
To a solution of BOC-4(S)-phenyl-pyrrolidine-2(S)-carboxylic acid
(4.0g; 13.73 mmol) in a solution of MeOH (35 mL) and CH2C12 (35 mL) at
0°C was
added over a 10 min period a solution of 1M trimethylsilydiazomethane in
hexanes
(13.73 mL, 27.46 mmol). The reaction was allowed to come to rt over a period
of 3 h.
The reaction was quenched with HOAc. Toluene was added and the reaction was
concentrated ira vacuo. The crude product was used in the subsequent reaction.
Step B N-BOC-2-methyl-4-(S)-phen 1-~proline, meth 1
To a solution of N-BOC-4-(S)-phenyl-(L)-proline, methyl ester from
Step A (2.0g, 6.56 mmol) in anhydrous THF (20 mL) at -78°C was added
over a 30
min period 1M LiHMDS in hexanes (9.83 mL, 9.83 mmol) and allowed to stir for
30
mins at -78°C. The reaction was then allowed to stir for 1 hr at
0°C. The reaction
was then cooled to -78°C and MeI (4.65 g, 32.79 mmol) was added
dropwise. The
reaction was allowed to warm to rt overnight. The reaction was quenched with
NH4Cl. The organic layer was collected and washed with 1M HCI, NaHC03, brine,
dried over anhydrous MgS04 and concentrated ire vacuo. The crude product was
purified by flash column chromatography on silica gel eluted with 75%
Et2Olhexanes
to give 1.85 g of desired product.
500 MHz 1H NMR (CDC13): 7.34 (t, 2H); 7.24 (m, 3H); 4.24 (t, 1H); 3.56(m, 2H);
2.56 (m, 1H); 1.62 (s, 3H); 1.4 (s, 9H)
Step C 2-methyl-4-(S)-pheny~L)-proline, meth, 1y ester.
To a solution of N-BOC-2-methyl-4-(S)-phenyl-(L)-proline, methyl
ester from Step B (1.85g, 5.8 mmol) in MeOH was bubbled HCl (g) over a 10 min
period. The HCl saturated solution was allowed to stir at rt for 5 h,
concentrated ire
vacuo and azeotroped with toluene. The crude product was used in the
subsequent
step.
Step D N-f(3,5-dichlorobenzene)sulfonyll-2-methyl-4-(S)-phenyl-(L)-proline
meth, l
To a solution of 2-methyl-4-(S)-phenyl-(L)-proline, methyl ester from
Step C (0.592g, 1.42 mmol) and 3,5-dichlorobenzenesulfonyl chloride ( 4.26 g,
17.3
mmol) in CH2C12 at 0°C was added DIPEA ( 10g; 57.9 mmol). The reaction
was
allowed to warm to rt over 3 days. The reaction was diluted with EtOAc and
washed
-44-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
with 1M HCI, NaHC03, brine, and dried over anhydrous MgS04 and concentrated in
vacLCO. The crude product was purified by flash column chromatography on
silica gel
eluted with 50% Et20/hexanes. A mixture of two diastereomers was noted.
Step E N-f(3,5-Dichlorobenzene)sulfonyll-2-methyl-4-(S)-phen,1-~proline.
To a solution of N-[(3,5-dichlorobenzene)sulfonyl]-2-methyl-4-(S)-
phenyl-(L)-proline, methyl ester from Step D (2.0g, 5.4mmo1) in MeOH was added
1M NaOH. The reaction was allowed to stir at reflux overnight. The reaction
was
diluted with EtOAc and then acidified with concentrated HCl to a pH of 4 and
extracted with EtOAc. The organic layers were then washed with brine, dried
over
anhydrous MgS04 and concentrated i~a vacuo.
Step F N-(N-f (3,5-dichlorobenzene)sulfonyll-2-methyl-4-(S)-phen 1-
prop)-4-f(3,5-dichloroisonicotinoyl)aminol-(L)-phenylalanine, methxl
ester.
To a solution of N-[(3,5-Dichlorobenzene)sulfonyl]-2-methyl-4-(S)-
phenyl-(L)-proline from Step E (0.2g, 0.483 mmol), ), 4-(3,5-
dichloroisonicotinoyl)-
amino-(L)- phenylalanine (0.195g, 0.483 mmol), and HOAt (0.1g, 0.725 mmol) in
CH2Cl2 (4m1) at 0°C was added DIPEA (0.125g, 0.966 mmol), and HATU
(0.184g,
0.483 mmol). The reaction was allowed to come to rt overnight. The reaction
was
diluted with EtOAc, and washed with 1M HCI, NaHC03, brine, dried over
anhydrous
MgS04 and concentrated in vacuo. The two diastereomers were separated by flash
column chromatography on chiral silica gel to yield 0.1583g of desired
product.
Step G N-(N-f(3,5-dichlorobenzene)sulfonyll-2-methyl-4-(S)-phen,1-
prop-4-f(3,5-dichloroisonicotino 1)aminol-(L)-phenylalanine.
To a solution of one isomer of N-(N-[(3,5-dichlorobenzene)sulfonyl]-
2-methyl-4-(S)-phenyl-(L)-prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-
phenylalanine, methyl ester from Step F (0.1583g, 0.21 mmol) in THF was added
1M
LiOH (1m1). The reaction was allowed to stir at rt for 4 h. The reaction was
diluted
with EtOAc and acidified with 2N HCl to a pH of 4 and~extracted with EtOAc.
The
organic layers were washed with brine, dried over anhydrous MgS04 and
concentrated ira vacuo.
- 45 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
500 MHz 1H NMR (CD30D): 8.6 (s, 1H); 7.8 (2H); 7.72 (d, 1H); 7.55(d, 2H); 7.1
(m,
6H);4.75 (m, 2H); 3.82 (t, 1H); 3.38 (dd, 1H); 3.16 (m, 1H); 2.5 (m, 1H); 1.68
(s, 3H)
MS rile 751.3 (M+)
EXAMPLE 12
N-(N-((3 5-dichlorobenzene)sulfonyll-2-methyl-3-(S)-phenyl-prolxl)-4-~(3,5-
dichloroisonicotinoyl)aminol-(L)-phenylalanine
Step A N-f(3,5-dichlorobenzene)sulfon~l-2-methyl-3-(S)-phen 1-y (L)-proline
methyl ester.
N-[(3,5-Dichlorobenzene)sulfonyl]-2-methyl-3-(S)-phenyl-(L)-proline
methyl ester vas prepared according to the procedures described in Example 11,
Steps
A-D substituting BOC-3(S)-phenyl-pyrrolidine-2(S)-carboxylic acid in place of
BOC-
4(S)-phenyl-pyrrolidine-2(S)-carboxylic acid in Step A.
Step B N-f(3,5-dichlorobenzene)sulfonyll-2-methyl-3-(S)-phen 1-y (L)-proline.
To N-[(3,5-dichlorobenzene)sulfonyl]-2-methyl-3-(S)-phenyl-(L)-
proline methyl ester from Step A (0.601g) was added TMSI neat (5 mL). The
reaction
was allow to stir at 95°C overnight. The blade solution was
concentrated i~z vacuo
and acidified with 2N HCI, and extracted with EtOAC, dried over anhydrous
MgS04
and concentrated iyZ vacuo. The residue was purified by flash column
chromatography
on silica gel elutd with graduated elutions starting from 25 % Et20lhexanes to
100%
Et20 to yield 0.180g of the title compound.
Step C N-f(3,5-dichlorobenzene)sulfonyll-2-methyl-3-(S)-phen~)-proline,
carboxylic acid chloride.
To a solution of N-[(3,5-dichlorobenzene)sulfonyl]-2-methyl-3-(S)-
phenyl-(L)-proline from Step B ( 0. I80g, 0.435 mmol) in CH2C12 was added
thionyl
chloride (1.03g, 8.7 mmol) and stirred at reflux overnight. The reaction was
concentrated ifa vacuo and then azeotroped with toluene. The crude acid
chloride was
used in the subsequent reaction.
Step D N-(N-f(3,5-dichlorobenzene)sulfonyll-2-meth-3-(S~phen ~~1-prop
4-f(3,5-dichloroisonicotinoyl)aminol-(L)-phenylalanine, meth 1 e~ster_.
-46-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
To the acid chloride, N-[(3,5-dichlorobenaene)sulfonyl]-2-methyl-3-
(S)-phenyl-(L)-proline, carboxylic acid chloride, from Step C (0.188, 0.4
mmol) in
CH2C12 at 0°C was added a solution of 4-(3,5-
dichloroisonicotinoyl)amino-(L)-
phenylalanine (0.11g, 0.27 mmol), and DIPEA (0.1g, 0.80 mmol) in CH2Cl2. The
reaction was allowed to warm to rt overnight. The reaction was diluted with
EtOAc
and washed with 1M HCI, NaHC03, brine, dried over anhydrous MgS04 and
concentrated in vacuo. The crude product was purified by flash column
chromatography on silica gel eluted with 75°loEt20/Hexanes and used in
the
subsequent reaction.
Step E ' N-(N-f(3,5-dichlorobenzene)sulfonyll-2-methyl-3(S)-phenyl-prolyl)-4
f (3,5-dichloroisonicotinoyl)aminol-(L)-phenylalanine.
To a solution of N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-methyl-3-(S)-
phenyl-prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl
ester
from Step D in THF was added 1M LiOH. The reaction was allowed to stir at rt
for 4
h. The reaction was acidified with 2N HCl to a pH of 4, extracted with EtOAc,
dried
over anhydrous MgS04 and concentrated isz vacuo to yield N-(N-[(3,5-dichloro-
benzene)sulfonyl]-2-methyl-3-(S)-phenyl-prolyl)-4-[(3,5-dichloroisonicotinoyl)-
amino]-(L)-phenylalanine as a pink solid.
MS mle 751.4 (M+)
EXAMPLE 13
N-(N-f(3,5-dichlorobenzene)sulfonyll-3(R)-cyclohex 1-~prol~l-4-X3,5-dichloro-
isonicotinoyl)aminol-(L)-phenylalanine
Step A N-BOC-3 R)-cyclohexyl-(L)-proline.
To a solution of BOC-3(R)-phenyl-pyrrolidine-2(S)-carboxylic acid
(1.0g, 0.30 mmol) in MeOH was added PtO~ (0.1g, 0.44mmol) which was shaken in
a
Parr shaker under 50 psi atmosphere of H2 overnight. The reaction was filtered
through a pad of celine and concentrated zn vacuo. The crude product was used
in the
subsequent reaction.
Step B 3(R)-cyclohexyl-(L)-proline.
To a solution of N-BOC-3(R)-cyclohexyl-(L)-proline from step A
(1.0g, 0.28 mmol) in CH2C12 (3 mL) at 0°C was added TFA (6 mL). The
reaction
-47-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
was allowed to warm to rt for 4 h. The reaction was then concentrated i~2
vacuo and
azeotroped with toluene. The crude product was used in the subsequent
reaction.
Step C N-(N-f(3,5-dichlorobenzene)sulfonyll-3(R)-cyclohex,1-~prol, l
~(3,5-dichloroisonicotino~)aminol-(L)-phenxlalanine.
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-3(R)-cyclohexyl-(L)-prolyl)-4-
[(3,5-dichloroisonicotinoyl)amino]-(L)-phenylalanine was prepared from 3(R)-
cyclohexyl-(L)-proline according to the procedures described in Example 9,
Steps B-
D.
MS mle 743.2 (M+)
EXAMPLE 14
N-(N-f(3,5-dichlorobenzene)sulfonyll-4-(S)-c cly ohexyl-(L)-prolyl)-4-f(3,5-
dichloro-
isonicotinoyl)aminol-(L -phenylalanine
N-(N-[(3,5-dichlorobenzene)sulfonyl]-4-(S)-cyclohexyl-(L)-prolyl)-4-
[(3,5-dichloroisonicotinoyl)amino]-(L)-phenylalanine was prepared according to
the
procedures described in Example 13 substituting BOC-4(S)-phenyl-pyTOlidine-
2(S)-
carboxylic acid (1.0g, 0.30 mmol) for BOC-3(R)-phenyl-pyrrolidine-2(S)-
carboxylic
acid in Step A.
MS m/s 743.2 (M+)
EXAMPLE 15
N-lN-f (3.5-dichlorobenzene)sulfonvll-3(S)-cvclohexvl-(L)-prolvl)-4-f (3.5-
dichloro
isonicotinoyl)aminol-(L)-phenylalanine
N-(N-[(3,5-dichlorobenzene)sulfonyl]-3(S)-cyclohexyl-(L)-prolyl)-4-
[(3,5-dichloroisonicotinoyl)amino]-(L)-phenylalanine was prepared according to
the
procedures described in Example 13 substituting racemic BOC-3-phenyl-
pyrrolidine-
2-carboxylic acid (1.0g, 0.30 mmol) for BOC-3(R)-phenyl-pyrrolidine-2(S)-
carboxylic acid in Step A. The cis (3S,2S) isomer was isolated by flash column
chromatography on silica gel at Step C after sulfonylation with the 3,5-
dichloro-
benzenesulfonyl chloride.
MS m/s 737.2 (M+)
- 48 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
EXAMPLE 16
N-(N-f (3,5-dichlorobenzene)sulfonyll-traps-3-carboxy-2-methyl-prolyl)-4-f (3'
, 5'-
dichloroisonicotinoyl)aminol-(L)-phenylalanine
Step A Alanineitert-butyl ester, tart-butyl imine.
To a mixture of 25.0 g (0.14 mol) alanine, tart-butyl ester
hydrochloride, 20.0g of 4A powdered molecular sieves, 21 mL (0.15 mol) of TEA
and
400 mL of dry CH2C12 was added 18 mL (0.16 mol) of pivaldehyde. The mixture
was stirred at rt for 17h, diluted with 1L of Et20, filtered through a pad of
celite and
concentrated in vacuo to give 30 g 0100%) of alanine, tart-butyl ester, tart-
butyl
imine as a colorlesss oil which was used without further purification.
500 MHz 1H NMR (CDCl3):S 7.55 (s, 1H); 3.78 (q, 1H); 1.42 (s, 9H); 1.39 (d,
3H);
1.08 (s, 9H).
Step B traps-3-Methoxycarbonyl-2-methyl-5-oxo-proline, oc-tart-but fester.
To a solution of 15 g (70.4 mmol) of the imine from Step A and 6.7 g
(77.5 mmol) of Liar in 300 mL of THF at -10°C was added 10.1 g (70.4
mmol) of
dimethyl fumarate in 25 mL of THF followed by 10.5 mL (70.4 mmol) of DBU.
After 75 min at -10°C, the reaction was quenched with saturated aqueous
solution of
NH4Cl and diluted with Et20 and the layers were separated. The organic layer
was
washed with NaHC03 (2 x 50 mL), brine (1 x 50 mL), dried over anhydrous MgS04
and concentrated i~z vacuo.
The crude residue was dissolved in 250 mL of MeOH and 80 mL of
H20 and 0.1 mL of HOAc was added. The reaction was refluxed for 16h, cooled
and
concentrated. The crude product was dissolved in EtOAc and washed with NaHC03
(2 x 50 mL), brine (1 x 50 mL), dried over anhydrous MgS04 and concentrated zn
vauco to give a solid. Trituration with hexanes provided 7.7g of traps-3-
methoxy-
carbonyl-2-methyl-5-oxo-proline, tart-butyl ester as a white solid,
homogeneous by
T.L.C. analysis.
500 MHz 1H NMR (CDC13): S 7.10 (brs, 1H, NH); 3.75 (s, 3h); 3.60 (dd, 1H);
2.78
(dd, ~H); 2.5 (dd, 1H); 1.42 (s, 9H); 1.39 (s, 3H).
Step C traps-3-Methoxycarbonyl-2-methyl-5-thiocarbonyl-proline, a-tert-
butyl ester.
-49-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
A mixture of 7.5g (25.8 mmol) of 2-methyl-3-methoxycarbonyl-5-oxo-
proline, tart-butyl ester from Step B and 6.2g (15.5 mmol) of Lawesson's
reagent in
100 mL of toluene was warmed to 70°C for 1h and then cooled and
concentrated to
give a solid. Trituration with Et20/hexanes gave 6.6 g of 2-methyl-3-methoxy-
carbonyl-5-thiocarbonyl-proline, tart-butyl ester as a white solid homogeneous
by
T.L.C. analysis that was used in the subsequent reaction without further
purification.
Step D traps-3-Methoxycarbonyl-2-meth~proline, a-tent-but.1
To a solution of 6.6 g (21.5 mmol) of 2-methyl-3-methoxycarbonyl-5-
thiocarbonyl-proline, tart-butyl ester from step C in 150 mL of MeOH was added
excess RaNiThe reaction was stirred at rt until the starting material was
consumed
as judged by T.L.C. analysis. The mixture was filtered through a pad of celite
and
concentrated isi vacuo to give 5 g of a colorless oil.
A mixture of this oil, PdIC and 100 mL of EtOAc was stirred under a
balloon of H2 for 26 h. The mixture was filtered through a pad of celite and
concentrated to give 4.5g of 2-methyl-3-methoxycarbonyl-proline, tart-butyl
ester (a
single diastereomer) as a colorless oil homogeneous by T.L.C. analysis that
was used
without further purification.
500 MHz 1H NMR (CDC13): 8 3.65 (s, 3H); 3.2 (m, 1H); 3.15 (m, 1H); 3.0 (m,
1H):
2.36 (s, 1H, NH); 2.2 (m, 1H); 1.95 (m, 1H); 1.42 (s, 9H); 1.25 (s, 3H).
Step E N-((3,5-dichlorobenzene)sulfonyll-traps-3-methox. c~nyl-2-methyl-
proline, a-tent-butfester.
To a solution of 2-methyl-3-methoxycarbonylrproline, tart-butyl ester
(2.0 g, 7.3 mmol) from Step D and 2.6 g (14.5 mmol) of 3,5-
dichlorobenzenesulfonyl
chloride in 20 mL of CH2C12 at 0°C was added 3.8 mL (21.8 mmol) of
DIPEA. The
reaction was allowed to warm to rt overnight. After 14 h, the reaction was
diluted
with 150 mL of Et20 and washed with 1M HCl (2 x 50 mL), saturated NaHC03 (2 x
50 mL), brine (1 x 50 mL), dried over anhydrous MgS04, and concentrated in
vacuo.
N-[(3,5-Dichlorobenzene)sulfonyl]-traps-3-methoxycarbonyl-2-methyl-proline,
tart-
butyl ester (3.8 g) was isolated as a light orange solid and was used without
further
purification.
-50-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
The test-butyl ester was dissolved in CH2Cl2 and treated with TFA
according to the procedure described in Example 8, Step C to afford N-[(3,5-
dichloro-
benzene)sulfonyl]-traps-3-methoxycarbonyl-2-methyl-proline.
Step F N-(N-f (3,5-dichlorobenzene)sulfonyll-traps-3-methoxycarbonyl-2-
meth ~~l-prolyl)-4-f(3',5'-dichloroisonicotinoyl)aminol-(L)-
phenylalanine, methyl ester.
A solution of 426 mg (1.08 mmol) of N-[(3,5-dichlorobenzene)-
sulfonyl]-2-methyl-3-methoxycarbonyl-proline, 435 mg (1.08 mmol) of 4-(3,5-
dichloroisonicotinoyl)amino-(L)-phenylalanine, methyl ester hydrochloride, 492
mg
(1.29 mmol) ~HATU, 220 mg (1.6 mmol) HOAt, and 348 mg (2.73 mmol) DIPEA in
THF (3 mL) was stirred at rt overnight. The desired product was purified by
flash
column chromatography on silica gel eluted with 3:1 hexanes-Et20 then 3:1 Et20-
.
hexanes then 100% Et20) to give 900 mg of the desired product as a colorless
oil.
Subsequent purification by preparative chiral HPLC (AS column,
15°l0
EtOH/hexanes) gave 400 mg of a less polar diastereomer and 386 mg of a more
polar
diastereomer.
Step G N-(N-f(3,5-dichlorobenzene)sulfonyll-traps-3-methoxycarbon
meth ~~1-prolyl)-4-f(3',5'-dichloroisonicotinoyl)aminol-(L)-
phen~alanine.
To a solution of 400mg of the less polar diastereomer from Step F in
10 mL of THF at 0°C was added 8 mL of 1M LiOH. The reaction was stirred
at 0°C
for 2h and was then warmed to rt, diluted with EtOAc and acidified with 2M HCl
until a pH ~4 was reached. The layers were separated and the aqueous layer was
extracted with EtOAc (3x). The combined organic layers were dried over
anhydrous
MgS04 and concentrated irz vacuo to give 355 mg of N-(N-[(3,5-dichlorobenzene)-
sulfonyl]-traps-3-methoxycarbonyl-2-methyl-prolyl)-4-[(3',5'-
dichloroisonicotinoyl)-
amino]-(L)-phenylalanine as a colorless foam.
HPLC:MS 719.1 (M+H).
EXAMPLE 17
N-(N-f(3,5-Dichlorobenzene)sulfonyll-traps-3-phenyl-prol, 1y )-4-f(3,5-
dichloro-
isonicotinoyl)aminol-(L)-phen lalanine
-51-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
N-(N-[(3,5-dichlorobenzene)sulfonyl]-trans-3-phenyl-prolyl)-4-[(3,5-
dichloroisonicotinoyl)amino]-(L)-phenylalanine was prepared by the procedures
described in Example 9 substituting racemic 3-phenylpyrrolidine-2-carboxylic
acid
for 4(S)-phenyl-(L)-proline in Step A. The cis and trans final products were
separated
by preparative thin layer chromatography by sequential multiple elutions with
Et20,
Et20/EtOAc, Et20/EtOAcIMeOH to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-
trans-3-phenyl-prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-phenylalanine
as a
1:1 mixture of diastereomers.
HPLC:MS 737.3 (M+H).
EXAMPLE 18
N-(N-f (3,5-dichlorobenzene)sulfonyll-trans-3-hydrox~yl-2-meth~prol 1»
f (3' ,5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine
Step A N-f(3,5-dichlorobenzene)sulfonyll-trans-3-carboxy-2-methyl-proline,
oc-tart-but, l
N-[(3,5-dichlorobenzene)sulfonyl]-trans-3-methoxycarbonyl-2-methy1-
proline, tart-butyl ester (3.8 g, 9.5 mmol) from Example 16, Step E was
hydrolyzed in
THF with 1M LiOH to give 3.3 g of N-[(3,5-dichlorobenzene)sulfonyl]-trans-3-
carboxy-2-methyl-proline, oc-tart-butyl ester as a light tan solid that was
used without
further purification.
Step B N-f(3,5-dichlorobenzene)sulfonyll-trans-3-hydrox~yl-2-meth
proline, cc-tart-but, l ester.
To a solution of N-[(3,5-dichlorobenzene)sulfonyl]-trans-3-carboxy-2-
methyl-proline, cc-tart-butyl ester (300 mg, 0.64 mmol) in 4 mL of THF was
added
0.091 mL 0.96 mmol) of BH3~DMS. The reaction was warmed to 45°C and
maintained there until the starting material was consumed as judged by T.L.C.
analysis (5-6 h). MeOH was added to quench excess reducing agent and the
reaction
was concentrated in vacuo to give a colorless oil which was used without
further
purification.
500 MHz 1H NMR (CDC13): b 7.78 (s, 2H); 7.58 (s, 1H); 3.75 (m, 1H); 3.62 (m,
1H); 3.48 (m, 1H); 3.37 (m, 1H), 2.65 (m, 1H); 2.08 (m, 1H); 1.8 (br s, 1H);
1.70 (m,
1H); 1.6 (s, 3H); 1.58 (s, 9H).
-52-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Step C ~N-f(3,5-dichlorobenzene)sulfonyll-trans-3-hydrox meth
meth ~~1-prolyl)-4-f(3',5'-dichloroisonicotinoyl)aminol-(L)-
phenylalanine.
N-[(3,5-dichlorobenzene)sulfonyl]-trans-3-hydroxymethyl-2-methyl-
proline, oc-tert-butyl ester (175 mg (0.38 mmol) from Step B was converted to
N-(N-
[(3,5-dichlorobenzene)sulfonyl]-trans-3-hydroxyxnethyl-2-methyl-prolyl)-4-
[(3',5'-
dichloroisonicotinoyl)amino]-(L)-phenylalanine according to the procedures
described
in Example 16, Steps E-G.
HPLC:MS 659.2 (M+H).
EXAMPLE 19
N-(N-f (3,5-dichlorobenzene)sulfonyll-3(R)-methox -~prolyl)-4-[(3,5-dichloro-
isonicotino~)aminol-(L)-phenylalanine
Step A N-BOC-3(R)-methox, -~proline, methyl ester.
To a solution of 500 mg (2.05 mmol) of N-BOC-3(R)-hydroxy-(L)-
proline, methyl ester and 1.7 g (12.2 mmol) of MeI in 10 mL of DMF was added
473
mg (2.05 mmol) of Ag20. The mixture was stirred for 18h and was then filtered
through a pad of celite and concentrated. The crude residue was dissolved in
EtOAc
and washed with 1M HCl (2 x 10 mL), saturated NaHC03 (2 x 10 mL), brine (1 x
10
mL), dried anhydrous MgS04, and concentrated to give 500 mg of N-BOC-3(R)-
methoxy-(L)-proline, methyl ester as a light yellow oil which was used without
further
purification.
500 MHz 1H NMR (CDC13): 8 4.4 (s, 0.4H); 4.22 (s, 0.6H); 3.9 (m, 0.6H); 3.8
(m,
0.4H), 3.7 (s, 3H), 3.7-3.62 (m, 2H), 3.0 (s, 3H), 2.0 (m, 2H), 1.63 (s, 4H),
1.60 (s,
5H).
Step B N-(3,5-dichlorobenzene)sulfon 1~-3(_R -methoxy~L)-proline meth
ester.
To a solution of N-BOC-3(R)-methoxy-(L)-proline, methyl ester (500
mg, 1.94 mmol) of the product from Step A in 5 mL of CH2C12 was added 5 mL of
TFA. The reaction was aged at rt until the starting material was consumed as
judged
by T.L.C. analysis. The reaction was concentrated, azeotroped with toluene,
and used
without further purification.
-53-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
To as solution of the crude amine in 5 mL of CH2C12 at 0°C was
added 682 mg (3.88 mmol) of 3,5-dichlorobenzenesulfonyl chloride followed by 1
mL 9.7 mmol) of DIPEA. The reaction was allowed to warm to rt over 12 h and
was
then diluted with EtOAc and washed with 1M HCl (2 x 50 mL), saturated NaHC03
(2
x 50 mL), brine (1 x 50 mL), dried anhydrous MgS04, and concentrated in vacuo.
The residue was purified by flash column chromatography on silica gel eluted
with
sequentially with a gradient of 3:1 hexanes-Et20 then 1:1 hexanes-Et20 then
3:1
hexanes-Et20 then 100% Et20 to give 150 mg of N-(3,5-dichlorobenzene)sulfonyl-
3-
(R)-methoxy-(L)-proline, methyl ester as a near colorless oil.
500 MHz 1H NMR (CDC13): 8 7.78 (s, 2H); 7.6 (s, 1H); 4.4 (s, 1H); 3.95 (s,
1H); 3.8
(s, 3H); 3.6 (t, 1H), 3.4 (m, 1H), 3.22 (s, 3H), 2.05 (m, 2H).
Step C N-(N-f (3,5-dichlorobenzene)sulfonyll-3-(R)-methox~!-~L)-prol~l)-4
f (3,5-dichloroisonicotinoyl)aminol-(L)-phenylalanine.
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-3-(R)-methoxy-(L)-prolyl)-4-
[(3,5-dichloroisonicotinoyl)amino]-(L)-phenylalanine was prepared from N-(3,5-
dichlorobenzene)sulfonyl-3(R)-methoxy-(L)-proline, methyl ester (150 mg)
according
to the procedures described in Example 9, (Step D (LiOH, MeOH), Step C (3,5-
(dichloroisonicotinoyl) amino-(L)-phenylalanine, methyl ester, HOAt, DIPEA,
and
HATU), and Step D again (LiOH, MeOH)) and was obtained as a white solid.
HPLC:MS 705.2 (M+H).
EXAMPLE 20
N-(N-f (3-Chlorobenzene)sulfonyll-4(R)-isopro~ylamino-2-methyl-(L~prolxl)-4-
j(3',5'-dichloroisonicotinoyl)aminol-fL)-phenylalanine, formic acid salt
Step A N-BOC-4(S)-h d~roxy-2-methyl-(L)-proline methyl ester
To a solution of N-BOC-4(S)-hydroxyproline, methyl ester (Bachem,
10 g, 40 mmol) and MeI (10 mL, 160 mmol) in 150 mL of anhydrous THF at -30
°C
was added LDA (Aldrich, 1.5 M in cyclohexane, 100 mL, 150 mmol). The reaction
was allowed to warm up to rt over 4 h. The reaction was cooled to -30
°C, and was
quenched with saturated aqueous ammonium chloride (50 mL). The resulting
mixture
was partitioned between EtOAc and brine, and the product was extracted with
EtOAc
(3x100 mL). The combined extracts were dried with anhydrous MgS04 and
-54-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
concentrated irZ vacuo. The residue was purified by silica gel chromatography
eluting
with 20:1 to 10:1 CH2C12lacetone to give the title compound (repeated
chromatography was necessary to obtain diastereomerically pure material;
faster
eluting and minor isomer, 2.2 g, 21%), which solidify upon standing. The
stereochemistry was assigned by NMR comparisons with literature reports (Noe,
CR
et al Pl2armazie 1996, SI, 800).
1H NMR (500 MHz, CD30D): 8 (mixture of two rotamers) 4.40-4.32 (m, 1H); 3.73-
3.66 (m, 1H); 3.70 (s, 3H); 3.34-3.28 (m, 1H); 2.17/2.12 (d, J=6.5 Hz, 1H);
1.534/1.529 (s, 3H); 1.44/1.40 (s, 9H).
Step B ' N-f(3-Chlorobenzene)sulfonyll-4(S)-hydroxy-2-methyl-(L)-proline
meth, l ester.
A solution of N-BOC-4(S)-hydroxy-2-methyl-(L)-proline, methyl ester
. (Step A, 2.2 g, 8.5 mmol), tert-butyldimethylsilyl chloride (1.8 g, 12
mmol), and
imidazole (1.1 g, 17 mmol) in 40 mL of anhydrous DMF was stirred at rt
overnight.
The reaction mixture was partitioned between Et20 and water, and the product
was
extracted with Et20 (2x200 mL). The combined extracts were dried with
anhydrous
MgS04, filtered and concentrated in vacuo. The residue was dissolved in
anhydrous
CH2C12 (15 mL), and was added with 2,6-dimethyllutidine (3.0 mL, 26 mmol) and
tent-butyldimethylsilyl trifluoromethanesulfonate (3.9 mL, 17 mmol) at rt.
After
stirring for 2 h, the reaction mixture was cooled by an ice-water bath, and
was added
potassium fluoride (1.4 g, 24 mmol) in 20 mL of water. After stirring at
0°C for 2 h,
the reaction mixture was partitioned between ether and water. The product was
extracted with Et20 (3x50 mL), and the organic extracts were dried over
anhydrous
MgS04, filtered and concentrated in vacuo. The residue was azeotroped with
toluene
and was dissolved in CH2C12 (10 mL) and THF (10 mL), The solution was cooled
with an ice water bath, and was added DIPEA (4.4 mL, 25 mmol), 4-DMAP (0.10 g,
0.85 mmol) and 3-chlorobenzenesulfonyl chloride (2.7 g, 12 mmol). The reaction
was allowed to warm to rt overnight. The reaction mixture was concentrated to
dryness, and the residue was purified by flash column chromatography on silica
gel
eluted with 4:1 hexane/EtOAc to give the tent-butyldimethylsilyl ether of the
title
compound, which was used immediately. Thus, to a solution of the silyl ether
in
acetonitrile (40 mL) at 0 °C was added aqueous HF (48%, 4 mL), and the
reaction was
allowed to warm up to rt overnight. The reaction mixture was partitioned
between 5
-55-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
N aqueous NaOH, brine and EtOAc, and the product was extracted with EtOAc
(3x100 mL). The combined extracts were dried over anhydrous sodium sulfate,
filtered and concentrated in vacuo to dryness to give the title compound (3.0
g, 96%
from the product of Step A).
1H NMR (500 MHz, CD30D): 8 7.87 (br t, J=1.8 Hz, 1H); 7.81 (br d, J=8.0 Hz,
1H);
7.65 (br d, J=8.0 Hz, 1H); 7.57 (apparent t, J=8.0 Hz, 1H); 4.36 (apparent
quintet,
J=5.3 Hz, 1H); 3.72 (s, 3H); 3.62 (dd, J=9.5, 6.0 Hz, 1H); 3.27 (dd, J=9.5,
4.5 Hz,
1H); 2.27 (dd, J=13.0, 5.5 Hz, 1H); 2.17 (dd, J=13.0, 5.5 Hz, 1H); 1.65 (s,
3H).
MS: calculated for C13H16C1N05S 333, observed m/e 334 (M + H)~".
Step C ~ N-f(3-Chlorobenzene)sulfonyll-4(R)-tert-butox. ca~rbon_ylamino-2-
meth.~(L)-proline, meth.1
To a solution of the product of Step B (3.1 g, 9.0 mmol) in anhydrous
CH2Cl2 (20 mL) was added pyridine (3.6 mL, 45 mmol) and mesyl chloride (1.4
mL,
18 mmol) at 0 °C. After stirring at rt overnight, the reaction mixture
was diluted with
EtOAc and was washed with dilute cupric sulfate (2x) and brine, dried over
anhydrous MgS04, filtered and concentrated to dryness to give the crude
product,
which was used without further purification. Thus, the residue was dissolved
in DMF
(20 mL) and sodium azide (3.0 g, 46 mmol) was added. After stirring at
50°C
overnight, another batch of sodium azide (3:0 g, 46 mmol) was added, and
stirring
was continued for another 4 h at 70°C. The reaction was cooled to rt,
and the
resulting mixture was partitioned between Et20 and water. The product was
extracted with Et20 (3x100 mL). The combined extracts were dried with
anhydrous
MgS04, filtered and concentrated in vacuo, and the residue was purified by
flash
column chromatography on silica gel eluted with 4:1 hexane/EtOAc to give the
product N-[(3-chlorobenzene)sulfonyl]-4(R)-azido-2-methyl-(L)-proline methyl
ester,
which was used without further purification.
Thus, a solution of the above azide (0.90 g, 2.5 mmol) and di-tert-butyl
Bicarbonate (0.70 g, 3.2 mmol) in EtOAc (10 mL) was added to a slurry of
platinum
dioxide (90 mg) in 2 mL of EtOAc (pre-degassed and charged with hydrogen). The
mixture was degassed and was stirred at rt overnight under a balloon
atmosphere of
hydrogen gas. The reaction mixture was concentrated to dryness, and the
residue was
purified by flash column chromatography on silica gel eluted with 4:1
hexane/EtOAc
-56-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
to give the product N-[(3-chlorobenzene)sulfonyl]-4(R)-tert-
butoxycarbonylamino-2-
methyl-(L)-proline, methyl ester (1.1 g, 28% from the product of Step B).
1H NMR (500 MHz, CD30D): ~ 7.81 (br t, J=1.8 Hz, 1H); 7.77 (br d, J=8.0 Hz,
1H);
7.65 (br d, J=8.0 Hz, 1H); 7.57 (apparent t, J=8.0 Hz, 1H); 4.28-4.20 (m, 1H);
3.87
(dd, J=9.5, 7.5 Hz, 1H); 3.76 (s, 3H); 3.15 (dd, J=9.5, 7.8 Hz, 1H); 2.44 (dd,
J=13.0,
7.0 Hz, 1H); 1.90 (dd, J=13.0, 9.5 Hz, 1H); 1.64 (s, 3H); 1.41 (s, 9H).
MS: calculated for C18H25C1N2O6S 432, observed m/e 455 (M + Na)+.
Step D N-f(3-Chlorobenzene)sulfonyll-4(R)-tert-butox c~onylamino-2-
meth,1-~proline.
To a solution of N-[(3-chlorobenzene)sulfonylJ-4(R)-tert-butoxy-
carbonylamino-2-methyl-(L)-proline, methyl ester from Step C (1.1 g, 2.5 mmol)
in
1:1:1 THF/MeOH/water (total 12 mL) was added LiOH monohydrate (1.1 g, 26
mmol). After stirring at rt overnight, the reaction mixture was partitioned
between
EtOAc/brine/0.5 M sodium hydrogen sulfate. The product was extracted with
EtOAc
(3x20 mL), and the organic extracts were dried with sodium sulfate, filtered
and
concentrated to dryness to give the product N-[(3-chlorobenzene)sulfonylJ-4(R)-
tert-
butoxycarbonylamino-2-methyl-(L)-proline (1.0 g, 91%).
~1H NMR (500 MHz , CD30D): 8 7.85 (br t, J=1.8 Hz, 1H); 7.79 (br d, J=8.0 Hz,
1H); 7.64 (br d, J=8.0 Hz, 1H); 7.56 (apparent t, J=8.0 Hz, 1H); 4.30-4.20 (m,
1H);
3.84 (dd, J=9.0, 7.5 Hz, 1H); 3.14 (dd, J=9.5, 9.0 Hz, 1H); 2.46 (dd, J=13.0,
7.0 Hz,
1H); 1.89 (dd, J=13.0, 9.5 Hz, 1H); 1.65 (s, 3H); 1.41 (s, 9H).
Step E N-(N-((3-Chlorobenzene)sulfon 1y 1-4(R)-tert-butox, carbonylamino-2-
meth 1-~prolyl)-4-f(3',5'-dichloroisonicotinoxl)aminol-(L)-
phenylalanine, meth, l
N-(N-[(3-Chlorobenzene)sulfonyl]-4-(R)-tert-butoxycarbonylamino-2-
methyl-(L)-proline (Step D, 0.75 g, 1.8 mmol) was coupled to 4-[(3',5'-
dichloro-
isonicotinoyl)amino]-(L)-phenylalanine, methyl ester by the procedure
described in
Example 1 (0.69 g, 50%).
1H NMR (400 MHz, CD30D): b 8.63 (s, 2H), 7.9-7.2 (m, 8H), 4.73 (m, 1H), 4.1-
4.0
(m, 1H), 3.8-3.7 (m, 1H), 3.78 (s, 3 H); 3.3-3.0 (m, 3H); 2.3-2.2 (m, 1H); 1.7-
1.6 (m,
1H); 1.6 (s, 3H); 1.4 (s, 9H).
MS: calculated for C33H36C13N5O8S 767, observed m/e 768 (M + H)+.
-57-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Step F N-(N-f(3-Chlorobenzene)sulfonyll-4(R)-tert-butoxycarbonylamino-2-
meth ~~1-~L)-prolyl)-4-f(3',5'-dichloroisonicotino~)aminol-(L)-
nhenylalanine.
To a solution of N-(N-[(3-chlorobenzene)sulfonyl]-4(R)-tent-butoxy-
carbonylamino-2-methyl-(L)-prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine, methyl ester (Step E, 0.69 g, 0.89 mmol) in AcCN (5 mL) and
water (2
mL) was added LiOH monohydrate (0.15 g, 3.6 mmol). After stirring at rt for 2
h, the
reaction mixture was partitioned between EtOAc/brine/0.5 M sodium hydrogen
sulfate. The product was extracted with EtOAc (2x50 mL), and the organic
extracts
were dried over anhydrous MgS04, filtered and concentrated to give the product
N-
(N-[(3-chlorobenzene)sulfonyl]-4(R)-tent-butoxycarbonylamino-2-methyl-(L)-
prolyl)-
4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine (0.67 g, 99%).
1H NMR (400 MHz, CD30D): ~ 8.63 (s, 2H); 7.9-7.3 (m, 8H); 4.8-4.7 (m, 1H); 4.1-
4.0 (m, 1H); 3.7 (dd, 1H); 3.3-3.0 (m, 2H); 2.3-2.2 (m, 1H); 1.7 (dd, 1H); 1.6
(s, 3H);
1.4 (s, 9H).
MS: calculated for C32H34C13N508S 753, observed m/e 754 (M + H)+.
Step G N-(N-f(3-Chlorobenzene)sulfonyll-4(R)-amino-2-meth,1-~prol, l
f~3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine, HCl.
A sample of N-(N-[(3-chlorobenzene)sulfonyl]-4(R)-tert-butoxy-
carbonylamino-2-methyl-(L)-prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine (Step F, 0.62 mg, 0.81 mmol) was treated with 7 mL of saturated
hydrogen chloride in EtOAc for 5 h at rt. Concentration in vacuo afforded N-(N-
[(3-
chlorobenzene)sulfonyl]-4(R)-amino-2-methyl-(L)-prolyl)-4-[(3',5'-dichloro-
isonicotinoyl)amino]-(L)-phenylalanine, hydrochloride as a solid (0.61 g,
100%).
1H NMR (400 MHz, CD30D): S 8.63 (s, 2H); 7.8-7.3 (m, 8H); 4.62 (m, 1H); 3.93
(dd, 1H); 3.75 (m, 1H); 3.45-3.30 (m, 2H); 3.06 (dd, 1H); 2.64 (dd, 1H)1.92
(dd,
1H); 1.75 (s, 3H).
MS: calculated for C27H26C13N5O6S 653, observed m/e 654 (M + H)+.
Step H N-(N-f(3-Chlorobenzene)sulfon, l~(R)-isopropylamino-2-meth 1-~)-
prolyl)-4-f (3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine,
formic acid salt
_58_
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
To a sample of N-(N-[(3-chlorobenzene)sulfonyl]-4(R)-amino-2-
methyl-(L)-prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine
hydrochloride salt (Step G, 0.10 mg, 0.14 mmol) in acetonitrile (1 mL) was
added
acetone (0.010 mL, 0.14 mmol), DIPEA (0.048 mL, 0.27 mmol) and sodium
triacetoxyborohydride (0.15 g, 0.70 mmol). After stirring at rt overnight, the
reaction
was quenched with formic acid (96%, 0.1 mL) and was diluted with DMSO (1 mL)
and water (1 mL). The resulting mixture was loaded onto a reverse phase
preparative
HPLC column eluted with acetonitrile and water (containing 0.1 % formic acid)
to
afford N-(N-[(3-chlorobenzene)sulfonyl]-4(R)-isopropylamino-2-methyl-(L)-
prolyl)-
4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, formic acid salt (74
mg,
72%).
1H NMR (400 MHz, CD30D): 8 8.63 (s, 2H); 8.20 (s, 1H); 7.85-7.30 (m, 8H); 4.65
(m, 1H); 4.00 (dd, 1H); 3.82 (m, 1H); 3.42-3.25 (m, 2H); 3.12 (dd, 1H); 2.7-
2.6 (m,
2H); 1.95 (dd, 1H);1.72 (s, 3H); 1.28 (d, 3H); 1.24 (d, 3H).
MS: calculated for C30H32C13N5O6S 695, observed m/e 696 (M + H)+.
EXAMPLE 21
N-(N-f (3-Chlorobenzene)sulfonyll-4(R)-dimethylamino-2-meth 1-~prol l
f (3' S'-dichloroisonicotinoxl)aminol-(L)-phenylalanine, formic acid salt
To a sample of N-(N-[(3-chlorobenzene)sulfonyl]-4(R)-amino-2-
methyl-(L)-prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine
hydrochloride salt from Example 20, Step G (0.30 mg, 0.41 mmol) in
acetonitrile (3
mL) was added aqueous formaldehyde (0.31 mL, 4.1 mmol), DIPEA (0.14 mL, 0.82
mmol) and sodium triacetoxyborohydride (0.44 g, 2.1 mmol). After stirring at
rt
overnight, the reaction was quenched with formic acid (96%, 0.2 mL) and was
diluted
with dimethylsulfoxide (3 mL) and water (3 mL). The resulting mixture was
loaded
onto a reverse phase preparative HPLC column eluted with acetonitrile and
water
(containing 0.1% formic acid) to yield N-(N-[(3-Chlorobenzene)sulfonyl]-4(R)-
dimethylamino-2-methyl-(L)-prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine, formic acid salt (0.21 g, 69%).
1H NMR (400 MHz, CD3OD): ~ 8.63 (s, 2H); 8.18 (s, 1H); 7.88-7.25 (m, 8H); 4.58
(m, 1H); 3.88 (dd, 1H); 3.42-3.00 (m, 3H); 2.5-2.5 (m, 7H); 1.98 (dd, 1H);
1.78 (s,
3H).
MS: calculated for C29H30C13N5O6S 681, observed m/e 682 (M + H)+.
-59-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
EXAMPLE 22
N-(N-f (3-Chlorobenzene)sulfonyll-4(R)-(1-azetidinyl)-2-methyl-(L)-prop)-4-
f(3',5'-
dichloroisonicotinoyl)aminol-(L)-phenylalanine, formic acid salt
Step A N-f(3-Chlorobenzene)sulfon 1Y 1-4(R)-(1-azetidinyl)-2-methyl-(L)-
proline, meths ester
To a solution of N-[(3-chlorobenzene)sulfonyl]-4(S)-hydroxy-2-
methyl-(L)-proline, methyl ester from Example 20, Step B (1.2 g, 3.3 mmol) and
DIPEA (1.4 mL, 8.3 mmol) in 10 mL of anhydrous CH2Cl2 at -20 °C
was added
trifluoromethanesulfonic anhydride (0.83 mL, 5.0 mmol) dropwise. After
stirring at
-20 °C for 45 min, half of the reaction mixture was transferred to a
separate flask, and
azetidine (1.0 g, 18 mmol) was added to the remaining solution. The reaction
was
allowed to warm up to rt overnight, and the resulting mixture was purified by
flash
column chromatography on silica gel eluted with 1:1:0.02 hexane/EtOAcl2 M
ammonia in MeOH to yield N-[(3-chlorobenzene)sulfonyl]-4(R)-(1-azetidinyl)-2-
methyl-(L)-proline, methyl ester (0.54 g, 97°l0).
1H NMR (500 MHz, CD30D): 8 7.91 (br t, J=1.8 Hz, 1H); 7.78 (br d, J=8.0 Hz,
1H);
7.65 (br d, J=8.0 Hz, 1H); 7.57 (apparent t, J=8.0 Hz, 1H); 3.69 (s, 3H); 3.64
(dd,
J=10.0, 6.0 Hz, 1H)3.22-3.16 (m, 4H); 3.08 (m, 1H); 2.28 (dd, J=13.5, 6.5 Hz,
1H);
2.04 (qintet, J=7.0 Hz); 1.76 (dd, J=13.5, 6.0 Hz, 1H); 1.66 (s, 3H).
Step B N-f (3-Chlorobenzene)sulfonyll-4(R)-(1-azetidinyl)-2-meth 1-
rop line.
To a solution of N-[(3-chlorobenzene)sulfonyl]-4(R)-(1-azetidinyl)-2-
methyl-(L)-proline, methyl ester (Step A, 0.54 g, 0.15 mmol) in THF/MeOH/water
(1
mL each) was added LiOH monohydrate (0.27 g, 6.4 mmol). After stirring at rt
overnight, the reaction was quenched with the addition of 0.5 M potassium
hydrogen
sulfate until pH=4. The precipitate formed was collected by filtration, which
was
washed with water and ether and air dried to afford N-[(3-
chlorobenzene)sulfonyl]-
4(R)-(1-azetidinyl)-2-methyl-(L)-proline (0.34 g, 64%).
-60-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Step C N-(N-f(3-Chlorobenzene)sulfonyll-4(R)-(1-azetidinyl)-2-meth~L)-
prolyl)-4-((3',5'-dichloroisonicotino~)aminol-(L)-phenylalanine,
meth.1
N-(N-[(3-Chlorobenzene)sulfonyl]-4(R)-(1-azetidinyl)-2-methyl-(L)-
proline (Step B, 0.10 g, 0.28 mmol) was coupled to 4-[(3',5'-
dichloroisonicotinoyl)-
amino]-(L)-phenylalanine, methyl ester by the procedure described in Example 1
to
yield N-(N-[(3-chlorobenzene)sulfonyl]-4(R)-(1-azetidinyl)-2-methyl-(L)-
prolyl)-4-
[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (0.13 g,
61%).
1H NMR (400 MHz, CD30D): 8 8.63 (s, 2H), 7.9-7.3 (m, 8H), 4.75 (dd, 1H), 3.79
(s, 3H); 3.7 (m, 1H), 3.58 (dd, 1H), 3.3-3.0 (m, 6H); 2.75 (m, 1H); 2.1-2.0
(m, 3H);
1.58 (s, 3H);1.48 (dd, 1H).
MS: calculated for C31H32C13N506S 707, observed m/e 708 (M + H)+.
Step D N-(N-f (3-Chlorobenzene)sulfonyll-4(R)-(1-azetidin~)-2-meth,-(L)-
prolyl)-4-f (3',5'-dichloroisonicotinovl)aminol-(L)-phenylalanine.
N-(N-[(3-Chlorobenzene)sulfonyl]-4(R)-(1-azetidinyl)-2-methyl-(L)-
prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine methyl ester
(Step
C, 0.12 g, 0.17 mmol) was dissolved in acetonitrile (1 mL) and water (0.5 mL)
containing LiOH monohydrate (28 mg, 0.68 mmol) and stirred at rt for 3 h. The
reaction was quenched with formic acid (96%, 0.2 mL) and was diluted with DMSO
(1 mL) and water (1 mL). The resulting mixture was loaded onto a reverse phase
preparative HPLC column and eluted with acetonitrile and water (containing 0.1
%
formic acid) to afford N-(N-[(3-chlorobenzene)sulfonyl]-4(R)-(1-azetidinyl)-2-
methyl-(L)-prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine
(55 mg,
47%).
1H NMR (400 MHz, CD30D): 8 8.63 (s, 2H); 8.15 (br s, 1H); 7.9-7.3 (m, 8H);
4.58
(dd, 1H); 3.88 (t, 4H); 3.82 (dd, 1H); 3.65 (dd, 1H); 3.28 (dd, 1H); 3.08 (dd,
1H); 2.50
(dd, 1H); 2.35 (m, 2H); 1.72 (dd, 1H); 1.68 (s, 3H).
MS: calculated for C30H30C13N5O6S 693, observed xn/e 694 (M + H)+.
EXAMPLE 23
N-(N-((3-Chlorobenzene)sulfonyll-4(R)-(1-pyrrolidinyl)-2-methyl-(L)-prolyl)-4-
f(3',5'-diehloroisonicotinoyl)aminol-(L)-phenylalanine, formic acid salt
-61-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Step A N-f(3-Chlorobenzene)sulfonyll-4(R)-(1-pyrrolidin~)-2-meth 1-
proline, methyl ester.
To a solution of N-[(3-chlorobenzene)sulfonyl]-4(S)-hydroxy-2-
methyl-(L)-proline methyl ester (Example 20, Step B) (1.2 g, 3.3 mmol) and
DIPEA
(1.4 mL, 8.3 mmol) in 10 mL of anhydrous CH2C12 at -20 °C was added
trifluoro-
methanesulfonic anhydride (0.83 mL, 5.0 mmol) dropwise. After stirnng at -20
°C
for 45 min, half of the reaction mixture was transferred to a separate flask
(pre-cooled
at -20 °C), and pyrrolidine (0.5 mL, 6.0 mmol) was added. The reaction
was allowed
to warm up to rt overnight, and the resulting mixture was loaded onto a silica
gel
column and eluted with 1:1:0 to 1:1:0.1 hexane/EtOAc/2 M ammonia in MeOH to
yield N-[(3-chlorobenzene)sulfonyl]-4(R)-(1-pyrrolidinyl)-2-methyl-(L)-
proline,
methyl ester (74 mg, 12%).
1H NMR (500 MHz, CD30D): 8 7.85 (br t, J=1.8 Hz, 1H); 7.84 (br d, J=8.0 Hz,
1H);
7.65 (br d, J=8.0 Hz, 1H); 7.57 (apparent t, J=8.0 Hz, 1H); 3.86 (dd, J=9.0,
7.5 Hz,
1H); 3.71 (s, 3H); 3.27 (dd, J=9.0, 9.0 Hz); 3.00 (m, 1H); 2.56-2.46 (m, 4H);
2.43 (dd,
J=13.0, 6.5 Hz, 1H); 1.91 (dd, J=13.0, 10.0 Hz, 1H); 1.82-1.74 (m, 4H); 1.63
(s, 3H).
Step B N-f(3-Chlorobenzene)sulfonyll-4(R)-(1-pyrrolidinyl -2-meth 1-y (L)-
rop line.
N-[(3-Chlorobenzene)sulfonyl]-4(R)-( 1-pyrrolidinyl)-2-methyl-(L)-
proline, methyl ester (74 mg, 0.19 mmol) was converted to N-[(3-chlorobenzene)-
sulfonyl]-4(R)-(1-pynolidinyl)-2-methyl-(L)-proline (35 mg, 50%) by the
procedure
described in Example 22, Step B.
Step C N-(N-f(3-Chlorobenzene)sulfonyll-4(R)-(1-pyrrolidinxl)-2-methyl-(L)-
prolyl)-4-f (3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine,
methyl ester
N-(N-[(3-Chlorobenzene)sulfonyl]-4(R)-(1-pyrrolidinyl)-2-methyl-(L)-
proline (Step B, 35 mg, 0.094 mmol) was coupled to 4-[(3',5'-
dichloroisonicotinoyl)-
amino]-(L)-phenylalanine, methyl ester by the procedure described in Example 1
to
yield N-(N-[(3-chlorobenzene)sulfonyl]-4(R)-(1-pyrrolidinyl)-2-methyl-(L)-
prolyl)-4-
[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (54 mg,
79%).
1H NMR (500 MHz, CD30D): 8 8.64 (s, 2H); 7.86 (t, J=1.8 Hz, 1H); 7.81 (d,
J=8.0
Hz, 1H); 7.69 (td, J=8.0, 1.8 Hz, 1H); 7.63 (d, J=13.5 Hz, 2H); 7.55 (t, J=8.0
Hz, 1H);
-62-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
7.32 (d, J=8.0 Hz, 2H); 4.69 (m, 1H); 4.06 (dd, J=13.5 Hz, 1H); 3.76 (s, 3H);
3.48
(dd, J=14.5, 9.0 Hz, 1H); 3.28 (dd, 1H); 3.20-3.12 (m, 4H); 3.09 (dd, J=9.5,
9.0 Hz,
1H); 2.61 (dd, J=13.0, 7.0 Hz, 1H); 2.10-1.82 (m, 6H); 1.72 (s, 3H).
MS: calculated for C32H34C13N5O6S 721, observed m/e 722 (M + H)+.
Step D N-(N-f(3-Chlorobenzene)sulfon~l-4(R)-(1-pyrrolidinyl)-2-meth~~
urolyl)-4-f (3',5'-dichloroisonicotino~~l)aminol-(L)-phenylalanine,
formic acid salt.
N-(N-[(3-Chlorobenzene)sulfonyl]-4(R)-(1-pyrrolidinyl)-2-methyl-(L)-
prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine methyl ester
(58 mg, 0.080 mmol) was converted to N-(N-[(3-chlorobenzene)sulfonyl]-4(R)-(1-
pyrrolidinyl)-2-methyl-(L)-prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine, formic acid salt (30 mg, 53°70) following the procedure
of Example 22,
Step D
1H NMR (500 MHz, CD30D): 8 8.64 (s, 2H); 7.88 (t, J=1.8 Hz, 1H); 7.74 (d,
J=8.0
Hz, 1H); 7.66 (td, J=8.0, 1.8 Hz, 1H); 7.60 (d, J=13.5 Hz, 2H); 7.55 (t, J=8.0
Hz, 1H);
7.31 (d, J=8.0 Hz, 2H); 4.54 (m, 1H); 3.94 (m, 1H); 3.39 (m, 1H); 3.2-3.1 (m,
2H),
3.10-3.04 (m, 4H); 2.60 (dd, 1H); 2.00-1.82 (m, 6H); 1.68 (s, 3H).
MS: calculated for C31H32C13N5O6S 707, observed m/e 708 (M + H)+.
EXAMPLE 24
N-(N-f (3,5-Dichlorobenzene)sulfonyll-4(R)-amino-2-methyl-(L)-prolyl)-4-[(3'
5'-
dichloroisonicotinoyl)aminol-(L)-phenylalanine, hydrochloride
Step A N-f(3,5-Dichlorobenzene)sulfon l~)-hydroxy-2-methyl-(L)-
proline, meth 1y ester.
N-BOC-4(S)-hydroxy-2-methyl-(L)-proline, methyl ester from
Example 20, Step A (1.4 g, 5.4 mmol) was converted to N-[(3,5-dichlorobenzene)-
sulfonyl]-4(S)-hydroxy-2-methyl-(L)-proline, methyl ester (1.1 g, 55%)
following the
procedures described in Example 20 substituting 3,5-dichlorobenzenesulfonyl
chloride for 3-chlorobenzenesulfonyl chloride in Step B.
1H NMR (500 MHz, CD30D): 8 7.82 (d, J=1.8 Hz, 1H); 7.75 (t, J=1.8 Hz, 1H);
4.37
(apparent quintet, J=5.0 Hz, 1H); 3.72 (s, 3H); 3.66 (dd, J=9.5, 5.5 Hz, 1H);
3.25 (dd,
J=9.5, 4.0 Hz, 1H); 2.88 (dd, J=13.0, 5.0 Hz, 1H); 2.18 (dd, J=13.0, 5.5 Hz,
1H); 1.67
(s, 3H).
-63-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Step B N-f(3,5-Dichlorobenzene)sulfonyll-4(R)-tart-butoxXcarbonylamino-2-
meth 1-~proline meth l
N-[(3,5-Dichlorobenzene)sulfonyl]-4(S)-hydroxy-2-methyl-(L)-
proline, methyl ester (Step A, 0.26 g, 0.70 mmol) was converted to N-[(3,5-
dichloro-
benzene)sulfonyl]-4(R)-tart-butoxycarbonylamino-2-methyl-(L)-proline, methyl
ester
(0.12 g, 36%) following the procedures described in Example 20, Step C.
1H NMR (500 MHz, CD30D): 8 7.78-7.75 (m, 3H); 4.22 (m, 1H); 3.86 (dd, J=9:0,
7.5 Hz, 1H); 3.71 (s, 3H); 3.19 (dd, J=9.0, 7.S Hz, 1H); 2.45 (dd, J=13.5, 7.0
Hz, 1H);
1.92 (dd, J=13.5, 10.0 Hz, 1H); 1.66 (s, 3H); 1.41 (s, 9H).
Step C N-f(3,5-Dichlorobenzene)sulfonyll-4(R)-tart-butoxycarbonylamino-2-
meth girl-(L)-proline.
N-j(3,5-Dichlorobenzene)sulfonyl]-4(R)-tart-butoxycarbonylamino-2-
methyl-(L)-proline, methyl ester (0.12 g, 0.26 mmol) was converted to N-[(3,5-
dichlorobenzene)sulfonyl]-4(R)-tart-butoxycarbonylamino-2-methyl-(L)-proline
following the procedures described in Example 20, Step D. (0.13 g, 100%).
1H NMR (500 MHz, CD30D): ~ 7.79 (d, J=1.8 Hz, 1H); 7.74 (t, J=1.8 Hz, 1H);
4.24
(m, 1H); 3.83 (dd, J=9.0, 8.0 Hz, 1H); 3.19 (dd, J=8.5, 8.5 Hz, 1H); 2.48 (dd,
J=13.0,
7.0 Hz, 1H); 1.92 (dd, J=13.0, 9.5 Hz, 1H); 1.66 (s, 3H); 1.41 (s, 9H).
Step D N-(N-f(3,5-Dichlorobenzene)sulfon,1~R~-tart-butox carbon
amino-2-meth 1-y (L)-prolyl)-4-~(3',5'-dichloroisonicotinoyl)aminol-
~L~phenylalanine, meth.~er.
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-4-(R)-tart-butoxycarbonyl-
amino-2-methyl-(L)-proline (Step C, 0.13 g, 0.28 mmol) was coupled to 4-
[(3',5'-
dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester by the procedure
described in Example 1 to yield N-(N-[(3,5-dichlorobenzene)sulfonyl]-4(R)-tert-
butoxycarbonylamino-2-methyl-(L)-prolyl)-4-[(3',5'-
dichloroisonicotinoyl)amino]-
(L)-phenylalanine, methyl ester (0.10 g, 45%).
1H NMR (400 MHz, CD30D): S 8.63 (s, 2H); 7.80-7.75 (m, 3H); 7.60 (d, 2H); 7.30
(d, 2H); 4.80-4.70 (m, 1H); 4.15-4.00 (m, 1H); 3.8-3.7 (m, 1H); 3.78 (s, 3 H);
3.3-3.0
(m, 3H); 2.3-2.2 (m, 1H); 1.75 (dd, 1H); 1.6 (s, 3H); 1.4 (s, 9H).
MS: calculated for C33H35C14N508S 801, observed m/e 802 (M + H)+.
-64-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Step E N-(N-f(3,5-Dichlorobenzene)sulfonyll-4(R)-tert-butoxycarbonyl-
amino-2-meth 1-~ (L)-prop)-4-f (3',5'-dichloroisonicotinoyl)aminol-
(L)-phenylalanine.
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-tert-butoxycarbonyl-
amino-2-methyl-(L)-prolyl)-4-[(3' ,5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine,
methyl ester (Step D, 0.10 g, 0.12 mmol) was converted to N-(N-[(3,5-
dichlorobenzene)sulfonyl]-4(R)-test-butoxycarbonylamino-2-methyl-(L)-prolyl)-4-
[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine (98 mg, 99%) by the
procedures described in Example 20, Step F.
1H NMR (400 MHz, CD30D): b 8.63 (s, 2H); 7.80-7.58 (m, 5H); 7.32 (d, 2H); 4.95
(m, 1H); 4.05 (m, 1H); 3.70 (m, 1H); 3.4-3.0 (rn, 3H); 2.25 (m, 1H); 1.75 (dd,
1H);
1.65 (s, 3H); 1.42 (s, 9H).
MS: calculated for C32H33C14N5O8S 787, observed m/e 788 (M + H)~.
Step F N-(N-x(3,5-Dichlorobenzene)sulfon 11-4 R)-amino-2-meth,~(L~
prop)-4-f (3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine
hydrochloride
A sample of N-(N-[(3,5-dichlorobenzene)sulfonyl]-4(R)-tert-butoxy-
carbonylamino-2-methyl-(L)-prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine (83 mg, 0.10 mmol) was treated with 3 mL of saturated HCl in
EtOAc
for 2 h at rt. Concentration irz vacuo afforded the title compound as a solid
(94 mg,
100%).
1H NMR (400 MHz, CD3OD): ~ 8.63 (s, 2H); 7.8-7.3 (m, 7H); 4.62 (m, 1H); 3.90
(dd, 1H); 3.75 (m, 1H); 3.40 (dd, 1H); 3.30 (dd, 1H); 3.08 (dd, 1H); 2.64 (dd,
1H);
1.95 (dd, 1H); 1.75 (s, 3H).
MS: calculated for C27H25C14N5O6S 687, observed mle 688 (M + H)+.
EXAMPLE 25
N-(N-f(3,5-Dichlorobenzene)sulfonyll-4(R)-c~propylamino-2-methxl-(L)-prolyl)-
4-x(3',5'-dichloroisonicotinoyl aminol- L)-phenylalanine
Step A N-f(3,5-Dichlorobenzene)sulfon 1y 14(R)-c ~~cl~ro~ylamino-2-meth
L - roline
-65-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-cyclopropylamino-2-methyl-
(L)-proline (0.27 g, 66%) was prepared according to the procedures described
in
Example 22, Step A, substituting N-[(3,5-dichlorobenzene)sulfonyl]-4(S)-
hydroxy-2-
methyl-(L)-proline, methyl ester (from Example 24, Step A) for N-[(3-chloro-
benzene)sulfonyl]-4(S)-hydroxy-2-methyl-(L)-proline, methyl ester and
cyclopropylamine for azetidine.
1H NMR (500 MHz, CD30D/CF3C02D): 8 7.83 (d, J=2.0 Hz, 2H); 7.79 (t, J=2.0
Hz, 1H); 3.90 dd, J=7.7, 7.0 Hz, 1H); 3.69 (m, 1H); 3.33 (dd, J=9.0, 7.5 Hz,
1H); 2.56
(dd, J=13.0, 6.5 Hz, 1H); 2.26 (m, 1H); 1.92 (dd, J=13.0, 9.0 Hz, 1H); 1.68
(s, 3H);
0.54 (m, 2H); 0.42 (m, 2H).
Step B N-(N-f(3,5-Dichlorobenzene)sulfonyll-4(R)-cyclopropylamino-2-
methyl-(L)-prolyl)-4- f (3' ,5' -dichloroisonicotinoyl)aminol-(L)-
phen~lalanine, meth.1
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-4-(R)-cyclopropylamino-2-
methyl-(L)-proline (0.026 g, 0.068 mmol) was coupled to 4-[(3',5'-dichloro-
isonicotinoyl)amino]-(L)-phenylalanine, methyl ester by the procedure
described in
Example 1 to yield N-(N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-cyclopropylamino-
2-
methyl-(L)-prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine,
methyl
ester (0.020 g, 40%).
MS: calculated for C31H31C14N5O6S 741, observed m/e 742 (M + H)+.
Step C N-(N-f (3,5-Dichlorobenzene)sulfonyll-4(R)-c~propylamino-2-
meth~L)-prolyl)-4-((3',5'-dichloroisonicotinoyl)aminol-~L)-
phenylalanine, trifluoroacetic acid salt
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-cyclopropylamino-2-
methyl-(L)-prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine,
methyl
ester (0.020 g, 0.027 mmol) was converted to N-(N-[(3,5-
dichlorobenzene)sulfonyl]-
4(R)-cyclopropylamino-2-methyl-(L)-prolyl)-4-[(3' ,5' -
dichloroisonicotinoyl)amino]-
(L)-phenylalanine, trifluoroacetic acid salt (0.012 g) following the procedure
described in Example 22, Step D, substituting 0.1 % TFA for formic acid in the
HPLC
eluent.
1H NMR (500 MHz , CD30D): 8 8.63 (s, 2H), 7.74 (m, 3H), 7.62 (d, J=8.5 Hz,
2H),
7.33 (d, J=8.5 Hz, 2H), 4.65 (m, 1H), 3.99 (t, J=8.9 Hz, 1H), 3.83 (m, 1H),
3.45 (t,
-66-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
J=8.7 Hz, 1H), 3.31 (m, 1H), 3.11 (dd, J=8.9, 14.2 Hz, 1H), 2.68 (m, 2H), 1.99
(dd,
J=10.7, 12.8 Hz, 2H), 1.73 (s, 3H), 0.88 (m, 2H), 0.78 (m, 2H).
MS: calculated for C30H29C14N506S 727, observed mle 728 (M + H)+.
EXAMPLE 26
N-(N-f (3,5-Dichlorobenzene)sulfonyll-4(R)-(1-azetidinyl)-2-meth 1-y (L)-prol
1
f (3' ,5'-diehloroisonicotinoyl)aminol-(L)-phenylalanine
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-(1-azetidinyl)-2-methyl-
(L)-prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine was
prepared by
the procedures described in Example 22, Steps A-D, substituting N-[(3,5-
dichloro-
benzene)sulfonyl]-4(S)-hydroxy-2-methyl-(L)-proline, methyl ester for N-[(3-
chloro-
benzene)sulfonyl]-4(S)-hydroxy-2-methyl-(L)-proline, methyl ester in Step A.
Characterization of intermediate compounds and the title compound are
provided:
Step A N-f(3,5-Dichlorobenzene)sulfonyll-4(R)-(1-azetidinyl)-2-meth 1-
proline, meth, l ester.
1H NMR (500 MHz, CD30D): ~ 7.86 (d, J=1.5 Hz, 2H); 7.75 (t, J=1.5 Hz, 1H);
3.72
(s, 3H), 3.62 (dd, J=10.0, 5.5 Hz, 1H); 3.26 (dd, J=10.0, 4.0 Hz, 1H); 3.20
(t, J=7.5
Hz, 4H); 3.07 (m, 1H); 2.28 (dd, J=13.5, 6.5 Hz, 1H); 2.05 (quintet, J=7.5 Hz,
2H),
1.80 (dd, J=13.5, 5.0 Hz, 1H); 1.66 (s, 3H).
Step B N-f(3,5-Dichlorobenzene)sulfonyll-4(R)-(1-azetidin~)-2-meth~L)-
rop line.
MS: calculated for C15H18C12N2O4S 392, observed m/e 393 (M + H)+.
Step C N-(N-f(3,5-Dichlorobenzene)sulfon.1~)-(1-azetidin~)-2-methyl-
(L)-prolyl)-4-f (3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine
meth, l
1H NMR (500 MHz, CD30D): 8 8.64 (s, 2H), 7.84 (d, J= 1.5 Hz, 2H), 7.76 (t, J=
1.5
Hz, 1H), 7.54 (d, J=8.5 Hz, 2H), 7.29 (d, J=8.5 Hz, 2H), 4.75 (dd, J=9.5, 5.0
Hz, 1H),
3.77 (s, 3H); 3.56 (dd, J=9.5, 5.5 Hz, 1H), 3.34-3.30 (m, 1H), 3.16-3.00 (m,
6H), 3.78
(quintet, J=7.5 Hz, 1H); 2.10-2.00 (m, 3H); 1.60 (s, 3H); 1.55 (dd, J=13.0,
5.0 Hz,
1H).
MS: calculated for C31H31C14N5O6S 743, observed m/e 744 (M + H)+.
-67-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Step D N-(N-f(3,5-Dichlorobenzene)sulfon 1~1-4(R)-(1-azetidinyl)-2-methyl-
~L)-prolyl)-4-f (3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine
formic acid salt.
1H NMR (500 MHz, CD30D): 8 8.64 (s, 2H), 7.78 (d, J= 2.0 Hz, 2H), 7.76 (br s,
1H), 7.61 (d, J=9.0 Hz, 2H), 7.32 (d, J=9.0 Hz, 2H), 4.57 (dd, J=7.0, 5.0 Hz,
1H),
3.78 (t, J=7.5 Hz, 4H); 3.54 (m, 1H); 3.34-3.30 (m, 3H), 3.11 (dd, J=14.0, 8.0
Hz,
1H), 2.45 (dd, J=13.0, 7.0 Hz, 1H), 2.31 (m, 2H); 1.74 (dd, J=13.0, 9.0 Hz,
1H); 1.69
(s, 3H).
MS: calculated for C30H29C14N5O6S 729, observed m/e 730 (M + H)+.
EXAMPLE 27
N-(N-f (3,5-Dichlorobenzene)sulfon 1~)-hydroxy-2-methyl-(L)-~rolyl)-4-f (3' 5'-
dichloroisonicotinoyl)aminol-(L)-phenylalanine, formic acid salt.
Step A N-f(3,5-Dichlorobenzene)sulfonyll-4(R)-hydroxy-2-meth.1-
rop line.
N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-hydroxy-2-methyl-(L)-
proline, methyl ester was prepared by the procedure described in Example 20
substituting N-BOC-4(R)-hydroxyproline, methyl ester for N-BOC-4(S)-hydroxy-
proline, methyl ester in Step A and 3,5-dichlorobenzenesulfonyl chloride for 3-
chloro-
benzenesulfonyl chloride in Step B; 0.50 g, 1.4 mmol) was treated with LiOH
according to the procedure described in Example 20, Step D to afford N-[(3,5-
dichlorobenzene)sulfonyl]-4(R)-hydroxy-2-methyl-(L)-proline (0.49 g, 100%).
1H NMR (400 MHz, CD30D): 8 8.63 (s, 2H), 7.74 (t, 1H), 4.42 (rn, 1H), 3.64
(dd,
1H), 3.40 (dd, 1H); 2.44 (dd, 1H), 2.00 (dd, 1H); 1.72 (s, 3H).
Step B N-(N-f(3,5-Dichlorobenzene)sulfonyll-4(R)-hydroxy-2-meth~L)-
prolyl)-4-f (3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine
meth.ly ester.
N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-hydroxy-2-methyl-(L)-proline
(0.080 g, 0.23 mmol) was coupled to 4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine, methyl ester by the procedure described in Example 1 to yield N-
(N-
[(3,5-dichlorobenzene)sulfonyl]-4(R)-hydroxy-2-methyl-(L)-prolyl)-4-[(3',5'-
dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (0.10 g, 64%).
-68-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
1H NMR (400 MHz, CD30D): S 8.63 (s, 2H), 7.78 (d, 2H); 7.74 (t, 1H); 7.61 (d,
2H); 7.28 (d, 2H); 4.72 (dd, 1H), 2.26 (m, 1H); 3.75 (s, 3H); 3.59 (dd, 1H),
3.38 (dd,
1H), 3.24 (dd, 1H); 3.08 (dd, 1H); 2.22 (dd, 1H); 1.84 (dd, 1H); 1.66 (s, 3H).
MS: calculated for C28H26C14N4O7S 702, observed m/e 703 (M + H)'~.
Step C N-(N-f(3,5-Dichlorobenzene sulfonyll-4(R)-hydroxy-2-meth~)-
prop)-4-f (3',5'-dichloroisonicotino~)aminol-(L)-phenylalanine.
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-hydroxy-2-methyl-(L)-
prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester
(0.23
g, 0.33 mmol) was reacted with LiOH according to the procedure described in
Example 22; Step F to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-4(R)-hydroxy-
2-
methyl-(L)-prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine
(0.10 g,
44°7o).
1H NMR (400 MHz, CD30D): 8 8.63 (s, 2H); 7.78 (d, 2H); 7.72 (t, 1H); 7.61 (d,
2H); 7.30 (d, 2H); 4.70 (dd, 1H); 4.24 (m, 1H); 3.56 (dd, 1H); 3.39 (dd, 1H);
3.28 (dd,
1H); 3.10 (dd, 1H); 2.25 (dd, 1H); 1.85 (dd, 1H); 1.70 (s, 3H).
MS: calculated for C27H24C14N4O7S 688, observed m/e 689 (M + H)~.
EXAMPLE 28
N-(N-f(3,5-Dichlorobenzene)sulfonyll-3(S)-meth~L~prolyl)-4-f(3',5'-dichloro-
isonicotinoyl)aminol-(L)-phenylalanine
Step A N-f(3,5-Dichlorobenzene)sulfonyll-3(S)-methyl-(L)-proline.
To a solution of 3(S)-methyl-(L)-proline (Acros, 0.50 g, 3.9 mmol) and
Na2C03 (0.81 g, 7.8 mmol) in 10 mL of water at 0 °C was added 3,5-
dichloro-
benzenesulfonyl chloride (1.1 g, 4.5 mmol). After stirring at rt overnight,
the reaction
mixture was acidified with concentrated HCl (pH=2), and the product was
extracted
with EtOAc (3x8 mL). The organic extracts were dried over anhydrous MgS04,
filtered and concentrated i~a vacuo to dryness to yield N-[(3,5-
dichlorobenzene)-
sulfonyl]-3(S)-methyl-(L)-proline (crude, 1.9 g).
1H NMR (500 MHz, CD3OD): b 7.83 (d, J=2.0 Hz, 2H); 7.51 (t, J=2.0 Hz, 1H);
3.82
(d, J=5.5 Hz, 1H), 3.52-3.38 (m, 2H), 2.39 (m, 1H); 2.08 (m, 1H); 1.46 (m,
1H); 0.98
(d, J=6.5 Hz, 3H).
-69-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Step B N-(N-f(3,5-Dichlorobenzene)sulfonyll-3(S)-meth,1-~prolyl)-4-
f(3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine, meth 1 ester.
N-[(3,5-Dichlorobenzene)sulfonyl]-3(S)-methyl-(L)-proline (0.080 g,
0.24 mmol) was coupled to 4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine,
methyl ester by the procedure described in Example 1 to yield N-(N-[(3,5-
dichloro-
benzene)sulfonyl]-3(S)-methyl-(L)-prolyl)-4-[(3',5'-
dichloroisonicotinoyl)amino]-(L)-
phenylalanine, methyl ester (0.076 g, 47%a).
1H NMR (400 MHz, CD30D): 8 8.62 (s, 2H); 7.79 (apparent s, 3H); 7.60 (d, 2H);
7.32(d, 2H); 4.77 (dd, 1H); 3.75 (s, 3H); 3.73 (d, 1H); 3.50-3.38 (m, 2H);
3.23 (dd,
1H); 3.04 (dd, 1H); 2.17 (m, 1H); 1.95 (m, 1H); 1.27(m, 1H); 0.82 (d, 3H).
MS: calculated for C28H26C14N4O7S 702, observed m/e 703 (M + H)+.
Step C NON-f(3,5-Dichlorobenzene sulfonyll-3(S)-methyl-2-meth.1-
prolYl)-4-f (3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine.
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-3(S)-methyl-2-methyl-(L)-
prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester
(Step
B, 0.070 g, 0.10 mmol) was reacted with LiOH according to the procedure
described
in Example 22, Step F. The reaction mixture was partitioned between brine, 0.5
M
sodium hydrogen sulfate and ethyl acetate. The product was extracted with
EtOAc
(3x), and the combined extracts were dried over anhydrous MgSO4 and
concentrated
ira vacuo to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-3(S)-methyl-2-methyl-
(L)-
prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine.
1H NMR (400 MHz, CD30D): ~ 8.62 (s, 2H); 7.79 (apparent s, 3H); 7.60 (d, 2H);
7.34 (d, 2H); 4.76 (dd, 1H); 3.74 (d, 1H); 3.50-3.35 (m, 2H); 3.28 (dd, 1H);
3.04 (dd,
1H); 2.20 (m, 1H); 1.94 (m, 1H); 1.26 (m, 1H); 0.82 (d, 3H).
MS: calculated for C27H24C14N4O6S 672, observed m/e 673 (M + H)+.
EXAMPLE 29
N-(N-f(3,5-Dichlorobenzene)sulfon ~~11-3(S)-h d~rox -~prolyl)-4-f(3',5'-
dichloro-
isonicotinoxl)aminol-(L)-~henylalanine.
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-3(S)-hydroxy-(L)-prolyl)-4-
[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine was prepared by the
procedures described in Example 20, substituting N-.[(3,5-
dichlorobenzene)sulfonyl]-
4(S)-hydroxy-2-methyl-(L)-proline, methyl ester for N-[(3-
chlorobenzene)sulfonyl]-
-70-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
4(R)-hydroxy-2-methyl-(L)-proline, methyl ester in Step B. Characterization of
intermediate compounds and the title compound are provided:
Step A N-(N-f(3,5-Dichlorobenzene)sulfonyll-3(S)-h droxy (L~-proline.
1H NMR (400 MHz, CD30D): b 7.82 (d, J=2.0 Hz, 2H); 7.76 (t, J=2.0 Hz, 1H);
4.38
(d, 1H); 4.15 (s, 1H); 3.57 (dd, 1H), 3.40 (m, 1H), 2.05 (m, 1H); 1.83 (m,
1H).
Step B N-(N-f (3,5-Dichlorobenzene)sulfonyll-3(S)-h day-(L)-prol.1
f(3',5'-dichloroisonicotino~)aminol-(L)-phenylalanine meth.ly ester.
1H NMR (400 MHz, CD30D): 8 8.62 (s, 2H); 7.79 (d, 2H); 7.76 (t, 1H); 7.60 (d,
2H); 7.26 (d,°2H); 4.75 (dd, 1H); 4.14 (d, 1H); 4.02 (s, 1H); 3.74 (s,
3H); 3.56 (dd,
1H); 3.24 (dd, 1H); 3.06 (dd, 1H); 1.80-1.64 (m, 2H); 1.26 (m, 1H).
MS: calculated for C27H24C14N4O7S 688, observed m/e 689 (M + H)+.
Step C N-(N-f(3,5-Dichlorobenzene)sulfony~-3(S)-h droxy -2-meth. l~- L)-
prolyl)-4-f (3' ,5'-dichloroisonicotino~)aminol-(L)-phenylalanine.
1H NMR (400 MHz, CD30D): b 8.62 (s, 2H); 7.78 (d, 2H); 7.74 (t, 1H); 7.60 (d,
2H); 7.28 (d, 2H); 4.72 (m, 1H); 4.16 (d, 1H); 4.04 (s, 1H); 3.54 (td, 1H),
3.36-3.26
(m, 2H); 3.08 (dd, 1H); 1.80-1.68 (m, 2H).
MS: calculated for C26H22C14N4O7S 674, observed m/e 675 (M + H)+.
EXAMPLE 30
N-(N-f(3,5-Dichlorobenzene)sulfon l~-4(R)-cyano-(L)-~rolyl)-4-f(3' S'-dichloro-
isonicotinoyl)aminol-(L)-phenylalanine.
Step A 4(R)-C a~L)-proline, hydrochloride
N-BOC-3(R)-cyano-(L)-proline (RSP, 2.0 g, 8.3 mmol) was treated
with HCl gas in EtOAC according to the procedure described in Example 20, Step
G
to yield 4(R)-cyano-(L)-proline, hydrochloride (1.5 g, 100%).
Step B N-f (3,5-Dichlorobenzene)sulfonyll-4~(R)-cyano-(L)-proline
4(R)-Cyano-(L)-proline, hydrochloride (1.5 g, 8.5 mmol) was reacted
with 3,5-dichlorobenzene sulfonyl chloride and Na2C03 (1.5 equiv.)according to
the
procedure described in Example 28, Step A to afford N-[(3,5-dichlorobenzene)-
sulfonyl]-4(R)-cyano-(L)-proline (2.6 g, 87%).
-71-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Step C N-(N-f (3,5-Dichlorobenzene)sulfonyll-4(R)-cyano-(L)-prolyl)-4-
f(3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine, meth 1y ester
N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-cyano-(L)-proline (Step B,
0.080 g, 0.26 mmol) was coupled to 4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine, methyl ester by the procedure described in Example 1 to yield N-
(N-
[(3,5-dichlorobenzene)sulfonyl]-4(R)-cyano-(L)-prolyl)-4-[(3',5'-dichloro-
isonicotinoyl)amino]-(L)-phenylalanine, methyl ester (0.13 g, 70%).
1H NMR (400 MHz, CD30D): 8 8.63 (s, 2H), 7.80 (d, 2H); 7.76 (t, 1H), 7.62 (d,
2H);
7.30 (d, 2H); 4.74 (dd, 1H), 4.42 (dd, 1H); 3.72 (s, 3H); 3.70 (m, 2H), 3.35
(m, 1H);
3.21 (dd, 1H); 3.05 (dd, 1H); 2.36 (m, 1H); 2.20 (m, 1H).
MS: calculated for C28H23C14N5O6S 697, observed mle 698 (M + H)+.
Step D N-(N-f(3,5-Dichlorobenzene)sulfonyll-4(R)-cyano-2-meth 1-
prolyl)-4-f (3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine.
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-cyano-(L)-prolyl)-4-
[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (0.13 g,
0.18
mmol) was reacted with LiOH according to the procedure described in Example
20,
Step F. The crude reaction mixture was partitioned between brine, 1 M HCl and
EtOAc. The product was extracted with EtOAc (3x), and the combined extracts
were
dried over anhydrous MgS04 and concentrated ire vaciio to afford N-(N-[(3,5-
dichlorobenzene)sulfonyl]-4(R)-cyano-2-methyl-(L)-prolyl)-4-[(3',5'-dichloro-
isonicotinoyl)amino]-(L)-phenylalanine (0.12 g, 100°7o).
1H NMR (400 MHz, CD30D): 8 8.63 (s, 2H), 7.79 (d, 2H); 7.77 (t, 1H), 7.62 (d,
2H);
7.34 (d, 2H); 4.72 (m, 1H), 4.42 (dd, 1H); 3.72 (m, 2H), 3.35 (m, 1H); 3.24
(dd, 1H);
3.08 (dd, 1H); 2.36 (m, 1H); 2.23 (m, 1H).
MS: calculated for C27H21C14N5O6S 683, observed m/e 684 (M + H)+.
EXAMPLE 31
N-(N-f (3,5-Dichlorobenzene)sulfonyll-4(R)-tart-butox -~prolxl)-4-f (3',5'-
dichloroisonicotinoyl)aminol-(L)-phenylalanine
Step A N-f(3,5-Dichlorobenzene)sulfonyll-4(R)-tart-butoxy-(L)-proline.
4(R)-tart-Butoxy-(L)-proline (Bachem, 5.0 g, 27 mmol) was reacted
with 3,5-dichlorobenzene sulfonyl chloride and Na2C03 according to the
procedure
-72-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
described in Example 28, Step A to afford N-[(3,5-dichlorobenzene)sulfonyl]-
4(R)-
teut-butoxy-(L)-proline (9.3 g, 88%)
1H NMR (400 MHz, CD30D): 8 7.82 (d, 2H); 7.78 (t, 1H); 4.25 (m, 1H), 4.19 (dd,
1H), 3.62 (dd, 1H); 3.25 (m, 1H); 2.16-2.00 (m, 2H); 0.98 (s, 9H).
Step B N-(N-f ~3,5-Dichlorobenzene)sulfonyll-4(R)-tert-butoxy-(L~ r~ol~
f(3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine, methyl ester.
N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-tert-butoxy-(L)-proline (0.080
g, 0.20 mmol) was coupled to 4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine, methyl ester by the procedure described in Example 1 to yield N-
(N-
[(3,5-dichlorobenzene)sulfonyl]-4(R)-tert-butoxy-(L)-prolyl)-4-[(3',5'-
dichloro-
isonicotinoyl)amino]-(L)-phenylalanine, methyl ester (0.11 g, 71%).
1H NMR (400 MHz, CD30D): b 8.64 (s, 2H), 7.79 (d, 2H); 7.76 (t, 1H), 7.62 (d,
2H); 7.32 (d, 2H); 4.77 (dd, 1H), 4.22-4.18 (m, 2H); 3.72 (s, 3H); 3.62 (dd,
1H), 3.24
(d, 1H); 3.21 (dd, 1H); 3.10 (dd, 1H); 2.0-1.82 (m, 2H); 0.94 (s, 9H).
MS: calculated for C31H32C14N4O7S 744, observed m/e 703 (M + H - C4H9)+.
Step C N-(N-f(3,5-Dichlorobenzene)sulfonyll-4(R)-tert-butoxy-2-meth.~~)-
prolyl)-4- f (3' ,5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine.
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-tert-butoxy-2-methyl-(L)-
prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester
(0.11
g, 0.14 mmol) was treated with LiOH according to the procedure described in
Example 20, Step F with THF added as a cosolvent. The reaction mixture was
partitioned between brine, 0.5 M sodium hydrogen sulfate and ethyl acetate.
The
product was extracted with EtOAc (3x), and the combined extracts were dried
over
anhydrous MgS04 and concentrated irZ vacuo to afford N-(N-[(3,5-
dichlorobenzene)-
sulfonyl]-4(R)-tert-butoxy-2-methyl-(L)-prolyl)-4-[(3',5'-
dichloroisonicotinoyl)-
amino]-(L)-phenylalanine. (99 mg, 95%).
1H NMR (400 MHz, CD30D): ~ 8.60 (s, 2H), 7.76 (d, 2H); 7.70 (t, IH), 7.62 (d,
2H);
7.34 (d, 2H); 4.77 (dd, 1H), 4.22-4.18 (m, 2H); 3.62 (dd, 1H), 3.23 (dd, 1H);
3.21 (d,
1H); 3.12 (dd, 1H); 2.0-1.82 (m, 2H); 0.94 (s, 9H).
MS: calculated for C30H30C14N4O7S 730, observed m/e 731 (M + H)+.
73 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
EXAMPLES 32 and 33
N-(N-f (3,5-Dichlorobenzene)sulfonyll-3-(4-hydroxyphenyl)prolyl)-4-f (3',5'-
dichloro-
isonicotinoyl)aminol-(L)-phenylalanine.
Step A N-Acetyl-2-ethoxycarbonyl-3-(4-methoxyphenyl)proline ethyl ester
N-Acetyl-2-ethoxycarbonyl-3-(4-methoxyphenyl)proline, ethyl ester
(13 g, 72°7o) was prepared from 4-methoxycinnamaldehyde (Lancaster, 8.1
g, 50
mmol) and diethyl acetamidomalonate (Aldrich, 9.9 g, 45 mmol) by the procedure
described by Chung et al (J. Org. Chew. 1990, 55, 270).
1H NMR (500 MHz, CD30D): 8 7.13 (d, J=9.0 Hz, 2H); 6.85 (d, J=9.0 Hz, 2H);
4.25 (q, J=7.0 Hz, 2H); 4.24 (q, J=7.0 Hz, 2H); 4.02 (t, 1H), 3.92-3.72 (m,
2H); 3.77
(s, 3H); 2.59~(m, 1H); 2.24 (m, 1H); 2.12 (s, 3H); 1.29 (t, J=7.0 Hz, 3H);
0.98 (t,
J=7.0 Hz, 3H).
MS: calculated for C19H25N06S 363, observed m/e 364 (M + H)~.
Step B 3-(4-Hydroxyphenyl)proline, ethyl ester, hydrobromide
A solution of N-acetyl-2-ethoxycarbonyl-3-(4-methoxyphenyl)proline
ethyl ester (10 g, 27 mmol) in aqueous HBr (48°70, 40 mL) and HOAc (10
mL) was
heated at 120 °C under a nitrogen atmoshere overnight. The reaction
mixture was
concentrated iTZ uacu~, and the residue was azeotroped with acetonitrile (2x)
and
Et2O/toluene ( 1 x) to yield 3-(4-hydroxyphenyl)proline, ethyl ester
hydrobromide as a
mixture of diastereomers.
MS: calculated for C11H13N03 207, observed m/e 208 (M + H)+.
Step C N-f(3,5-Dichlorobenzenelsulfonyll-3-~4-hydroxyphenyl)-proline.
3-(4-Hydroxyphenyl)proline ethyl ester hydrobromide salt (Step B, 3.3
g, 11 mmol) was reacted with 3,5-dichlorobenzene sulfonyl chloride and Na2CO3
(1.5 equiv.) according to the procedure described in Example 28, Step A to
afford N-
[(3,5-dichlorobenzene)sulfonyl]-3-(4-hydroxyphenyl)-(L)-proline (3.7 g,
79°70)
MS: calculated for C17H15C12N05S 415, observed m/e 416 (M + H)+.
Step D N-(N-((3,5-Dichlorobenzene)sulfon. ly_1-3-(4-h. d~Xphen,~prolyl)-
f(3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine meth ly_ester
N-[(3,5-Dichlorobenzene)sulfonyl]-3-(4-hydroxyphenyl)-(L)-proline
(0.4 g, 0.96 mol) was coupled to 4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
-74-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
phenylalanine, methyl ester by the procedure described in Example 1 to yield N-
(N-
[(3,5-dichlorobenzene)sulfonyl]-3-(4-hydroxyphenyl)-prolyl)-[(3',5'-dichloro-
isonicotinoyl)amino]-(L)-phenylalanine, methyl ester. The four possible
isomers were
partially separated into two components, which were further purified by
reverse phase
HPLC. Each component was shown to be a ca 1:1 mixture of two isomers by NMR.
MS: calculated for C33H28C14N4O7S 764, observed m/e 765 (M + H)+.
Faster eluting component:
1H NMR (400 MHz, CD30D): 8 (selected peaks) 8.63/8.65 (s, 2H), 4.57/4.59 (d,
1H), 3.62/3.56 (s, 3H).
Slower eluting component:
1H NMR (400 MHz, CD3OD): 8 (selected peaks) 8.64/8.62 (s, 2H), 4.24/4.16 (d,
1H), 3.78/3.58 (s, 3H).
Step E N-(N-f(3,5-Dichlorobenzene)sulfonyll-3-(4-hydroxyphen~)-prolxl)-
f (3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine.
Each of the two components from Step D of N-(N-[(3,5-dichloro-
benzene)sulfonyl]-3-(4-hydroxyphenyl)-prolyl)-[(3',5'-
dichloroisonicotinoyl)amino]-
' (L)-phenylalanine, methyl ester was separately treated with LiOH according
to the
procedure described in Example 20, Step F. The reaction mixture was
partitioned
between brine, 1 M HCl and EtOAc. The product was extracted with EtOAc (3x),
and the combined extracts were dried over anhydrous MgSO4 and concentrated in
vacaco to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-3-(4-hydroxyphenyl)-
prolyl)-
[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, each component as a
1:1
mixture of diastereomers.
Isomers from the faster eluting component of Step D:
1H NMR (400 MHz, CD30D): 8 (selected peaks) 8.64/8.62 (s, ZH).
MS: calculated for C32H26C14N4O7S 750, observed m/e 751 (M + H)+.
Isomers from the slower eluting component of Step D:
1H NMR (400 MHz, CD30D): 8 (selected peaks) 8.64/8.62 (s, 2H), 4.18/4.12 (d,
1H).
-75-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
MS: calculated for C32H26C14N4O7S 750, observed m/e 751 (M + H)+.
EXAMPLES 34 and 35
N-(N-f(3,5-Dichlorobenzene)sulfonyll-3,3-dimeth ~~1-prol ly_)-4-j(3' S'-
dichloro-
isonicotinoyl)aminol-(L)-phenylalanine.
Step A N-Acetyl-2-ethoxycarbonyl-3,3-dimeth,~proline, ethyl ester
N-Acetyl-2-ethoxycarbonyl-3,3-dimethylproline, ethyl ester (6.4 g,
38%) was prepared from 3-methyl-2-butenal (Aldrich, 5.0 g, 59 mmol) and
diethyl
acetamidomalonate (Aldrich, 12 g, 54 mmol) by the procedure described by Chung
et
al (J. Org. Chena. 1990, SS, 270).
1H NMR (400 MHz, CD30D): 8 4.16 (q, 4H); 3.78 (t, 2H), 2.08 (s, 3H); 2.02 (t,
2H); 1.242 (t, 6H); 1.12 (s, 6H).
MS: calculated for C14H23N05 285, observed m/e 308 (M + Na)+.
Step B 3,3-Dimethylproline, ethyl ester, hydrobromide.
A solution of N-acetyl-2-ethoxycarbonyl-3-(4-methoxyphenyl)proline,
ethyl ester (6.4 g, 22 mmol) in aqueous HBr (48%, 35 mL) and HOAc (5 mL) was
heated at 120 °C under nitrogen for 65 h. The reaction mixture was
concentrated ira
vacuo, and the residue was azeotroped with AcCN (2x) and Et20/toluene (lx) to
yield
3,3-dimethylproline, ethyl ester hydrobromide (4.8 g, 96%).
MS: calculated for C7H13N02 143, observed mle 144 (M + H)+.
Step C N-f(3,5-Dichlorobenzene)sulfonyll-3,3-dimeth ~~1-proline.
3,3-Dimethylproline ethyl ester hydrobromide salt (2.4 g, 11 mmol)
was reacted with 3,5-dichlorobenzene sulfonyl chloride and Na2C03 (1.5 equiv.)
according to the procedure described in Example 28, Step A to afford N-[(3,5-
dichlorobenzene)sulfonyl]-3,3-dimethyl-proline (2.9 g, 76%)
Step D N-(N-f(3,5-Dichlorobenzene)sulfonyll-3,3-dimethyl- rolyl)-f(3' S'-
dichloroisonicotinoyl)aminol-(L)-phenxlalanine meth 1y ester.
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-3,3-dimethyl-proline (0.080 g,
0.23 mol) was coupled to 4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine,
methyl ester by the procedure described in Example 1 to yield N-(N-[(3,5-
dichloro-
-76-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
benzene)sulfonyl]-3,3-dimethyl-prolyl)-[(3',5'-dichloroisonicotinoyl)amino]-
(L)-
phenylalanine, methyl ester as a 1:1 mixture of isomers, 0.13 g, 80%).
1H NMR (400 MHz, CD30D): 8 (selected peaks) 8.64/8.63 (s, 2H); 3.84/3.76 (s,
1H); 3.78/3.70 (s, 3H); 0.88/0.69 (s, 3H); 0.72/0.67(s, 3H).
MS: calculated for C29H28C14N4O6S 700, observed m/e 701 (M + H)+.
A portion of the mixture of diastereomers obtained above was separated into
two pure
isomers on a Chiralcel OD column eluting with 15% ethanol/hexane.
Faster eluting isomer:
1H NMR (500 MHz, CD30D): 8 8.63 (s, 2H); 7.77 (t, J=2.0 Hz, 1H); 7.74 (d,
J=2.0
Hz, 2H); 7.61 (d, J=8.5 Hz, 2H); 7.31 (d, J=8.5 Hz, 2H); 4.74 (dd, J=8.0, 5.5
Hz, 1H);
3.84 (s, 1H); 3.70 (s, 3H); 3.55 (ddd, J=9.0, 9.0, 1.5 Hz, 1 H); 3.28 (ddd,
J=9.5, 9.5,
7.0 Hz, 1H); 3.18 (dd, J=14.0, 6.0 Hz, 1H); 3.08 (dd, J=14.0, 8.0 Hz, 1H);
1.92 (ddd,
J=12.0, 9.5, 9.5 Hz, 1H); 1.53 (ddd, J=I2.0, 5.5, 2.0 Hz, 1H); 0.88 (s, 3H);
0.72 (s,
3H).
MS: calculated for C29H28C14N4O6S 700, observed m/e 701 (M + H)+.
Slower eluting isomer:
1H NMR (500 MHz, CD30D): 8 8.64 (s, 2H); 7.80 (d, J=2.0 Hz, 2H); 7.77 (t,
J=2.0
Hz, 1H); 7.58 (d, J=8.5 Hz, 2H); 7.29 (d, J=8.5 Hz, 2H); 4.63 (dd, J=9.5, 5.0
Hz, 1H);
3.79 (s, 1H); 3.76 (s, 3H); 3.53 (ddd, 1H); 3.29 (ddd, 1H); 3.22 (dd, J=14.0,
5.0 Hz,
1H); 3.00 (dd, J=14.0, 10.0 Hz, 1H); 1.89 (ddd, J=12.0, 9.0, 9.0 Hz, 1H); 1.50
(ddd,
J=12.0, 6.5, 2.0 Hz, 1H); 0.69 (s, 3H); 0.67 (s, 3H).
MS: calculated for C29H28C14N4O6S 700, observed m/e 701 (M + H)+.
Step E N-(N-f(3,5-Dichlorobenzene)sulfonyll-3 3-dimeth ~~1-prolyl)-f(3' 5'-
dichloroisonicotinoyl)aminol-(L)-phe~lalanine.
Each of the isomers from Step D of N-(N-[(3,5-dichlorobenzene)-
sulfonyl]-3,3-dimethyl-prolyl)-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine, methyl ester was separately treated with LiOH according to the
procedure described in Example 20, Step F. The reaction mixture was
partitioned
between brine, 1 M HCl and EtOAc. The product was extracted with EtOAc (3x),
and the combined extracts were dried overanhydrous MgS04 and concentrated i~c
_77_
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
vacuo to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-3,3-dimethyl-prolyl)-
[(3',5'-
dichloroisonicotinoyl)amino]-(L)-phenylalanine.
Isomer from the faster eluting isomer of Step D:
1H NMR (500 MHz, CD30D): 8 8.63 (s, 2H); 7.76 (t, J=1.5 Hz, 1H); 7.74 (d,
J=1.5
Hz, 2H); 7.60 (d, J=8.5 Hz, 2H); 7.35 (d, J=8.5 Hz, 2H); 4.72 (m, 1H); 3.87
(s, 1H);
3.55 (ddd, J=8.5, 8.5, 0.5 Hz, 1 H); 3.27 (m, 1H); 3.20 (dd, J=14.0, 5.5 Hz,
1H); 3.09
(dd, J=14.0, 7.5 Hz, 1H); 1.94 (ddd, 1H); 1.53 (ddd, J=12.5, 7.0, 1.5 Hz, 1H);
0.91 (s,
3H); 0.72 (s, 3H).
MS: calculated for C28H26C14N4O6S 686, observed m/e 687 (M + H)'~.
Isomer from the slower eluting isomer of Step D:
1H NMR (500 MHz, CD30D): 8 8.64 (s, 2H); 7.81 (d, J=2.0 Hz, 2H); 7.68 (t,
J=2.0
Hz, 1H); 7.57 (d, J=8.5 Hz, 2H); 7.32 (d, J=8.5 Hz, 2H); 4.72 (dd, J=9.0, 5.0
Hz, 1H);
3.78 (s, 1H); 3.54 (ddd, J=9.0, 9.0, 2.5 Hz, 1H); 3.28 (m, 1H); 3.22 (dd,
J=14.0, 5.0
Hz, 1H); 3.02 (dd, J=14.0, 9.0 Hz, 1H); 1.89 (ddd, J=12.0, 9.0, 9.0 Hz, 1H);
1.49
(ddd, J=12.0, 6.5, 2.0 Hz, 1H); 0.69 (s, 6H).
MS: calculated for C28H26C14N4O6S 686, observed m/e 687 (M + H)+.
EXAMPLE 36
N-(N-f (3, 5-dichlorobenzene)sulfonyll-2-methyl-(L)-prolyl)-4- f (3' ,5'-
dichloro-
isonicotinoyl-N-oxide)aminol-(L)-phenylalanine.
To a solution of N-(N-(3,5-Dichlorobenzenesulfonyl)-2-methyl-(L)-
prolyl)-4-((3',5'-dichloroisonicotinoyl)amino)-(L)-phenylalanine from Example
2
(0.050 g, 0.074 mmol) in EtOAc (0.25 mL) was added DMF dropwise until a
solution
was formed. mCPBA (100 mg) was added and reaction was stiiTed at 50°C
for 18 h.
T'he reaction was concentrated in vacuo then dissolved in AcCN/water 70:30 and
purified by preparative HPLC to afford N-(N-(3,5-dichlorobenzenesulfonyl)-2-
methyl-(L)-prolyl)-4-((3',5'-dichloroisonicotinoyl-N-oxide)amino)-(L)-
phenylalanine
as a white solid (0.041 g, 0.059 mmol, 80%).
1H NMR (500 MHz , CD3OD): ~ 8.58 (s, 2H), 7.77 (d, J=2Hz, 2H), 7.73 (t,
J=l.9Hz,
1H), 7.59 (d, J=8.5Hz, 2H), 7.53 (d, J=7.8Hz, 1H), 7.29 (d, J=8.5Hz, 2H), 4.71
(m,
1H), 3.41 (m, 1H), 3.30 (m, 2H), 3.10 (dd, J=8.2, l4Hz, 1H), 2.15 (m, 1H),
1.81 (m,
2H), 1.70 (m, 1H), 1.60 (s, 3H); MS mle 689.18 (M+).
_78_
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
The following compounds were prepared according to the procedures
described in Example 9, substituting the appropriate arylsulfonyl chloride in
Step B:
ExampleName mass
No. spectrum
(m/e)
37 N-(N-[benzenesulfonyl]-4-(S)-phenyl-(L)-prolyl)-4-[(3,5-667.2
dichloroisonicotinoyl)amino]-(L)-phenylalanine
38 N-(N-[(3-trifluoromethoxybenzene)sulfonyl]-4-(S)-phenyl-751.1
(L)-prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-
phenylalanine
39 N-(N-[(4-chlorobenzene)sulfonyl]-4-(S)-phenyl-(L)-703.1
prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-
phenylalanine
40 N-(N-[(3-bromobenzene)sulfonyl]-4-(S)-phenyl-(L)-747.0
prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-
phenylalanine
41 N-(N-[(3,4-dichlorobenzene)sulfonyl]-4-(S)-phenyl-(L)-736.9
prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-
phenylalanine
42. N-(N-[(3,5-bis-(trifluoromethyl)benzene)sulfonyl]-4-(S)-803.0
phenyl-(L)-prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-
(L)-phenylalanine
43 N-(N-[(3-methylbenzene)sulfonyl]-4-(S)-phenyl-(L)-681.1
prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-
phenylalanine
44 N-(N-[(3,5-dimethylbenzene)sulfonyl]-4-(S)-phenyl-(L)-695.1
prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-
phenylalanine
45 N-(N-[(3-fluorobenzene)sulfonyl]-4-(S)-phenyl-(L)-685.1
prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-
phenylalanine
46 N-(N-[(3-chlorobenzene)sulfonyl]-4-(S)-phenyl-(L)-703.1
-79-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-
phenylalanine
47 N-(N-[(3-(trifluoromethyl)benzene)sulfonyl]-4-(S)-phenyl- 735.1
(L)-prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-
phenylalanine
48 N-(N-[(3-biphenyl)sulfonyl]-4-(S)-phenyl-(L)-prolyl)-4- 743.1
[(3,5-dichloroisonicotinoyl)amino]-(L)-phenylalanine
49 N-(N-[(5-methyl-3-pyridyl)sulfonyl]-4-(S)-phenyl-(L)- 682.1
prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-
phenylalanine
EXAMPLE 50N-(N-f(3,5-dichlorobenzene)sulfonyll-2-methyl-(L)-prol 1~~3' 5'-
dichloro-2'-h day-isonicotinoyl)aminol-(L)-phenylalanine
Step A N-(N-f 3,5-dichlorobenzene)sulfonyll-2-meth,1-~prolyl)-4-1(3',5'-
dichloroisonicotinoyl-N-oxide)aminol-(L)-phenylalanine, meth, l~r.
N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-methyl-(L)-prolyl)-4-[(3',5'-
dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (113 mg, 0.164
mmol)
from Example 1 was treated with mCPBA according to the procedure described in
Example 36 to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-methyl-(L)-prolyl)-
4-
[(3',5'-dichloroisonicotinoyl-N-oxide)amino]-(L)-phenylalanine, methyl ester
(66 mg,
57%).
400 MHz 1H NMR (CDC13) 8 8.52 (br, 1H); 8.29 (br, 2H); 7.76 (d, J = 2.0 Hz,
2H);
7.59 (d, J = 8.8 Hz, 2H); 7.24 (d, J = 8.4 Hz, 2H); 4.88 (m, 1H); 3.82 (s,
3H); 3.57 (m,
1H); 3.36-3.31 (m, 2H); 3.36 (m, 2H); 3.13 (m, 1H); 1.84 (m, 1H); 1.74 (m,
3H); 1.61
(s, 3H).
Step B N-(N-f(3,5-Dichlorobenzene)sulfonyll-2-meth~(L)-prolyl)-4-f(2'-
acetoxy-3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine methyl
ester
A solution of N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-methyl-(L)-
prolyl)-4-[(3',5'-dichloroisonicotinoyl-N-oxide)amino]-(L)-phenylalanine,
methyl
ester (24 mg, 0.0341 mmol) in 0.5 mL of acetic anhydride was heated to 100
°C for 3
h. After cooling to rt, the reaction mixture was diluted with EtOAc (25 mL),
washed
-80-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
with saturated NH4C1 solution (10 mL) and extracted with EtOAc (1X). The
combined extracts were washed with brine, dried over anhydrous MgS04 and
concentrated in vacaao to yield 182 mg of light yellow oil. The product was
purified
by preparative HPLC eluted with 30-95% AcCN in water (0.1% TFA) at 10 mL/min
for 15 min to yield N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-methyl-(L)-prolyl)-
4-[(2'-
acetoxy-3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (20
mg)
as a white solid.
400 MHz 1H NMR (CDC13) ~ 8.27 (br, 2H); 7.76 (d, J = 1.6 Hz, 2H); 7.59 (m,
1H);
7.40 (br, 1H); 7.27 (d, J = 2.0 Hz, 2H); 7.22 (br, 1H); 4.90 (m, 1H); 3.82 (s,
3H); 3.57
(m, 1H); 3.34 (m, 3H); 3.19 (m, 1H); 2.37 (m, 1H); 1.84 (m, 1H); 1.67 (m, 2H);
1.61
(s, 3H).
Step C N-(N-f(3,5-Dichlorobenzene)sulfonyll-2-meth 1-~prolyl)-4-[(3' 5'-
dichloro-2'-h day-isonicotin~l)aminol-(L)-phenylalanine
To a solution of N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-methyl-(L)-
prolyl)-4-[(2'-acetoxy-3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine,
methyl
ester (15 mg, 0.0201 mmol) in 0.5 mL of MeOH was added 1M KOH (0.060 mL).
The reaction mixture was stirred at rt overnight. The reaction was quenched by
addition of TFA (1 drop). The product was purified by preparative HPLC eluted
with
30-90% AcCN in water (0.1 % TFA) at 10 mL/min over 15 min to afford N-(N-[(3,5-
dichlorobenzene)sulfonyl]-2-methyl-(L)-prolyl)-4-[(3',5'-dichloro-2'-hydroxy-
isonicotinoyl)amino]-(L)-phenylalanine (9 mg) as a white solid.
400 MHz 1H NMR (CDCl3) S 9.36 (br, 1H); 8.38 (br, 2H); 7.75 (br, J = 2 Hz,
2H);
7.57 (s, 1H); 7.40 (d, J = 7.6, 2H); 7.17 (d, J = 7.2 Hz, 2H); 4.89 (m, 1H);
3.60 (m,
1H); 3.41 (m, 1H); 3.22 (m, 2H); 2.39 (m, 1H); 1.94 (m, 2H); 1.82 (m, 1H);
1.65 (s,
3H).
EXAMPLE 51
N-(N-f(3,5-Dichlorobenzene)sulfonyll-2-meth 1-y_ (L)-prolyl)-4-f(3' 5'-
dichloro-2'-
methoxy-isonicotinoyl)aminol-(L)-phenylalanine
Step A N-(N-f(3,5-Dichlorobenzene)sulfonyll-2-methyl-(L)-prolyl)-4-f(3' S'-
dichloro-2'-methoxy-isonicotinoyl)aminol-(L)-phenylalanine meth
ester.
-81-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
To a ice-cooled solution of N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-
methyl-(L)-prolyl)-4-[(3',5'-dichloroisonicotinoyl-N-oxide)amino]-(L)-
phenylalanine,
methyl ester from Example 50, Step A (40 mg, 0.0577 mmol) in MeOH (0.5 mL) was
added acetyl chloride (0.005 mL, 0.075 mmol) and TEA (0.016 mL, 0.115 mmol).
Ethyl chloroformate (0.016 mL, 0.150 mmol) and TEA (0.016 mL, 0.115 mmol) were
added and the reaction mixture was allowed to stirred overnight. The solvent
was
removed ifi wacuo and the residue purifed by preparative HPLC eluted with 30-
90%
AcCN in water (0.1 % TFA) at 10 mL/min over 15 min to afford N-(N-[(3,5-
dichlorobenzene)sulfonyl]-2-methyl-(L)-prolyl)-4-[(3',5'-dichloro-2'-methoxy-
isonicotinoyl)amino]-(L)-phenylalanine, methyl ester (15 mg, 36%) as a white
solid.
400 MHz 1H NMR (CDCl3) ~ 8.14 (s, 1H); 7.76 (d, J = 2 Hz, 2H); 7.57 (d, J =
8.8
Hz, 2H); 7.41 (m, 1H); 7.23 (d, J = 8.0 Hz, 2H); 4.88 (m, 1H); 4.06 (s, 3H);
3.82 (s,
3H); 3.56 (m, 1H); 3.31 (m, 2H)3.12 (m, 1H); 2.33 (m, 1H); 1.82 (m, 3H); 1.68
(s,
3H).
Step B N-(N-f(3,5-Dichlorobenzene)sulfonyll-2-meth 1-~~rolyl)-4-[(3',5'-
dichloro-2'-methoxy-isonicotinoyl)aminol-(L)-phen~lalanine.
N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-methyl-(L)-prolyl)-4-[(3',5'-
dichloro-2'-methoxy-isonicotinoyl)amino]-(L)-phenylalanine, methyl ester (15
mg)
was treated with MeOHic KOH according to the procedure described in Example
50,
Step C. The product was purified by preparative HPLC eluted with 30-90% AcCN
in
water (0.1% TFA) at 10 mL/min over 15 min to afford N-(N-[(3,5-
dichlorobenzene)-
sulfonyl]-2-methyl-(L)-prolyl)-4-[(3',5'-dichloro-2'-methoxy-
isonicotinoyl)amino]-
(L)-phenylalanine (12 mg, 82% yd) as white solid.
400 MHz 1H NMR (CD30D) ~ 8.18 (s, 1H); 7.79 (d, J = 1.6 Hz, 2H); 7.74 (s, 1H);
7.58 (d, J = 8.0 Hz, 2H); 7.27 (d, J = 8.4 Hz, 2H); 4.71 (m, 1H); 4.02 (s,
3H); 3.45-
3.32 (m, 3H); 3.13 (m, 1H); 2.15 (m, H); 1.83 (m, 3H); 1.60 (s, 3H).
EXAMPLE 52
N-(N-L(3,5-Dichlorobenzene)sulfonyll-2-meth 1-~pro~l)-4-[(2'-amino-3',5'-
dichloroisonicotinoyl)aminol-(L)-phenylalanine.
Step A N-(N-f(3,5-Dichlorobenzene)sulfonyll-2-meth 1~- L)-~rolyl)-4-[(2'-
amino-3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine, meth
ester
-82-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
To a solution of N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-methyl-(L)-
prolyl)-4-[(3',5'-dichloroisonicotinoyl-N-oxide)amino]-(L)-phenylalanine,
methyl
ester from Example 50, Step A (40 mg, 0.0568 mmol), in CH2C12 (1 mL) at 0
°C was
added TsCI (12 mg, 0.0624 mmol). The reaction was stirred for 20 min. A 10%
NH40H solution (1 mL) was added and the ice bath was removed. The reaction
mixture was stirred at rt for 4 h. The reaction mixture was then diluted with
EtOAc
(20 mL), washed with brine, dried over anhydrous Na2S04 and concentrated in
vacuo
to yield 44 mg of the crude product mixture which was purified by preparative
HPLC
eluted with 30-90% AcCN in water (0.1 % TFA) at 10 mL/min over 15 min to yield
N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-methyl-(L)-prolyl)-4-[(2'-amino-3',5'-
dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (4.7 mg, 12% yd).
400 MHz 1H NMR (CDCl3) 8 7.91 (br, 1H); 7.76 (s, 2H); 7.58 (d, J = 6.8 Hz,
2H);
7.23 (d, J = 6.0 Hz, 2H); 7.15 (m, 1H); 4.88 (m, 1H); 3.82 (s, 3H); 3.56 (m,
1H); 3.31
(m, 2H); 3.12 (m, 1H); 2.33 (m, 1H); 1.82 (m, 3H); 1.63 (s, 3H).
Step B N-(N-f(3,5-Dichlorobenzene)sulfonyll-2-methyl-(L)-prolyl)-4-f(2'-
amino-3',5'-dichloroisonicotinoyl)aminol-(L~-phenylalanine.
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-2-methyl-(L)-prolyl)-4-[(2'-
amino-3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester (4.7
mg)
was treated with MeOHic NaOH according to the procedure described in Example
50,
Step C. The product was purified by preparative HPLC eluted with 30-90% AcCN
in
water (0.1% TFA) at 10 mL/min over 15 min. to afford N-(N-[(3,5-
Dichlorobenzene)sulfonyl]-2-methyl-(L)-prolyl)-4-[(2'-amino-3',5'-dichloro-
isonicotinoyl)amino]-(L)-phenylalanine (3 mg, 63% yd).
400 MHz 1H NMR (CD30D) 8 7.99 (s, 1H); 7.79 (d, J = 1.6 Hz, 2H); 7.73 (m, 1H);
7.58 (d, J = 6.8 Hz, 2H); 7.26 (d, J = 6.8 Hz, 2H); 4.70 (m, IH); 3.43 (m,
2H); 3.31
(m, 1H); 3.10 (m, 1H); 2.13 (m, 1H); 1.79 (m, 2H); .1.68 (m, 1H); 1.59 (s,
3H).
EXAMPLE 53
N-(N-f(3-chlorobenzene)sulfonyll-3,3-dimethyl-prop)-4-f(3' 5'-dichloro-
isonicotinoyl)aminol-(L)-phenylalanine
Step A N-(3-chlorobenzene)sulfonyl-3,3-dimeth~proline
To a solution of 3,3-dimethylproline, hydrobromide (0.050 g,
0.223mmo1) in water (0.25 mL) was added 3-chlorobenzene sulfonyl chloride
(0.061
g, 0.291 mmol) and Na2C03 (0.060 g, 0.558 mmol). After being stirred for 5 h
at rt,
-83-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
the reaction was washed with EtOAc (1 mL) which was discarded. The aqueous
layer
was diluted with 2N HCl (5 mL) and the product extracted into EtOAc (5 mL).
The
organic layer was washed with saturated aqueous NaCl (5 mL), dried over
anhydrous
MgS04 and concentrated in vaeuo to yield N-(3-chlorobenzene)sulfonyl-3,3-
dimethylproline as a white foam.
1H NMR (500 MHz , CD30D): 8 7.84 (s, 1H), 7.79 (d, J=7.3Hz, 1H), 7.66 (d,
J=7.6Hz, 1H), 7.59 (t, J=7.7Hz, 1H), 5.0 (br, 1H), 3.76 (s, 1H), 3.55 (t,
J=7.9Hz, 1H),
3.31 (q, J=8.2Hz, 1H), 1.93 (q, J=9.4Hz, 1 H), 1.56 (m, 1H), 1.03 (s, 3H),
0.76 (s,
3H).
Step B ~ N-(N-f (3-chlorobenzene)sulfonyll-3,3-dimeth ~~l-prol~l)-41(3' S'-
dichloroisonicotinoyl)aminol-(L)-phenylalanine meth 1 ester
N-(3-Chlorobenzene)sulfonyl-3,3-dimethylproline (0.031 g, 0.098
mmol) was coupled to 4-[(3,5-dichloroisonicotinoyl)amino]-(L)-phenylalanine,
methyl ester hydrochloride (0.048 g, 0.107 mmol) in the presence of PyBOP
(0.061 g,
0.117 mmol) and DIPEA (0.055 mL, 0.292 mmol) in CH2Cl2 (0.3 mL) according to
the procedure described in Example 1 to yield N-(N-[(3-chlorobenzene)sulfonyl]-
3,3-
dimethyl-prolyl)-4-[(3',5'-dichloroisonicotinoyl) amino]-(L)-phenylalanine,
methyl
ester which was used without purification in the subsequent reaction.
Step C N-(N-f(3-chlorobenzene)sulfonyll-3,3-dimeth~prolyl)-4-[~3' S'-
dichloroisonicotinoyl)aminol-(L)- henylalanine.
The N-(N-[(3-chlorobenzene)sulfonyl]-3,3-dimethyl-prolyl)-4-[(3',5'-
dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester from Step B was
dissolved in MeOH (0.3 mL) and treated with 1N NaOH (0.2 mL). After stirring
at 3
h at rt, the reaction was acidified with TFA and and concentrated ih vaeuo.
The
residue was purified by preparative HPLC to yield N-(N-[(3-
chlorobenzene)sulfonyl]-
3,3-dimethylprolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine
as a
white solid:
MS m/e 688.2 (M+).
EXAMPLE 54
N-(N-f3-chlorobenzene)sulfonyll-3,3-dimethyl-prolyl)-4-[(3' S'-dichloro-
isonicotinoyl-N-oxide)aminol-(L)-phenylalanine
- 84 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Step A N-(BOC)-4-f(3',5'-Dichloroisonicotinoyl-N-oxide)-aminol-(L)-
phenylalanine, methyl ester.
N-(BOC)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine,
methyl ester (Reference Example 2, 0.3 g, 0.64 mmol) and mCPBA (0.22 g; 1.28
mmol) were dissolved in CH2C12. The reaction mixture was stirred at rt
overnight.
TLC showed starting material remaining so an additional amount of mCPBA (0.22
g;
1.28 mmol) was added. The reaction was heated at 45°C for 2 hours. The
solution
was concentrated in vaeuo and the residue purified by flash column
chromatography
on silica gel eluted with 90% EtOAc/hexane to afford N-(BOC)-4-[(3',5'-
dichloro-
isonicotinoyl-N-oxide)-amino]-(L)-phenylalanine, methyl ester (0.34 g).
MS mle 428.0 (mass spectrum shows the desired product minus the mass of BOC as
the parent peak).
Step B 4-f(3',5'-Dichloroisonicotinoyl-N-oxide)-aminol-(L)-phenylalanine,
methyl ester, hydrochloride
N-(BOC)-4-[(3',5'-dichloroisonicotinoyl-N-oxide)-amino]-(L)-
phenylalanine, methyl ester (0.7 mmol) was dissolved in EtOAc. HCl gas was
bubbled through the reaction mixture for several minutes. The resulting
solution was
stirred at rt for 15 min. The reaction mixture was concentrated ifz vacuo to
afford 4-
[(3',5'-dichloroisonicotinoyl-N-oxide)-amino]-(L)-phenylalanine, methyl ester,
hydrochloride (0.25 g).
Step C N-(N-f3-chlorobenzene)sulfon~l-3,3-dimeth ~~l-prolyl)-4-f(3',5'-
dichloroisonicotinoyl-N-oxide~aminol-(L)-phenylalanine, meth,1~.
4-(3',5'-Dichloroisonicotinoyl-N-oxide)-amino-(L)-phenylalanine,
methyl ester (0.48 g; 0.125 mmol) and N-(3-chlorobenzene)sulfonyl-3,3-dimethyl-
proline from Example 53, Step A (0.036 g; 0.11 mmol) were coupled in the
presence
of PyBOP (0.069 g; 0.132 mmol) and DIPEA (0.06 mL; 0.33 mmol) in CH2C12 (1
mL) according to the procedure described in Example 1 to yield N-(N-[3-chloro-
benzene)sulfonyl]-3,3-dimethyl-prolyl)-4-[(3',5'-dichloroisonicotinoyl-N-
oxide)-
amino]-(L)-phenylalanine, methyl ester after purification by preparative HPLC.
Step D N-(N-f3-chlorobenzene)sulfonyll-3,3-dimeth~prol,~)-4-f(3',5'-
dichloroisonicotinoyl-N-oxide)aminol-(L)~henylalanine
-85-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
N-(N-[3-Chlorobenzene)sulfonyl]-3,3-dimethyl-prolyl)-4-[(3',5'-
dichloroisonicotinoyl-N-oxide)amino]-(L)-phenylalanine, methyl ester was
dissolved
in MeOH (0.5 mL) and 1N NaOH (~lmL) was added. The reaction mixture was
stirred at rt for 2 h. The reaction was acidified with TFA and purified by
preparative
HPLC to afford N-(N-[3-chlorobenzene)sulfonyl]-3,3-dimethyl-prolyl)-4-[(3',5'-
dichloroisonicotinoyl-N-oxide)amino]-(L)-phenylalanine (0.035 g).
MS mle 671.1
EXAMPLE 55
N-(N-f3,5-Dichlorobenzene)sulfonyll-3,3-dimethyl-prolyl)-3-(6-(3' S'-dichloro-
isonicotinoyl)amino-3-~ r~idyl)-alanine
Step A 2-f (N-Diphen l~ylene)aminol-3-(4-bromo-3-p,~,~propanoic
acid, ethyl ester
To a solution of N-(diphenylmethylene)glycine ethyl ester (4.30 g,
16.11 mmol) in THF (25 mL) at -78°C was added dropwise a 2.0M solution
of
lithium diisopropylamide in THF (8.0 mL, 16.0 mmol). After being stirred for
30 min
at -78°C, a solution of 2-bromo-5-bromomethyl-pyridine in 10 mL of THF
was added
and the reaction allowed to warm to rt. After 1 h, the reaction was diluted
with
EtOAc (50 mL) and washed with water (50 mL) and saturated aqueous NaCl (50
mL).
The organic layer was dried over anhydrous MgS04 and concentrated in vacaco to
give a yellow oil. The oil was purified by flash column chromatography on
silica gel
eluted with hexane/EtOAc (10:1) to yield 2-[(N-diphenylmethylene)amino]-3-(4-
bromo-3-pyridinyl) propanoic acid, ethyl ester as a pale yellow oil.
Step B 3-(6-f3',5'-dichloroisonicotinoyl)aminol-3-pyridinyl)-2-f (N-
diphen l~ylene)aminol propanoic acid eth 1
A solution of 2-[(N-diphenylmethylene)amino]-3-(4-bromo-3-
pyridinyl)propanoic acid, ethyl ester (2.00 g, 4.57 mmol), 3,5-
dichloropyridine-4-
carboxamide (0.96 g, 5.03 mmol), tris(dibenzylideneacetone)dipalladium(0)
(0.083 g,
0.091 mmol), Cs2C03 (2.00 g, 6.40 mmol), and 9,9-dimethyl-4,5-bis(diphenyl-
phosphino)xanthene (0.16 g, 0.27 mmol) in THF (10 mL) was heated to
75°C under
nitrogen. After 16 h, the reaction was cooled, diluted with EtOAc (50 mL),
washed
with saturated aqueous NaCI (50 mL) and dried over anhydrous MgS04. The
solvent
was removed ifz vacuo followed by purification by flash column chromatography
on
silica gel eluted with hexane/EtOAc (10:1) to yield 3-(6-[3',5'-
dichloroisonicotinoyl)-
-86-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
amino]-3-pyridinyl)-2-[(N-diphenylmethylene)amino] propanoic acid, ethyl ester
as a
colorless oil (2 g).
MS nz/e 574.2 (M+).
Step C 3-(6-(3',5'-Dichloroisonicotinoyl)amino-3-p~yl)alanine, ethyl ester
To a solution of 3-(6-[3',5'-dichloroisonicotinoyl)amino]-3-pyridinyl)-
2-[(N-diphenylmethylene)amino] propanoic acid, ethyl ester in THF (10 mL) and
water (5 mL) was added glacial HOAc (5 mL). After 3 h, the reaction was
diluted
with 2N HCl (25 mL) and extracted with EtOAc (2 x 25 mL) which were discarded.
The aqueous layer was made basic (pH = 12) with 1N NaOH and the product
extracted into EtOAc (3 x 25 mL). The combined organics were washed with brine
(50 mL), dried over anhydrous MgS04 and concentrated i~z vacuo to yield 3-(6-
(3',5'-
dichloroisonicotinoyl)amino-3-pyridyl)-alanine, ethyl ester as a white foam:
MS hale
383.1 (M+).
1H NMR (500 MHz , CDCl3): 8 8.48 (s, 1H), 8.27 (d, J=8.7Hz, 1H), 7.62 (dd,
J=2.3,
8.5Hz, 1H), 7.42 (d, J=2Hz, 1H), 4.16 (q, J=7.lHz, 2H), 3.57 (dd, J=4.6, 7.8
Hz, 1H),
2.88 (dd, J=5.3, l4Hz, 1H), 2.69 (dd, J=7.8, 13.9Hz, 1H), 1.26 (t, J=7.lHz,
3H).
Step D N-(N-[3,5-Dichlorobenzene)sulfonyll-3,3-dimeth ~~l-prolyl)-3-(6-(3',5'-
dichloroisonicotinoyl)amino-3-p.~yl)-alanine, ethyl ester.
3-(6-(3',5'-dichloroisonicotinoyl)amino-3-pyridyl)-alanine, ethyl ester
(0.027 g, 0.071 mmol) and (3,5-dichlorobenzene)sulfonyl-3,3-dimethylproline
(0.025
g, 0.071 mmol) were coupled in the presence of PyBOP (0.036 g, 0.085 mmol) and
DIEA (0.020 mL, 0.106 mmol) in CH2C12 (0.25 mL) according to the procedure
described in Example 1. The crude product was purified by prepartive HPLC to
yield
N-(N-[3,5-dichlorobenzene)sulfonyl]-3,3-dimethyl-prolyl)-3-(6-(3',5'-dichloro
isonicotinoyl)amino-3-pyridyl)-alanine, ethyl ester as an oil.
Sep E N-(N-f3,5-Dichlorobenzene)sulfonyll-3 3-dimethylprolyl)-3-(6-(3' 5'-
dichloroisonicotinoyl)amino-3-~yridyl)-alanine.
N-(N-[3,5-Dichlorobenzene)sulfonyl]-3,3-dimethyl-prolyl)-3-(6-(3',5'-
dichloroisonicotinoyl)amino-3-pyridyl)-alanine, ethyl ester was dissolved in
MeOH
(1.0 mL) and treated with 1N NaOH (0.2 mL). After stirring at rt for 18 h, the
reaction was acidified with TFA. The solvent was removed ifz vacaao and the
residue
_87_
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
purified by preparative HPLC to yield N-(N-[3,5-dichlorobenzene)sulfonyl]-3,3-
dimethylprolyl)-3-(6-(3',5'-dichloroisonicotinoyl)amino-3-pyridyl)-alanine as
a white
solid: MS rfzle 688.2 (M+).
EXAMPLE 56
N-(N-f (3,5-Dichlorobenzene)sulfonyll-4(R)-(1-azetidin l~prolyl)-4-f(3',5'-
dichloroisonicotinoyl)aminol-(L)-phenylalanine
Step A N-(BOC)-4(R)-(1-azetidin l~proline, meth 1
N-(BOC)-4(R)-(1-Azetidinyl)-(L)-proline, methyl ester was prepared
according to the procedures described in Example 22, Step A substituting N-
(BOC)-
4(S)-hydroxy-(L)-proline, methyl ester (Bachem, 2.5 g, 10 mmol) for N-[(3-
chloro-
benzene)sulfonyl]-4(S)-hydroxy-2-methyl-(L)-proline, methyl ester. The product
was
a mixture of rotamers by 1H-NMR, and was used in the subsequent step.
Step B N-f(3,5-Dichlorobenzene)sulfonyll-4(R)-(1-azetidin, l~proline,
methyl ester
N-(BOC)-4(R)-(1-azetidinyl)-(L)-proline, methyl ester was treated
with HCl(g) in dioxane (Aldrich, 4 M, 80 mL) for 2 h at rt. The reaction
mixture was
concentrated in vacuo to dryness and the residue was azeotroped with
Et20/heptane.
The crude product was dissolved in CH2Cl2 (10 mL) and THF (10 mL) at 0
°C, and
4-DMAP (61 mg, 0.50 mmol), DIPEA (3.5 mL, 20 mmol) and 3,5-dichlorobenzene
sulfonyl chloride (1.8 g, 7.5 mmol) were added. The reaction was allowed to
warm to
rt overnight, and the resulting mixture was concentrated ire vacuo to dryness.
The
residue was purified by flash column chromatography on silica gel eluted with
1:1
hexane/EtOAc to 1:1:0.01 hexane/EtOAc/2 M NH3 in MeOH to afford N-[(3,5-
dichlorobenzene)sulfonyl]-4(R)-(1-azetidinyl)-(L)-proline, methyl ester (2.5
g).
1H NMR (500 MHz, CD30D): 8 7.83 (d, J=2.0 Hz, 2H); 7.80 (t, J=2.0 Hz, 1H);
4.29
(t, J=7.8 Hz, 1H); 3.74 (s, 3H), 3.41 (dd, J=11.0, 5.0 Hz, 1H); 3.19 (br d,
J=11.0 Hz,
1H); 3.06-2.90 (m, 5H); 2.08-1.80 (m, 4H).
MS: calculated for C15H18C12N2O4S 392, observed m/e 393 (M + H)+.
Step C N-f(3,5-Dichlorobenzene)sulfonyll-4(R)-(1-azetidin, l~proline
_88_
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-(1-azetidinyl)-(L)-proline was
prepared from N-[(3,5-dichlorobenzene)sulfonyl]-4(R)-(1-azetidinyl)-(L)-
proline,
methyl ester by the procedure described in Example 20, Step D.
Step D N-(N-f(3,5-Dichlorobenzene)sulfonyll-4(R)-(1-azetidin, l~~rol,
4-((3',5'-dichloroisonicotino~)aminol-(L)-phenylalanine methyl ester
N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-(1-azetidinyl)-(L)-proline was
coupled to 4-[(3,5-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl
ester
hydrochloride in the presence of PyBOP and D1PEA according to the procedure
described in Example 1.
1H NMR (500 MHz, CD30D): 8 8.64 (s, 2H), 7.82 (d, J= 1.5 Hz, 2H), 7.80 (t, J=
1.5
Hz, 1H), 7.62 (d, J=8.5 Hz, 2H), 7.31 (d, J=8.5 Hz, 2H), 4.78 (dd, J=8.0, 5.5
Hz, 1H),
4.20 (t, J=7.5 Hz, 1H); 3.73 (s, 3H); 3.43 (dd, J=11.0, 4.5 Hz, 1H), 3.22 (dd,
J=14.0,
5.5 Hz, 1H), 3.38-2.78 (m, 7H); 1.88-1.66 (m, 4H).
MS: calculated for C30H29C14N5O6S 727, observed m/e 728 (M + H)+.
Step E: N-(N-[(3,5-Dichlorobenzene)sulfonyll-4(R)-(1-azetidin l~prol~)-
4-[(3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine formic acid
salt
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-(1-azetidinyl)-(L)-prolyl)-
4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester was
treated
with LiOH according to Example 20, Step F to afford N-(N-[(3,5-
dichlorobenzene)-
sulfonyl]-4(R)-(1-azetidinyl)-(L)-prolyl)-4-[(3',5'-
dichloroisonicotinoyl)amino]-(L)-
phenylalanine.
1H NMR (500 MHz, CD30D): ~ 8.63 (s, 2H), 7.78-7.76 (m, 3H), 7.60 (d, J=8.5 Hz,
2H), 7.34 (d, J=8.5 Hz, 2H), 4.60 (dd, J=8.0, 5.5 Hz, 1H), 4.44 (dd, J=8.0,
6.6 Hz,
1H); 3.68-3.50 (m, 7H); 3.24 (dd, J=14.0, 5.0 Hz, 1H), 3.07 (dd, J=14.0, 7.5
Hz, 1H),
2.20-2.00 (m, 4H).
MS: calculated for C29H27C14N5O6S 713, observed m/e 714 (M + H)+.
EXAMPLE 57
N-(N-_ f(3 5-Dichlorobenzene)sulfon l~~R)-(4-carbo~phenyl)-(L)-prolyl)-4-f(3'
S'-
dich]oroisonicotinoyl)aminol-(L)-phenylalanine
Step A N-f (3,5-Dichlorobenzene)sulfonyll-3(R)-(4-iodophen, l~proline
-89-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
A mixture of 3(R)-phenyl-pyrrolidine-2(S)-carboxylic acid (Acros,
0.50 g, 2.6 mmol), iodine (0.27 g, 1.0 mmol), sodium iodate (0.10 g, 0.52
mmol) and
concentrated H2S04 (95%, 0.32 ml, 5.7 mmol) in HOAc (5 mL) was heated to 70
°C
overnight. After cooling to rt, the reaction mixture was diluted with water (1
mL) and
was concentrated in vacuo to dryness, and the residue was azeotroped with
heptane
(2x). The residue was then suspended in dilute aqueous Na2C03 (1.1 g, 10.4
mmol,
in 30 mL of water), and 3,5-dichlorobenzenesulfonyl chloride (0.96 g, 3.9
mmol) was
added. After stirring at rt overnight, the reaction mixture was poured into
brine/2 M
HCl/BtOAc, and the product was extracted with EtOAc (2x50 mL). The organic
extracts were dried over anhydrous Na2S04, filtered and concentrated isz vacuo
to
dryness to yield N-[(3,5-dichlorobenzene)sulfonyl]-3(R)-(4-iodophenyl)-(L)-
proline
which was used immediately in the subsequent reaction.
Step B N-(N-f(3,5-Dichlorobenzene)sulfonyll-3(R)-(4-iodophenyl)-(L)-
prolyl)-f(3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine meth
ester
N-[(3,5-Dichlorobenzene)sulfonyl]-3(R)-(4-iodophenyl)-(L)-proline
was coupled to 4-[(3,5-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl
ester
hydrochloride in the presence of PyBOP and DIPEA according to the procedure
described in Example 1.
MS: calculated for C33H27C14IN4O6S 874, observed m/e 875 (M + H)+.
Step C N-(N-f(3,5-Dichlorobenzene)sulfonyll-3(R)-(4-methoxycarbonyl-
phen l~prolyl)-f (3',5'-dichloroisonicotinoyl)aminol-(L)-
phenylalanine, methyl ester
To a solution of N-(N-[(3,5-dichlorobenzene)sulfonyl]-3(R)-(4-
iodophenyl)-(L)-prolyl)-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine,
methyl ester (0.79 g, 0.90 mmol) in DMF (5 mL) was added palladium acetate
(6.0
mg, 0.027 mmol) and l,1'-bis(diphenylphosphino)ferrocene (22 mg, 0.041 mmol).
A
stream of carbon monoxide was bubbled through the reaction for 5 min, MeOH
(0.73
mL, 18 mmol) and TEA (0.25 mL, 1.8 mmol) were added, and the resulting mixture
was charged with a carbon monoxide balloon and was heated at 60 °C for
18 h. The
reaction mixture was cooled to rt and concentrated if2 vacuo, and the residue
was
purified by flash column chromatography on silica gel eluted with 1:1
hexane/EtOAc
-90-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-3(R)-(4-methoxycarbonylphenyl)-
(L)-
prolyl)-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester
along
with some starting material (0.77 g).
MS: calculated for C35H30C14N408S 806, observed m/e 807 (M + H)~.
Step D N-(N-f(3,5-Dichlorobenzene)sulfon 1~R)-(4-carboxy~hen, l
prolyl)- f (3' ,5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine
N-(N-[(3,5-dichlorobenzene)sulfonyl]-3(R)-(4-methoxycarbonyl-
phenyl)-(L)-prolyl)-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine,
methyl
ester (0.77 g) was treated with LiOH according to the procedure described in
Example
20, Step F. The crude product was purified by preparative HPLC (YM-Pack ProCl8
column, 150x20 mm, eluted with 0.1 % aq. formic acid in an acetonitrile
gradient of
50% to 0% over 12 min; Flow rate=20 ml/min). The fastest moving component on
preparative HPLC was identified as the desired product N-(N-[(3,5-
dichlorobenzene)-
sulfonyl]-3(R)-(4-carboxyphenyl)-(L)-prolyl)-[(3',5'-
dichloroisonicotinoyl)amino]-
(L)-phenylalanine_(0.18 g) by NMR and LC-MS.
1H NMR (500 MHz, CD30D): 8 8.62 (s, 2H), 7.81 (d, J=8.5 Hz, 2H); 7.68 (d,
J=2.0
Hz, 2H); 7.63 (t, J=2.0 Hz, 1H); 7.60 (d, J=8.5 Hz, 2H), 7.33 (d, J=8.5 Hz,
2H), 7.03
(d, J=8.5 Hz, 2H); 4.73 (dd, J=8.5, 5.0 Hz, 1H); 4.26 (d, J=4.0 Hz, 1H); 4.01
(m, 1H);
3.70-3.60 (m, 1H); 3.41 (m, 1H), 3.29 (dd, 1H); 3.07 (dd, J=14.0, 9.0 Hz, 1H);
2.20-
2.10 (m, 1H), 2.02-1.92 (m, 1H).
MS: calculated for C33H26C14N408S 778, observed m/e 779 (M + H)+.
The second eluted compound was N-(N-[(3,5-dichlorobenzene)-
sulfonyl]-3(R)-phenyl-(L)-prolyl)-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine (0.11 g).
1H NMR (400 MHz, CD30D): 8 8.63 (s, 2H), 7.73 (d, 2H); 7.71 (t, 1H); 7.60 (d,
2H); 7.32 (d, 2H), 7.20-7.10 (m, 3H), 6.90 (d, 2H); 4.73 (m, 1H); 4.20 (d,
1H); 3.70-
3.54 (m, 2H); 3.35-3.20 (m, 2H); 3.05 (dd, 1H); 2.20-2.10 (m, 1H), 1.90-1.80
(m,
1H).
MS: calculated for C32H26C14N406S 734, observed m/e 735 (M + H)+.
The slowest eluted compound was N-(N-[(3,5-dichlorobenzene)-
sulfonyl]-3(R)-(4-iodophenyl)-(L)-prolyl)-[(3',5'-dichloroisonicotinoyl)amino]-
(L)-
phenylalanine (0.042 g).
-91-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
1H NMR (400 MHz, CD30D): 8 8.63 (s, 2H), 7.70 (t, 1H); 7.68 (d, 2H); 7.60 (d,
2H); 7.50 (d, 2H), 7.34 (d, 2H), 6.72 (d, 2H); 4.73 (m, 1H); 4.18 (d, 1H);
3.70-3.54
(m, 2H); 3.35-3.20 (m, 2H); 3.05 (dd, 1H); 2.15-2.04 (m, 1H), 2.00-1.88 (m,
1H).
MS: calculated for C32H25C14IN4O6S 860, observed m/e 861 (M + H)+.
EXAMPLE 5 8
N-(N-f (3,5-Dichlorobenzene)sulfonyll-3(S)-(4-carboxyphen 1y )-(L)-~rolyl)-4-f
(3',5'-
dichloroisonicotino,~)aminol-(L)-phenylalanine
Step A N-Trityl-3(S)-h dr~rox -~L)-proline, benzyl ester
A mixture trans-3-hydroxy-(L)-proline (Acros, 5.0 g, 38 mmol), benzyl
alcohol (8.0 rizL, 76 mmol) and pTSA (7.4 g, 38 mmol) in 30 mL of benzene was
refluxed overnight while water was removed using a Dean-Stark apparatus. The
reaction mixture was cooled to rt, and was concentrated in vacLCO to dryness.
The
residue was dissolved in CH2C12 (50 mL) and TEA (32 mL, 0.23 mol) and
chlorotrimethylsilane (19 mL, 0.15 mol) were added. After heating the solution
at 70
°C for 1 h, the reaction mixture was cooled to 0 °C, and MeOH
(3.1 mL, 76 mmol)
was added. After stirring at rt for 1 h, a solution of trityl chloride (16 g,
57 mmol) in
50 mL of CH2C12 followed by TEA (8.0 mL, 57 mmol) were added. After stirring
at
rt for 3 days, the reaction mixture was diluted with 150 mL of EtOAc, and the
precipitates were removed by filtering through celite. The filtrate was
concentrated in
vacuo to dryness, and the residue was stirred with I~2C03 (2.2 g) in MeOH (100
mL)
for 5 h at rt. The reaction mixture was diluted with THF (100 mL), and a
solution of
KF (8.8 g) in 40 mL of water was added. After stirring at rt overnight, the
reaction
mixture was cooled to 0 °C, diluted with EtOAc (100 mL), and
neutralized by careful
addition of 0.5 M aqueous sodium bisulfate. The reaction mixture was
partitioned in
water and EtOAc, and the product was extracted with EtOAc (3x) . The combined
extracts were dried over anhydrous MgS04, filtered and concentrated in vacuo
to
dryness. The residue was purified by flash column chromatography on silica gel
eluted with 4:1 to 1:4 hexane/Et20 to afford N-trityl-3(S)-hydroxy-(L)-
proline, benzyl
ester (8.8 g, 49 %).
1H NMR (400 MHz, CD3OD): b 7.50-7.10 (m, 20 H); 4.98 (ABq, 2H); 4.10 (m, 1H);
3.76 (d, 1H); 3.24 (m, 1H); 2,79 (dd, 1H); 1.95-1.84 (m, 1H), 1.15-1.05 (m,
1H).
Step B N-Trityl-3-keto-(L)-proline, benzyl ester
-92-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
To a solution of N-trityl-3-hydroxy-(L)-proline, benzyl ester (8.8 g, 19
mmol) in CH2C12 (50 mL) was added 4-methylmorpholine N-oxide (4.5 g, 38 mmol)
0
and 3A molecular sieves (3 g). After stirring at rt for 15 min, the reaction
mixture
was cooled to 0 °C, and was added tetrapropylammonium perruthenate (1.0
g, 2.8
mmol). After stirring at rt for 15 min, the reaction mixture was filtered
through a pad
of silica gel, and the filtrate was concentrated isz vacuo to dryness. The
residue was
dissolved in Et20 (100 mL), and was filtered through a pad of silica gel. The
filtrate
was concentrated ira vacuo to dryness to yield N-trityl-3-keto-(L)-proline,
benzyl ester
(6.5 g, 74%).
1H NMR (400 MHz, CD30D): 8 7.60-7.16 (m, 20 H); 5.22 (ABq, 2H); 4.18 (br s,
1H); 3.78 (m; 1H); 3.36 (m, 1H); 1.84 (dd, 1H); 1.74 (ddd, 1H).
Step C N-Trityl-3-trifluoromethanesulfon~y-3 4-dehydro-(L)-proline
benzyl ester
To a solution of N-trityl-3-keto-(L)-proline, benzyl ester (6.5 g, 14
mmol) in THF (30 mL) at -78 °C was added sodium
bis(trimethylsilyl)amide (1.0 M
in THF, 14 mL,, 14 mmol). After stirring at -78 °C for 45 min, a
solution of N-
phenylbis(trifluoromethanesulfonimide) (6.5 g, 18 mmol) in THF (20 mL) was
added,
and the reaction was allowed to warm to -10 °C over 5 h. The reaction
was quenched
with saturated NH4C1 solution (50 mL), and the product was extracted with
EtOAc
(2x50 mL). The combined extracts were dried over anhydrous Na2SO4 and
concentrated in vacuo to dryness. The residue was purified by flash column
chromatography on silica gel eluted with 20:1 hexane/Et20 to yield N-trityl-3-
trifluoromethanesulfonyloxy-3,4-dehydro-(L)-proline, benzyl ester (0.99 g,
12%).
1H NMR (400 MHz, CD30D): S 7.70-6.98 (m, 20 H); 5.54 (br s, 1H); 5.18 (ABq,
ZH); 4.45 (s, 1H); 4.20 (dd, 1H); 3.68 (dd, 1H).
Step D N-f(3,5-Dichlorobenzene)sulfonyll-3(S)-(4-methoxvcarbonvlnhenvl
L - roline
To a solution of N-trityl-3-trifluoromethanesulfonyloxy-3,4-dehydro-
(L)-proline, benzyl ester (0.76 g, 1.3 mmol) in dimethoxyethane (15 mL) was
added
4-carboxyphenyl boronic acid (0.42 g, 2.6 mmol), LiCI (0.27 g, 6.4 mmol),
palladium
tetrakis(triphenylphosphine) (0.15 g, 0.13 mmol) and aqueous Na2C03 (2M, 5.3
mL,
11 mmol). After stirnng at 80 °C overnight, the reaction mixture was
cooled to rt and
-93-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
a solution of aqueous sodium bisulfate was added until pH=4. The reaction
mixture
was then diluted with CH2C12 (20 mL), and was treated with trimethylsilyldiazo-
methane (2 M in hexane) until TLC indicated complete consumption of the
carboxylic
acid intermediate. The resulting mixture was partitioned between water and
EtOAc
and the product was extracted with EtOAc. The combined extracts were dried
over
anhydrous MS04 and concentrated in vacuo to dryness. The residue was purified
by
flash column chromatography on silica gel eluted with 10:1 hexane/Et2O to
yield N-
trityl-3-(4-methoxycarbonylphenyl)-3,4-dehydro-(L)-proline, benzyl ester (0.30
g)
which was used immediately in the next reaction.
Thus, to a solution of N-trityl-3-(4-methoxycarbonylphenyl)-3,4-
dehydro-(L)-proline, benzyl ester (0.30 g) in 5 mL of MeOH was added palladium
hydroxide on carbon (10%, 0.10 g), and the resulting mixture was hydrogenated
at rt
overnight under a balloon filled with hydrogen. The resulting mixture was
concentrated in vacuo to dryness and the residue was suspended in aqueous
Na2C03
(0.13 g in IO mL of water), which was filtered through celite, and the celite
calee was
washed with additional 10 mL of water. To the combined filtrate was added 3,5-
dichlorobenzenesulfonyl chloride (1.2 g, 0.73 mL) and the reaction was stirred
at rt
overnight. The resulting mixture was washed with Et20 (15 mL) and acidified
with 2
M HCI. The product was extracted with EtOAc (2x 15 mL), and the combined
extracts were dried over anhydrous Na2S04, filtered and concentrated ira vacuo
to
dryness to yield N-[(3,5-dichlorobenzene)sulfonyl]-3(S)-(4-
methoxycarbonylphenyl)-
(L)-proline (80 mg).
1H NMR (500 MHz, CD3OD): b 7.94 (d, J=8.5 Hz, 2H); 7.83 (d, J=1.5 Hz, 2H);
7.78 (t, J=1.5 Hz, 1H); 7.39 (d, J=8.5 Hz, 2H), 4.57 (d, J=8.5 Hz, 1H); 3.88
(s, 3H);
3.77 (dd, J=8.5, 8.0 Hz, 1H); 3.71 (m, 1H); 3.48 (m, 1H), 2.65 (m, 1H), 2.23
(m, 1H).
MS: calculated for C19H17C12N06S 457, observed m/e 458 (M + H)+.
Step E N-(N-x(3,5-Dichlorobenzene)sulfon l~S)-(4-methoxycarbonyl-
phenyl)-(L)-prolyl)-fW3',5'-dichloroisonicotinoyI)aminol-(L~
phenylalanine, meth l
A mixture of N-[(3,5-dichlorobenzene)sulfonyl]-3(S)-(4-methoxy-
carbonylphenyl)-(L)-proline (80 mg) and thionyl chloride (0.5 mL) in CH2C12 (1
mL)
was heated to 40 °C for 3 h. After cooling to rt, the reaction mixture
was diluted with
toluene (5 mL) and was concentrated ire vacuo to dryness. To the residue was
added a
-94-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
mixture of 3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester
hydrochloride (75 mg, 0.17 mmol) and TEA (0.061 mL, 0.43 mmol) in CH2C12 (1
mL) at 0 °C. After stirring at 0 °C for 1 h and at rt for 1 h,
the reaction mixture was
loaded onto a flash column chromatography of silica gel and eluted with 1:l
hexane/ethyl acetate to yield N-(N-[(3,5-dichlorobenzene)sulfonyl]-3(S)-(4-
methoxy-
carbonylphenyl)-(L)-prolyl)-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine,
methyl ester (30 mg).
1H NMR (500 MHz, CD3OD): 8 8.62 (s, 2H), 7.86-7.78 (m, 5H); 7.56 (d, J=8.5 Hz,
2H); 7.21 (d, J=8.0 Hz, 2H); 7.18 (d, J=8.5 Hz, 2H), 4.62 (d, J=9.0 Hz, 1H);
4.30 (t,
J=6.5 Hz, 1H); 3.86 (s, 3H); 3.78 (dd, J=9.0, 9.0 Hz, 1H); 3.42 (s, 3H); 3.40-
3.34 (m,
2H), 2.91 (ABq d, 2H); 2.64 (m, 1H), 2.11 (m, 1H).
MS: calculated for C35H30C14N4O8S 806, observed m/e 807 (M + H)+.
Step F N-(N-f(3,5-Dichlorobenzene)sulfonyll-3(S)-(4-carboxyphen. l
prolyl)-f (3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-3(S)-(4-methoxycarbonyl-
phenyl)-(L)-prolyl)-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine,
methyl
ester (30 m g) was treated with LiOH according to the procedure described in
Example 20, Step F to yield N-(N-[(3,5-dichlorobenzene)sulfonyl]-3(S)-(4-
carboxy-
phenyl)-(L)-prolyl)-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine.
1H NMR (500 MHz, CD3OD): b 8.62 (s, 2H), 7.87 (d, J=1.5 Hz, 2H); 7.85 (d,
J=8.5
Hz, 2H); 7.80 (t, J=1.5 Hz, 1H); 7.56 (d, J=8.5 Hz, 2H); 7.24 (d, J=8.S Hz,
2H); 7.20
(d, J=8.5 Hz, 2H), 4.66 (d, J=9.0 Hz, 1H); 4.24 (t, J=5.8 Hz, 1H); 3.81 (dd,
J=8.5, 8.5
Hz, IH); 3.46-3.33 (m, 2H), 2.99 (ABq d, 2H); 2.64 (m, 1H), 2.12 (m, 1H).
MS: calculated for C33H26C14N4O8S 778, observed m/e 779 (M + H)'".
EXAMPLE 59
N-(N-f (3,5-dichlorobenzene)sulfonyll-4(R)-(4-carboxyphen l~prolyl)-4-f (3 5-
dichloroisonicotinoyl)aminol-(L)-phenylalanine
Step A 4-H drox -~L)-proline, methyl ester hydrochloride.
To a solution of 33.0 g (0.25 mol) of 4-hydroxy-(L)-proline and 200
mL of MeOH was added 20 mL (0.27 mol) of thionyl chloride. The reaction was
warmed to reflux for 16 h and then cooled to rt and concentrated zrZ vacuo.
Trituration
- 95 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
with Et20 afforded 44g (97%) of 4-hydroxy-(L)-proline, methyl ester
hydrochloride
as a white solid which was used without further purification.
500 MHz 1H NMR (MeOH): b 4.60 (m, 2H); 3.85 (s, 3H); 3.50 (dd, 1H); 3.35 (m,
1H); 2.41 (m, 1H); 2.22 (m, 1H).
Step B N-Trite-4hydrox -y (L)-proline, methyl ester.
To a solution of 10 g (55 mmol) of 4-hydroxy-(L)-proline, methyl ester
hydrochloride in CH2C12 (150mL) was added 21 mL (276 mmol) of TEA followed by
21 mL (166 mmol) TMSCI. The reaction mixture was refluxed for 1 h, cooled to
0°C,
and treated with 4.5 mL (110 mmol) of MeOH. The reaction was warmed to rt and
stirred for 1 h'. A solution of 18.5g (66 mmol) of trityl chloride and 11 mL
(77 mmol)
of TEA in CH2C12 (30 mL) was added and the reaction was stirred for 18 h. The
mixture was concentrated in vacuo, diluted with EtOAc, filtered through
celite, and
concentrated to give 30a°a g of a pale yellow oil.
To a solution of this residue in MeOH was added I~2C03 (5g) and the
slurry was stirred at rt until completed as assessed by T.L.C. The reaction
was
concentrated irz vacuo to remove the MeOH, redissolved in EtOAc, washed with
brine
(3x), dried over anhydrous MgS04 and concentrated ira vacuo to give 25g of a
pale
yellow oil which slowly crystallized. This crude residue was used without
further
purification.
500 MHz 1H NMR (CDC13): 8 7.600-7.10 (m, 15H); 4.40 (m, 1H); 3.98 (dd, 1H);
3.79 (dd, 1H); 3.60 (s, 3H); 3.08 (s, OH); 2.70 (dd, 1H); 1.95 (dd, 1H); 1.20
(m, 1H).
Step C N-(L)-Trityl-4-oxoproline, methyl ester.
To a mixture of 5g (12.9 mmol) of N-trityl-4-hydroxy-(L)-proline,
methyl ester, 2.3g (19.3 mmol) of NMO, 5g of powdered 3A molecular sieves and
100 mL of CH2C12 at 0°C was added TPAP (~0.3g). The mixture was stirred
at 0°C
for 45 min, at rt for 1h, and then concentrated ira vacuo. The residue was
dissolved in
Et20 and filtered through a pad of silica gel and concentrated in vacuo. The
residue
was purified by flash column chromatography on silica gel eluted with 2:1
hexanes:Et20 to yield 3.5g (70%) of N-(L)-trityl-4-oxoproline, methyl ester as
a
white solid.
500 MHz 1H NMR (CDCl3): b 7.60 (d, 6H); 7.30 (m, 6H); 7.20 (t, 3H); 4.21 (d,
1H);
3.81 (d, 1H); 3.79 (s, 3H); 3.55 (d, 1H); 1.90 (d, 1H); 1.10 (dd, 1H).
-96-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Step D N-Trityl-3,4-dehydro-4-ff(trifluromethyl)sulfon lloxy~-(L)-proline,
meth. l ester.
To a solution of 5.5 g (14.2 mmol) of N-(L)-trityl-4-oxoproline,
methyl ester in 60 mL of THF at-78°C was added a 1M hexane solution of
sodium
hexamethyldisilylamide (17.1 mL) dropwise over 20 min. After 1h at -
78°C, a
solution of 6.6g (18.5 mmol) of N-phenyl trifluromethansulfonamide in THF
(l5mL)
was added. The solution was stirred at -78°C for 2.5 h and then
quenched with
saturated aq. NaHC03 and warmed to rt. The reaction was diluted with Et~O and
the
layers were separated. The aqueous layer was extracted with Et20 (3x) and the
combined organic layers were dried over anhydrous MgS04 and concentrated in
vacuo. The residue was purified by flash column chromatography on silica gel
eluted
with 3:1 hexanes:Et20 to give 5.9g of N-trityl-3,4-dehydro-4-
[[(trifluromethyl)-
sulfonyl]oxy]-(L)-proline, methyl ester as a white foam.
500 MHz 1H NMR (CDCl3): 8 7.62 (d, 6H); 7.30 (m, 6H); 7.21 (t, 3H); 5.33 (br
s,
1H); 4.62 (m, 1H); 4.35 (m, 1H); 3.78 (d, 1H); 3.76 (s, 3H).
Step E N-Trityl-3,4-dehydro-4-(4-carboxyphen 1y )(L)-proline meth,1~.
To a solution of 1.5 g (2.9 mmol) of N-trityl-3,4-dehydro-4-
[[(trifluromethyl)sulfonyl]oxy]-(L)-proline, methyl ester and 0.62 g (14.5
mmol) of
LiCI in 20 mL of DME was added 0.96 g (5.8 mmol) of p-carboxyphenylboronic
acid,
0.33g (0.29mmo1) of tetrakis(triphenylphosphine)palladium(0), and 6.0 mL of a
2M
Na2C03 solution. The reaction was warmed to 80°C for 10 h and then
cooled. The
mixture was diluted with EtOAc and brine and the layers were separated. The
organic
layer was dried over anhydrous MgS04 and concentrated ih vacuo to give a pale
yellow solid. The residue was purified by flash column chromatography on
silica gel
eluted with a stepwise gradient of 3:1 hexanes:Et20, 3:1 Et20:hexanes, 100%
Et20,
100% EtOAc, 10% MeOH in EtOAc to yield 0.90g of N-trityl-3,4-dehydro-4-(4-
carboxyphenyl)-(L)-proline, methyl ester as a pale yellow foam.
500 MHz 1H NMR (d4-MeOH): b 7.62 (m, 2H); 7.60 (d, 6H); 7.25 (m, 6H); 7.11 (m,
3H); 7.05 (d, 2H); 5.76 (s, 1H); 4.79 (s, 1H); 4.43 (m, 1H); 4.05 (d, 1H);
3.70 (s, 3H).
Step F N-Trityl-3,4-dehydro-4-(4-tart-butylcarbox~phenyl)-(L)-proline meth
ester.
-97-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
To a solution of 0.80 g (1.64 mmol) of N-trityl-3,4-dehydro-4-(4-
carboxyphenyl)-(L)-proline, methyl ester in CH2Cl2 was added N,N'-disiopropyl-
O-
tert-butylisourea (0.5mL). After stirring for 12 h, an additional 0.5mL of
N,N'-
disiopropyl-O-tart-butylisourea was added and the reaction was stirred at rt
for an
additional 48 h. The mixture was diluted with Et20 and filtered through a pad
of
silica gel and then concentrated ira vacuo. The residue was purified by flash
column
chromatography on silica gel eluted with 3:1 hexanes:Et20 to yield 0.30g of N-
trityl-
3,4-dehydro-4-(4-tart-butylcarboxyphenyl)-(L)-proline methyl ester as a
colorless
foam, slightly impure by 1H-NMR analysis.
500 MHz 1H NMR (MeOH): 8 7.80 (d, 2H); 7.60 (d, 6H); 7.305 (m, 6H); 7.19 (m,
3H); 7.08 (d, '2H); 5.76 (s, 1H); 4.80 (m, 1H); 4.58 (m, 1H); 4.05 (d, 1H);
3.75 (s,
3H); 1.60 (s, 9H).
Step G 4(R)-(4-tart-butylcarboxyphen l~proline, methyl ester.
A mixture of N-trityl-3,4-dehydro-4-(4-tart-butylcarboxyphenyl)-(L)-
proline methyl ester, 10% Pd/C and EtOH was stirred under latm H2 until trityl
removal was complete as judged by T.L.C. analysis. The reaction was filtered
through a pad of celite and concentrated i~2 vacuo. The residue was purified
by flash
column chromatography on silica gel eluted with a stepwise gradient of 3:1
hexanes:Et20, 3:1 Et20:hexanes, 100% Et20, 100% EtOAc, 10% MeOH in EtOAc
to yield 4(R)-(4-tart-butylcarboxyphenyl)-(L)-proline, methyl ester as a near
colorless
oil.
500 MHz 1H NMR (MeOH): 8 7.85 (d, 2H); 7.37 (d, 2H); 4.05 (t, 1H); 3.79 (s,
3H);
3.40 (m, 2H); 3.0 (t, 1H); 2.62 (m, 1H); 1.95 (m, 1H).
Step H N-f (3 5-Dichlorobenzene)sulfonyll-4(R)-(4-ter-t-butylcarboxyphenyl)-
(L~proline, meth. l ester.
To a solution of 40 mg (0.123 mmol) of 4(R)-(4-tart-butylcarboxy-
phenyl)-(L)-proline, methyl ester and 46 mg (0.26 mmol) of 3,5-dichlorobenzene-
sulfonyl chloride in CH2C12 (3mL) was added 0.07 mL (0.39 mmol) of DIPEA.
After stirring at rt for 15 h, Triamine-3 scavenging resin (Silicycle) was
added and
stirring was continued for an additional 2 h. The reaction was filtered and
concentrated ira vacuo. The residue was purified by flash column
chromatography on
silica gel eluted with a stepwise gradient of 3:1 hexanes:Et20, 3:1
Et20:hexanes,
-98-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
100% Et20 to afford N-[(3,5-dichlorobenzene)sulfonyl]-4(R)-(4-tert-
butylcarboxyphenyl)-(L)-proline, methyl ester (50 mg).
Step I N-f(3,5-Dichlorobenzene)sulfonyll-4(R)-(4-tent-butylcarboxyphenyl)-
L - roline.
N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-(4-tart-butylcarboxyphenyl)-
(L)-proline, methyl ester (50 mg, 0.097 mmol) was treated with 1M LiOH in THF
according to the procedure described in Example 20, Step D to afford N-[(3,5-
dichlorobenzene)sulfonyl]-4(R)-(4-tart-butylcarboxyphenyl)-(L)-proline (47
mg).
Step J ° N-(N-f(3,5-dichlorobenzene)sulfon l~R)-(4-tart-
butylcarbox~
phen l~prolyl)-4-f(3,5-dichloroisonicotinoyl)aminol-(L)-
phenylalanine, meth 1~.
N-((3,5-Dichlorobenzene)sulfonyl]-4(R)-(4-tent-butylcarboxyphenyl)-
(L)-proline (47 mg, 0.1 mmol) was coupled to 3,5-(dichloroisonicotinoyl) amino-
(L)-
phenylalanine, methyl ester hydrochloride (51 mg (0.13 mmol), in the presene
of
HATU (44 mg, 1.2 mmol), HOAt (20 mg, 0.15 mmol), and DIPEA (31 mg, 0.24
mmol) according to the procedure described in Example 8, Step D. The crude
product
was purified by flash column chromatography on silica gel eluted with a
stepwise
gradient of 3:1 hexanes:Et20, 3:1 Et20:hexanes, 100% Et20, 100% EtOAc to
afford
N-(N-[(3,5-dichlorobenzene)sulfonyl]-4(R)-(4-tart-butylcarboxyphenyl)-(L)-
prolyl)-
4-((3,5-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester 0100 mg)
as a
colorless oil.
Step K N-(N-f(3,5-dichlorobenzene)sulfonyll- 4(R)-(4-tart-butylcarboxy-
phen 1~L)-prolyl)-4-f(3,5-dichloroisonicotino,~~l)aminol-(L~
phenylalanine.
To a solution of N-(N-[(3,5-dichlorobenzene)sulfonyl]-4(R)-(4-tert-
butylcarboxyphenyl)-(L)-prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-
phenylalanine, methyl ester (100 mg) in 3 mL of THF at 0°C was added 3
mL of 1M
LiOH. The reaction was stirred at 0°C for 2 h and was then warmed to
rt, diluted with
EtOAc and acidified with 2M HCl until pH ~4. The layers were separated and the
aqueous layer was extracted with EtOAc (3x). The combined organic layers were
dried over anhydrous MgSO4 and concentrated i12 vacuo to yield N-(N-[(3,5-
dichloro-
-99-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
benzene)sulfonyl]-4(R)-(4-tart-butylcarboxyphenyl)-(L)-prolyl)-4-[(3,5-
dichloro-
isonicotinoyl)amino]-(L)-phenylalanine (70 mg) as a colorless foam.
HPLC:MS 835.1 (M+H).
Step L N-(N-[(3,5-dichlorobenzene)sulfonyll- 4(R)-(4-carboxyphenyl)-(L)-
prolyl)-4- f (3,5-dichloroisonicotinoyl)aminol-(L)-phenylalanine.
To a solution of N-(N-[(3,5-dichlorobenzene)sulfonyl]-4(R)-(4-tert-
butylcarboxyphenyl)-(L)-prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-
phenylalanine (70 mg) in 2 mL of CH2C12 at 0°C was added 2 mL of TFA.
The
reaction was stirred at 0°C for 1 h and then warmed to rt. When the
reaction was
done as judged by reverse-phase HPLC, the reaction was concentrated in vacuo.
Trituration with Et20 afforded N-(N-[(3,5-dichlorobenzene)sulfonyl]-4(R)-(4-
carboxyphenyl)-(L)-prolyl)-4-[(3,5-dichloroisonicotinoyl)amino]-(L)-
phenylalanine as
an off white solid.
HPLC:MS 779.1 (M+H).
EXAMPLE 60
N-(N-f(3,5-dichlorobenzene)sulfonyll-4(R)-tart-but~ycarboxy-(L~prol l
f (3' ,5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine
Step A N-Trityl-3,4-dehydro-4-carboxy-(L)-proline meth 1 ester.
Anhydrous CO gas was bubbled through a mixture of of N-trityl-3,4-
dehydro-4-[[(trifluromethyl)sulfonyl]oxy]-(L)-proline, methyl ester from
Example 59,
Step D (1.5 g, 2.9 mmol), KOAc (1.l g, 11.6 mmol), Ph3P (0.158, 0.58 mmol),
and
Pd(OAc)2 (0.068, 0.29 mmol) in DMF (20 mL) for 25 min. The reaction was then
warmed to 50°C under a balloon of CO for 18 h. The reaction was cooled,
diluted
with EtOAc, brine and the layers were separated. The aqueous layer was
extracted
with EtOAc (3x). The combined organic layers were washed with brine (5x),
dried
over anhydrous MgS04 and concentrated in vacuo. The residue was purified by
flash
column chromatography on silica gel eluted with a stepwise gradient of 3:1
hexanes:Et20, 3:1 Et20:hexanes, 100% Et20, 100% EtOAc, 10% MeOH in EtOAc
to yield N-trityl-3,4-dehydro-4-carboxy-(L)-proline, methyl ester (0.60g).
500 MHz 1H NMR (CDCl3): 8 7.40-7.10 (m, 15H); 6.10 (s, 1H); 4.80 (br s, 1H);
4.30
(m, 1H); 3.82 (d, 1H); 3.70 (s, 3H).
- 100 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Step B N-(N-f(3,5-dichlorobenzene)sulfonyll-4(R)-text-butxlox c~xy-(L~
prolyl)-4-[(3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine.
N-Trityl-3,4-dehydro-4-carboxy-(L)-proline, methyl ester (450 mg, 1.1
mmol) was converted to N-(N-[(3,5-dichlorobenzene)sulfonyl]-4(R)-ter-t-butyl-
carboxy-(L)-prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine
by the
procedures described in Example 59, Steps F-K. The product was isolated as a
white
solid.
HPLC:MS 759.7 (M+H).
EXAMPLE 61
N-(N-f (3,5-Dichlorobenzene)sulfonyll-4(R)-phen~L)-prolyl)-4-f (3' 5'-dichloro-
isonicotinoyl)aminol-(L)-phen~alanine
Step A N-BOC-3,4-Dehydro-4-phenyl-(L)-proline, meth,1~.
To a solution of 600 mg (1.6 mmol) of N-BOC-3,4-dehydro-4-
[[(trifluromethyl)sulfonyl]oxy]-(L)-proline, methyl ester (W. Lubell et al.
Tet. Lett.
(1998), 39(12), 1595-8), 336 mg (8.0 mmol) of LiCI in 20 mL of DME was added
390
mg (3.2 mmol) of phenylboronic acid, 184 mg (0.16mmo1) of Pd(Ph3P)4, and 5.0
mL
of a 2M Na2C03 solution. The reaction was warmed to 80°C for 8 h and
then
cooled. The mixture was diluted with EtOAc and brine and the layers were
separated.
The aqueous layer was extracted with EtOAc (3x). The combined organic layers
were
dried over MgSO4 and concentrated ira vacuo. The residue was purified by flash
column chromatography on silica gel eluted withl:l hexanes:Et20 to yield N-BOC-
3,4-dehydro-4-phenyl-(L)-proline, methyl ester as a pale yellow oil which
crystallized.
Step B N-BOC-4(R)-phen 1-y (L)-proline, meth 1 ester.
A mixture of 260 mg (0.86 mmol) of N-BOC-3,4-dehydro-4-phenyl-
(L)-proline, methyl ester, 10% Pd/C and EtOH was stirred under latm H2 for 24
h.
The reaction was filtered through a pad of celite and concentrated in vacuo.
The
crude product was used in the subsequent reaction without further
purification.
500 MHz 1H NMR (CDC13): 8 7.35 (m, 2H); 7.28 (m, 3H); 4.40 (m, 1H); 4.05 (m,
1H); 3.78 (s, 3H); 3.45-3.35 (m, 2H); 2.67 (m, 1H); 2.10 (m, 1H); 1.44 (s,
9H).
Step C N-f(3 5-dichlorobenzene~sulfonyll-4(R)-phenyl-(L)-proline meth
ester.
- 101 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
To a solution of 62mg of N-BOC-4(R)-phenyl-(L)-proline, methyl
ester in 2 mL of CH2C12 at 0°C was added 2 mL of TFA. The reaction was
stirred at
0°C for 1 h and was then warmed to rt. When the reaction was done as
assessed by
T.L.C. analysis, the reaction was concentrated irz vacuo.
To a solution of this crude residue in CH2C12 (4 mL) at 0°C was
added
0.18 mL (1 mmol) of DIPEA followed by 72 mg (0.41 mmol) of 3,5-dichloro-
benzene)sulfonyl chloride. The reaction was allowed to warm to rt overnight.
After
16 h, the reaction was diluted with EtOAc and washed with 1M HCl (3x), brine
(lx),
dried over anhydrous MgS04 and concentrated i~z vacuo to yield N-[(3,5-
dichloro-
benzene)sulfonyl]-4(R)-phenyl-(L)-proline, methyl ester as a pale yellow oil
which
was used without further purification.
Step D N-(N-f(3,5-dichlorobenzene)sulfonyll-4(R)-phen 1-~prol 1y )4-
f (3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine.
N-[(3,5-Dichlorobenzene)sulfonyl]-4(R)-phenyl-(L)-proline, methyl
ester (0.20mmo1) was reacted according to the procedures described in Example
59,
Steps I-K to yield N-(N-[(3,5-dichlorobenzene)sulfonyl]-4(R)-phenyl-(L)-
prolyl)-4-
[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine as a white solid.
HPLC:MS 735.2 (M+H).
EXAMPLE 62
N-(N-f(3,5-dichlorobenzene)sulfonyll-4(R)-(4-p~ l~prolyl)-4-f(3',5'-dichloro-
isonicotinoyl)aminol-(L)-phenylalaninc.
N-BOC-3,4-Dehydro-4-[[(trifluromethyl)sulfonyl]oxy]-(L)-proline,
methyl ester from Example 61, Step A (440 mg, 1.2 mmol) was reacted according
to
the procedures described in Example 61, Steps A-D substituting 4-
pyridylboronic acid
for phenylboronic acid in Step A to afford N-(N-[(3,5-
dichlorobenzene)sulfonyl]-
4(R)-(4-pyridyl)-(L)-prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine.
HPLC:MS 736.1 (M+H).
EXAMPLE 63
N-(N-f(3,5-dichlorobenzene)sulfonyll-3(R)-(allylox carbonylamino)-2-meth 1-
prolyl)-4-f(3' S'-dichloroisonicotinoyl)aminol-(L)-phenylalanine
- 102 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
Step A N-f(3,5-dichlorobenzene)sulfonyll-3(R)-(all.. c~nylamino)-2-
methyl-(L)-proline, text-but luster.
To a solution of 1.0 g (2.6 mmol) of N-[(3,5-dichlorobenzene)-
sulfonyl]-2-methyl-3(R)-carboxy-(L)-proline, tent-butyl ester (obtained from
LiOH
hydrolysis of the methyl ester prepared in Example 16, Step E) and 1.4 mL
(10.3
mmol) of TEA in THF (10 mL) at 0°C was added 0.74 mL (7.7 mmol) of
C1C02Et.
The reaction mixture was allowed to warm to rt, stirred for an additional
0.5h, and
then re-cooled to 0°C and treated with 0.84 g (12.9 mmol) of NaN3 in
water. The
reaction was allowed to warm to rt over 75min and then was diluted with EtOAc
and
brine. The layers were separated and the aqueous layer was extracted with
EtOAc
(3x). The combined organic layers were dried over anhydrous MgS04 and
concentrated in vacuo to give an oil which crystallized.
To a solution of this crude residue in toluene (10 mL) was added allyl
alcohol (IOmL) and the reaction was warmed to reflux overnight. After 16 h,
the
reaction was concentrated in vacuo to afford N-[(3,5-dichlorobenzene)sulfonyl]-
3(R)-
(allyloxycarbonylamino)-2-methyl-(L)-proline, tert-butyl ester as a pale
yellow oil
which crystalized. The crude residue was used without further purification.
500 MHz 1H NMR (CDC13): 8 7.80 (s, 2H); 7.58 (s, 1H); 5.90 (rn, 1H); 5.30 (d,
1H);
5.18 (d, 1H); 4.85 (d, 1H); 4.55 (br s, 2H); 3.40 (m, 2H); 2.24 (m, 1H); 1.80
(m, 1H);
1.55 (s, 3H); 1.75 (s, 9H).
Step B N-(N-x(3,5-Dichlorobenzene)sulfon 1v 13(R)-(allyloxycarbonyl-amino)-
2-meth 1-~prol 1y )-4-f(3',5'-dichloroisonicotinoyl)anninol-(L)-
phenylalanine.
To a solution of 515 mg (1.1 mmol) of N-[(3,5-dichlorobenzene)-
sulfonyl]-3(R)-(allyloxycarbonylamino)-2-methyl-(L)-proline, tert-butyl ester
in 5 mL
of CH2C12 at 0°C was added 5 mL of TFA. The reaction was stirred at
0°C for 1 h
and was then warmed to rt. When the reaction was done as judged by T.L.C.
analysis,
the reaction was concentrated iu vacuo. The crude acid was reacted according
to the
procedures described in Example 59, Steps J and K to yield N-(N-[(3,5-dichloro-
benzene)sulfonyl]-3(R)-(allyloxycarbonyl-amino)-2-methyl-(L)-prolyl)-4-[(3',5'-
dichloroisonicotinoyl)amino]-(L)-phenylalanine as a light tan solid.
HPLC:MS 772.1 (M+H).
-103-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
EXAMPLE 64
N-(N-f (3,5-Dichlorobenzene)sulfonyll-3(R)-(N,N-dimethylamino)-2-meth,1-
prop)-4-f (3' ,5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine.
Step A N-(N-f(3,5-dichlorobenzene)sulfon l~R)-amino-2-meth~L)-
prolyl)-4-f (3' ,5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine.
To a solution of 650 mg (0.83 mmol) of N-(N-[(3,5-dichlorobenzene)-
sulfonyl]-3 (R)-(allyloxycarbonyl-amino)-2-methyl-(L)-prolyl)-4-[(3',5'-
dichloro-
isonicotinoyl)amino]-(L)-phenylalanine, methyl ester from Example 63, Step B
(prior
to final LiOH hydrolysis) and Pd(Ph3P)4 (~20 mg) in 5 mL of THF was added 0.20
mL (1.6 mrnol) of PhSiH3. After stirring for ~90 min, 0.20 mL of water was
added
and the reaction was concentrated if2 vacuo to afford N-(N-[(3,5-
dichlorobenzene)-
sulfonyl]-3(R)-amino-2-methyl-(L)-prolyl)-4-[(3',5'-
dichloroisonicotinoyl)amino]-
(L)-phenylalanine, methyl estex which was used in subsequent reaction without
further
purification.
N-(N-[(3,5-Dichlorobenzene)sulfonyl]-3(R)-amino-2-methyl-(L)-
prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, methyl ester
was
hydrolyzed in THF with 1M LiOH according to the procedure described in Example
20, Step F to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-3(R)-amino-2-methyl-
(L)-
prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine.
HPLC:MS 688.1 (M+H).
Step B N-(N-f(3,5-dichlorobenzene)sulfonyll-3(R)-(N N-dimethylamino)-2-
meth~L)-prolyl)-4-f (3',5'-dichloroisonicotinoyl)aminol-(L)-
phenylalanine.
To a solution of 100 mg (0.15 mmol) of N-(N-[(3,5-dichlorobenzene)-
sulfonyl]-3(R)-amino-2-methyl-(L)-prolyl)-4-[(3',5'-
dichloroisonicotinoyl)amino]-
(L)-phenylalanine in CH3CN (1mL) was added 1 mL aqueous HCHO (37% in H20)
followed by 154 mg (0.73 mmol) of NaBH(OAc)3. The mixture was stirred at rt
for
40 h and was then diluted with EtOAc and 1M NaOH and stirred 10 min. The
aqueous layer was acidified with 2M HCl and the layers were separated. The
aqueous
phase extracted with EtOAc (3x) and the combined organic layers were dried
over
anhydrous MgSO4 and concentrated. The crude residue was purified by
preparative
reverse-phase-HPLC to yield N-(N-[(3,5-dichlorobenzene)sulfonyl]-3(R)-(N,N-
- 104 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
dimethylamino)-2-methyl-(L)-prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-
phenylalanine as a white solid. HPLC:MS 716.2 (M+H).
EXAMPLE 65
N-(N-f(3,5-Dichlorobenzene)sulfon 1v 13-phenyl-3-carboxy-2-meth ~~l-prol 1y )4-
f (3',5'-dichloroisonicotino~)aminol-(L)-phenylalanine
Step A 2-Amino-3-phenyl-succinic acid, dimethyl ester hydrochloride.
To a solution of 13 g (0.053 mol) of 2-amino-3-phenyl-succinic acid
(J. Med Cher~z. (1973), 1277) in MeOH (150 mL) was added 20 mL of thionyl
chloride. The solution was refluxed for 21 h, cooled, and concentrated in
vacuo. The
crude residue~was azeotroped with toluene to yield 2-amino-3-phenyl-succinic
acid,
dimethyl ester hydrochloride as a white solid which was used without further
purification. The product was a 1:1 mixture of diastereomers as judged by 1H-
NMR
analysis.
Step B 2-(N-trityl-amino)-3-phenyl-succinic acid, dimeth, lester.
To a solution of 5 g (18.5 mmol) of 2-amino-3-phenyl-succinic acid,
dimethyl ester hydrochloride in CH2C12 (100 mL) was added 7.7 mL (56 mmol) of
TEA followed by 6.2g (22.1 mmol) of trityl chloride. The reaction was stirred
at rt
for 2 days and then concentrated ica vacaco. Et20 was added to the crude
residue and
the mixture was filtered through a pad of silica gel and concentrated ice
vacuo to give a
colorless oil. The residue was purified by flash column chromatography on
silica gel
eluted with 3:1 hexanes:Et20 to yield 6g of 2-(N-tritylamino)-3-phenyl-
succinic acid,
dimethyl ester as a white solid. The product was a 1.3:1 mixture of
diastereomers as
judged by 1H-NMR analysis.
500 MHz 1H NMR (CDC13): 8 7.5-7.1 (m, 40H); 4.15 (dd, 1H); 3.85 (dd, 1H); 3.80
(s, 3H); 3.67 (s, 3H); 3.15 (s, 3H); 2.90 (d, 1H); 2.80 (s, 3H); 2.68 (d, 1H).
Step C 2-(N-Trityl-amino)-3-carbox~~henyl-hex-5-enoic acid, dimeth~
ester.
To a solution of 4.0 g (8.4 mmol) of 2-(N-trityl-amino)-3-phenyl-
succinic acid, dimethyl ester in THF (40 mL) at -78°C was added 40.0 mL
(20 mmol)
of KPM~S (0.5M in toluene) over 15 minutes. Once the addition was complete,
the
resulting enolate was stirred at-78°C for 30 minutes. 2.2mL (25.1 mmol)
of allyl
-105-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
bromide (neat) was added and the reaction was stirred at -78°C for 0.5
h and then at
-30°C for 23 h. The reaction was quenched with sat. aqueous NH4Cl
solution and
diluted with EtOAc. The layers were separated and the aqueous layer was
extracted
with EtOAc (2x). The combined organic layers were dried over anhydrous MgS04
and concentrated. This residue was purified by flash column chromatography on
silica gel eluted with 1:1 hexanes:Et20 to yield 3.8g of 2-(N-trityl-amino)-3-
carboxy-
3-phenyl-hex-5-enoic acid, dimethyl ester as a 7:1 mixture of diastereomers as
judged
by H NMR analysis.
(Major diastereomer) 500 MHz 1H NMR (CDC13): 8 7.5-7.1 (m, 20H); 5.35 (m, 1H);
4.85 (m, 2H); 4.05 (d, 1H); 3.79 (s, 3H); 3.35 (d, 1H); 3.05 (s, 3H); 2.80 (m,
2H).
Step D 2-(N-Benzyloxycarbonyl-amino)-3-carboxy-3-phenyl-hex-5-enoic
acid, dimethyl ester.
To a solution of 3.7 g (7.1 mmol) of 2-(N-trityl-amino)-3-carboxy-3-
phenyl-hex-5-enoic acid, dimethyl ester in MeOH (100 mL) at 0°C was
added 3.0 mL
of thionyl chloride. The reaction was allowed to warm to rt. When the starting
material was consumed as judged by T.L.C. analysis, the reaction was
concentrated in
vacuo and azeotroped with toluene (2x). To a solution of this crude residue in
CH2C12 (50 mL) at 0°C was added 1.7 mL (21.4 mmol) of pyridine followed
by 1.6
mL (10.7 mmol) of CbzCl. The reaction was allowed to warm to rt overnight and
after 20 h was concentrated i~z vacuo. The residue was redissolved in EtOAc
and
washed with 1M HCl (3x), dried over anhydrous MgS04 and concentrated in vacuo.
This residue was purified by flash column chromatography on silica gel eluted
with
1:1 hexanes:Et20 to yield 2-(N-benzyloxycarbonyl-amino)-3-carboxy-3-phenyl-hex-
5-enoic acid, dimethyl ester as a 7:1 mixture of diastereomers as judged by 1H
NMR
analysis.
Step E N-x(3,5-dichlorobenzene)sulfon l~phenyl-3-methox. cad,
methyl-proline, meth 1 ester.
A stream of 03 was bubbled through a solution of 1.0 g (2.4 mmol) of
2-(N-benzyloxycarbonyl-amino)-3-carboxy-3-phenyl-hex-5-enoic acid, dimethyl
ester
and 0.15g (2.4 mmol) of HOAc in CH2C12/MeOH (1:1 v/v, 10 mL) at -78°C
until the
solution turned blue. Excess ozone was purged with a stream on 02 until the
reaction
was colorless. Excess dimethylsulfide (2mL) was added and the reaction was
allowed
- 106 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
to warm to rt and stir for 60 h. The reaction was diluted with EtOAc and
washed with
NaHC03 (3x), dried over anhydrous MgS04 and concentrated in vacuo.
A mixture of this crude residue, 10%a Pd/C, and MeOH (5 mL,) was
stirred under 1 atm of H2 for 18 h. The mixture was then filtered through a
pad of
celite and concentrated to give 700mg of the amine as a 10:1 mixture of
diastereomers
i
as judged by H NMR analysis.
To a 0°C solution of this crude material and 1.5 mL (8.5 mmol) of
DIPEA in CH2C12 (5 mL) was added 1.0 g (5.7 mmol) of 3,5-dichlorobenzene-
sulfonyl chloride. The reaction was allowed to warm to rt overnight. After 16
h, the
reaction was diluted with EtOAc and washed with 1M HCl (3x), dried over
anhydrous
MgS04 and concentrated ifa vacuo. This residue was purified by flash column
chromatography on silica gel eluted with a stepwise gradient of 3:1
hexanes:Et20
then 1:1 hexanes:Et2O then 3:1 Et2O:hexanes to give a pale yellow solid.
Trituration
with hexanes afforded 750 mg of N-[(3,5-dichlorobenzene)sulfonyl]-3-phenyl-3-
methoxycarbonyl-2-methyl-proline, methyl ester as a white solid as a >15:1
mixture
i
of diastereomers as judged by H NMR analysis.
500 MHz 1H NMR (CDCl3): ~ 7.78 (s, 2H); 7.60 (s, 1H); 7.30 (m, 5H); 5.25 (s,
1H);
3.85 (t, 1H); 3.55 (s, 3H); 3.25 (m, 1H); 3.24 (s, 3H); 3.0 (m, 1H); 2.82 (dd,
1H).
Step F N-(N-f(3,5-dichlorobenzene)sulfon l~phenyl-3-carboxy-2-methyl-
prolyl)-4-f (3',5'-dichloroisonicotinoyl)aminol-(L~phenylalanine.
105 mg (0.22 mmol) of N-[(3,5-dichlorobenzene)sulfonyl]-3-phenyl-3-
methoxycarbonyl-2-methyl-proline, methyl ester in MeOH (2 mL), THF (2 mL), and
1M NaOH was stirred vigorously at 80°C overnight. The reaction was
cooled, diluted
with EtOAc and acidified with 2M HCI. The layers were separated and the
aqueous
layer was extracted with EtOAc (3x). The combined organic layers were dried
over
anhydrous MgS04 and concentrated in vacuo. The crude N-[(3,5-dichlorobenzene)-
sulfonyl]-3-phenyl-3-carboxy-2-methyl-proline was used without further
purification.
500 MHz 1H NMR (MeOH): 8 7.80 (s, 2H); 7.76 (s, 1H); 7.40-7.20 (m, 5H); 5.18
(s,
1H); 3.80 (m, 1H); 3.25 (m, 1H); 2.94 (m, 1H); 2.80 (m, 1H).
The N-[(3,5-dichlorobenzene)sulfonyl]-3-phenyl-3-carboxy-2-methyl-
proline was reacted according to the procedures described in Example 59, Steps
J and
K to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-3-phenyl-3-carboxy-2-methyl-
prolyl)-4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine as a white
solid.
- 107 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
HPLC:MS 779.0 (M+H).
EXAMPLES 66-67
N-(N-f(3,5-dichlorobenzene)sulfonyll-4-methyl-4-carbox~prol, 1y )4-f~3',5'-
dichloro-
isonicotinoyl)aminol-(L)-phenylalanine
Step A N-Benzyl-4-Methoxycarbonyl-4-methyl-proline, eth~ester (A) and N-
Benzyl-3-Methoxycarbonyl-3-methyl-proline, ethyl ester (B).
A mixture of 500 mg (2.6 mmol) of N-benzyl glycine, ethyl ester, 388
mg (13 mmol) of paraformaldehyde, 390 mg (3.9 mmol) of 2-methylmethacrylate,
357 mg (2.6 mmol) of K2CO3 and 10 mL of toluene was heated to 180°C in
a sealed
tube for 14 h 'and then cooled. The reaction was diluted with EtOAc and brine
and the
layers were separated. The organic layer was dried over anhydrous MgS04 and
concentrated ira vacLCO. This residue was purified by flash column
chromatography on
silica gel eluted with a stepwise gradient of 3:1 hexanes:Et20 then 1:1
hexanes:Et20
then 3:1 Et2O:hexanes then Et20 to afford 325 mg of a pale yellow oil which
was a
3:1 mixture of N-benzyl-4-methoxycarbonyl-4-methyl-proline, ethyl ester (A)
and N-
benzyl-3-methoxycarbonyl-3-methyl-proline, ethyl ester (B) as judged by 1H NMR
analysis. HPLC:MS 306.2 (M+H).
Step B 4-Methox~arbonyl-4-meth ~~1-proline, ethyl ester (A) and 3-Methox
carbonyl-3-meth ~~1-proline, ethyl ester (B).
A mixture of 325 mg (1.1 mmol) of N-benzyl-4-methoxycarbonyl-4-
methyl-proline, ethyl ester (A) and N-benzyl-3-methoxycarbonyl-3-methyl-
proline,
ethyl ester (B), 10% Pd/C and MeOH was stirred under latm H2 for 17 h. The
reaction was filtered through celite and concentrated ira vacuo to give 240 mg
of a
colorless oil which was a ~4:1 mixture of 4-methoxycarbonyl-4-methyl-proline,
ethyl
ester (A) and 3-methoxycarbonyl-3-methyl-proline, ethyl ester (B) as judged by
1H-
NMR analysis. The crude mixture was used without further purification.
Step C N-f(3,5-dichlorobenzene)sulfonyll-4- methoxycarbonyl-4-methyl-
proline, ethyl ester (A) and N-f(3,5-dichlorobenzene)sulfon, l
methoxycarbonyl -3-methyl-proline, ethyl ester (B).
A mixture of 230 mg (1.07 mmol) of 4-methoxycarbonyl-4-methyl-
proline, ethyl ester (A) and 3-methoxycarbonyl-3-methyl-proline, ethyl ester
(B) were
- 108 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
reacted with N-(3,5-dichlorobenzene sulfonyl chloride according to the
procedure
described in Example 20, Step B to afford 310 mg of N-[(3,5-dichlorobenzene)-
sulfonyl]-4- methoxycarbonyl -4-methyl-proline, ethyl ester (A) and N-[(3,5-
dichloro-
benzene)sulfonyl]-3- methoxycarbonyl -3-methyl-proline, ethyl ester (B) as a
2.5:1
mixture of compounds as judged by 1H NMR analysis.
Step D N-f (3,5-dichlorobenzene)sulfonyll-4-carboxy-4-meth,~~l-proline (A) and
N-f(3,5-dichlorobenzene)sulfonyll-3-carboxy-3-methyl-proline (B)
A mixture of 315 mg (0.74 mmol) of N-[(3,5-dichlorobenzene)-
sulfonyl]-4- methoxycarbonyl -4-methyl-proline, ethyl ester (A) and N-[(3,5-
dichloro-
benzene)sulfonyl]-3- methoxycarbonyl -3-methyl-proline, ethyl ester (B) were
hydrolyzed with LiOH acording to the procedure described in Example 20, Step D
to
yield 300 mg of the title compounds as a ~3:1 mixture of compounds as judged
by
H-NMR analysis. The mixture was purified by preparative reverse-phase HPLC to
give 63 mg of a white solid which was assigned as N-[(3,5-dichlorobenzene)-
sulfonyl]-4-carboxy-4-methyl-proline (A) and 225 mg of a colorless oil
assigned as N-
[(3,5-dichlorobenzene)sulfonyl]-3-carboxy-3-methyl-proline (B) as judged by 1H-
NMR and HPLC/MS analysis. Compound A was a single diastereomer and
compound B was an 1 1:l mixture of diastereomers.
Regioisomer A: 500 MHz 1H NMR (CDCl3): 8 7.80 (s, 2H); 7.60 (s, 1H); 4.44 (t,
1H); 4.0 (d, 1H); 3.40 (d, 1H); 2.95 (m, 1H); 2.02 (m, 1H); 1.42 (s, 3H).
Regioisomer B: 500 MHz 1H NMR (CDCl3): b 7.75 (s, 2H); 7.58 (s, 1H); 4.70 (s,
1H); 4.25 (m, 2H); 3.67 (t, 1H); 3.30 (m, 1H); 2.45 (m, 1H); 2.15 (m, 1H);
1.35 (t,
3H).
Step E N-(N-f(3,5-dichlorobenzene)sulfonyll-4-methyl-4-carboxy-prol,1
[(3' ,5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine.
N-[(3,5-Dichlorobenzene)sulfonyl]-4-carboxy-4-methyl-proline (A)
(63 mg) was reacted according to the procedures described in Example 59, Steps
J and
K to yield N-(N-[(3,5-dichlorobenzene)sulfonyl]-4-methyl-4-carboxy-prolyl)-4-
[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine as two individual
diastereomers, both as white solids.
HPLC:MS 717.0 (M+H).
-109-
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
EXAMPLE 68
N-(N-f (3.5-dichlorobenzene)sulfonvll-4(R)-cvclohexvl-(L)-prolvl)-4-f (3,5-
dichloro-
isonicotinoyl)aminol-(L)-phenylalanine
Step A N-BOC-4(R)-c~lohex.1-~proline, meth. l~.
A mixture of N-BOC-4(R)-phenyl-(L)-proline, methyl ester from
Example 61, Step B (0.165 g, 0.54 mmol), PtO2 (0.1g, 0.44mmol) and MeOH was
shaken under 50 psi of H2 overnight. The reaction was filtered through a pad
of celite
and concentrated iT2 vacuo to afford N-BOC-4(R)-cyclohexyl-(L)-proline, methyl
ester
which was used without further purification..
Step B ~ N-(N-f (3,5-dichlorobenzene)sulfonyll-4(R)-cyclohex.1-~prol,rl)=4
L(3,5-dichloroisonicotinoyl)aminol-(L)-phenylalanine.
N-BOC-4(R)-cyclohexyl-(L)-proline, methyl ester (0.16 g, 0.54 mmol)
was reacted according to the procedures described in Example 61, Steps C and D
to
yield N-(N-[(3,5-dichlorobenzene)sulfonyl]-4(R)-cyclohexyl-(L)-prolyl)-4-[(3,5-
dichloroisonicotinoyl)amino]-(L)-phenylalanine as a solid.
MS mle 743.2 (M+).
EXAMPLE 69
N-(N-f(3,5-dichlorobenzene)sulfonyll-3(S)-(allylox cad rbonyl-amino)-2-meth 1-
prolyl)-4-f (3' ,5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine
Step A N-f (3,5-dichlorobenzene)sulfon, l~~ )-methoxycarbonyl-2-methyl-
(L)-proline, tart-butyl ester.
N-[(3,5-dichlorobenzene)sulfonyl]-3(S)-methoxycarbonyl-2-methyl-
(L)-proline, tart-butyl ester was prepared according to the procedures
described in
Example 63, Step A for the 3(R) isomer but substituting dimethyl maleate for
dimethyl fumarate in Example 16, Step B.
Step B N-(N-f(3,5-dichlorobenzene)sulfon.1~~1-3(S)-(allylox, ca~xl-amino)-
2-methyl-(L)-prolyl)-4- f (3' ,5'-dichloroisonicotinoyl)aminol-(L)-
phenylalanine.
N-[(3,5-Dichlorobenzene)sulfonyl]-3(S)-methoxycarbonyl-2-methyl-
(L)-proline, tart-butyl ester (1.63g, 3.6 mmol) was reacted according to the
procedures
described in Example 63, Steps A and B for the 4(R) isomer to afford N-(N-
[(3,5-
- 110 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
dichlorobenzene)sulfonyl]-3 (S)-(allyloxycarbonyl-amino)-2-methyl-(L)-prolyl)-
4-
[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine as a solid. MS rf2/e
772.5
(M+).
EXAMPLE 70N-(N-f(3,5-dichlorobenzene)sulfonyll-3 3 -diall 1-4-oxo- L)-prol,1
f (3' ,5'-dichloroisonicotino~)aminol-(L)-phenylalanine
Step A N-(BOC)-3,3-diallyl-4-oxo-(L)-proline meth" 1 ester
To a solution of N-(BOC)-4-oxo-(L)-proline, methyl ester (0.197 g) in
3 mL of THF and 1 mL of DMPU was added 0.28 mL of allyl bromide. The reaction
mixture was cooled to -78°C and 2.0 mL of LiHMDS was dropwise
introduced into
the reaction. 'The mixture was stirred at -78°C for 2 h and then was
allowed to warm
to rt and stirred under nitrogen overnight. The reaction was quenched with
saturated
NH4Cl and diluted with water. The mixture was extracted with Et20 (3X). The
combined Et20 layers were washed with water, brine and dried over anhydrous
Na2S04. After concentration in vacuo, the crude product was purified by flash
column chomatography on silica gel eluted with 5%-10% EtOAc/Hexane to isolate
0.099g of N-(BOC)-3,3-diallyl-4-oxoproline, methyl ester,
1H NMR 500 MHz (ppm): ~ 1.43 and 1.45 (2s, 9H), 2.07 (m, 1H), 2.32-2.43 (m
3H),
3.69 (s, 3H), 2.39 (m,2H), 4.43 and 4.55 (2s, 1H), 5.09 (m, 4H), 5.63 (m, 1H),
5.80
(m, 1H).
Step B 3,3-Dially-4-oxo-(L)-proline
To a solution of 0.099 g of N-(BOC)-3,3-diallyl-4-oxo-(L)-proline,
methyl ester in 0.4 mL of water and 1.6 mL of MaOH was added 25.7 mg of
LiOH.monohydrate. The reaction mixture was sonicated for 2 h. The mixture was
acidified with 1.2N HCl to pH = 2 and concentrated ifa vacuo. The residue was
treated with 1.5 mL of HCl/dioxane solution for 1.5 h. After concentration in
vacuo,
0.12g of 3,3-diallyl-4-oxo-(L)-proline was obtained which was used in the next
step
without purification.
1NMR 500MHz(ppm): 8 2.24 (m, 1H), 2.43 (m, 1H), 2.51 (m, 1H),
2.73 (m, 1H), 3.89 (m 2H), 5.20 (m, 4H), 5.70 (m 1H). LCMS: Retention Time =
1.1
min, m/e = 209.9 (M+1).
Step C N-f(3,5-dichlorobenzene)sulfonyll-3 3 -diallyl-4-oxo-(L)-proline
- 111 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
To a solution of 3,3-diallyl-4-oxo-(L)-proline in 2 mL of water and 2
mL of dioxane was added 110 mg of Na2C03 and 192 mg of 3,5-dichlorobenzene
sulfonyl chloride. The reaction was stirred at rt under nitrogen overnight.
The
mixture was diluted with water and extracted with Et20 (1X). The aqueous phase
was acidified to pH = 2 and extracted with EtOAc (3X). The combined EtOAc
layers
were washed with brine, dried over Na2S04 and concentrated i~z vacuo. The
residue
was purified by preparative T.L.C. on silica gel eluted with 1% HOAc in 50%
EtOAc/hexane to yield N-[(3,5-dichlorobenzene)sulfonyl]-3,3,-diallyl-4-oxo-(L)-
proline (20 mg).
1H NMR 500MHz (ppm): b 2.30 (m, 4H), 3.82 (m, 1H), 4.15 (m, 1H), 4.50 (m, 1H),
5.10 (m, 4H)', 6.55 (m, 1H), 6.90 (m, 1H), 7.G0 (s, 1H), 7.75 (bs, 2H).
Step D N-(N-f(3,5-dichlorobenzene)sulfonyll-3,3,-diallyl-4-oxo-(L)-prolx1)-4-
f(3',5'-dichloroisonicotinoyl)aminol-(L)-phenylalanine, t-but l~.
A solution of 20 mg of N-[(3,5-dichlorobenzene)sulfonyl]-3,3,-diallyl-
4-oxo-(L)-proline in 1 mL of CH~C12 was treated with 0.01 mL of oxalyl
chloride in
presence of catalytic amount of DMF. After 1 h, the mixture was concentrated
under
vacuum. The residue was dissolved into 0.5 mL of CH2C12 and added to a
solution
of 4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, t-butyl ester in
presence
of 0.025 mL of DIPEA. The mixture was stirred at rt overnight. The reaction
was
quenched with water and purified by preparative T.L.C. eluted with 50% EtOAc/
hexane to afford N-(N-[(3,5-dichlorobenzene)sulfonyl]-3,3,-diallyl-4-oxo-(L)-
prolyl)-
4-[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, t-butyl ester (10
mg).
HPLC-MS: m/e = 810.9 (M+1).
Step E N-(N-f(3,5-dichlorobenzene)sulfonyll-3,3,-diallyl-4-oxo-(L)-prol,1
f(3',5'-dichloroisonicotinoyl)aminol-(L)-phen lade
N-(N-[(3,5-dichlorobenzene)sulfonyl]-3,3,-diallyl-4-oxo-(L)-proly1)-4-
[(3',5'-dichloroisonicotinoyl)amino]-(L)-phenylalanine, t-butyl ester (10 mg)
was
treated with 1 mL of TFA for 1h. The mixture was concentrated isa vacuo to
afford N-
(N-[(3,5-dichlorobenzene)sulfonyl]-3,3,-diallyl-4-oxo-(L)-prolyl)-4-[(3',5'-
dichloro-
isonicotinoyl)amino]-(L)-phenylalanine (7 mg).
HPLC-MS: Retention time = 3.6 min, m/e = 754.9 (M+1).
- 112 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
EXAMPLE 71
Anta~~onism of VLA-4 Dependent Binding to VCAM-I~ Fusion Protein.
Step A Preparation of VCAM-I~.
EXAMPLE 71
Antagonism of VLA-4 Dependent Binding to VCAM-I~ Fusion Protein.
Step A Preparation of VCAM-I~.
The signal peptide as well as domains 1 and 2 of human VCAM
(GenBank Accession no. M30257) were amplified by PCR using the human VCAM
cDNA (R & D Systems) as a template. The resulting PCR product of 650 by was
digested with EcoRI and BcII and ligated to expression vector pIg-Tail (R & D
Systems, Minneapolis, MN) digested with EcoRI and BamHI. The pIg-Tail vector
contains the genomic fragment which encodes the hinge region, CH2 and CH3 of
human IgGl (GenBank Accession no. 217370). The DNA sequence of the resulting
VCAM fragment was verified using Sequenase (US Biochemical, Cleveland, OH).
The fragment encoding the entire VCAM-Ig fusion was subsequently excised from
pIg-Tail with EcoRI and NotI and ligated to pCI-neo (Promega, Madison, WI)
digested with EcoRI and NotI. The resulting vector, designated pCI-neo/VCAM-Ig
was transfected into CHO-I~l (ATCC CCL 61) cells using calcium-phosphate DNA
precipitation (Specialty Media, Lavalette, NJ). Stable VCAM-Ig producing
clones
were selected according to standard protocols using 0.2-0.8 mg/ml active 6418
(Gibco, Grand Island, NY), expanded, and cell supernatants were screened for
their
ability to mediate Jurkat adhesion to wells previously coated with 1.5 mg/ml
(total
protein) goat anti-human IgG (Sigma, St. Louis, MO). A positive CHO-I~IIVCAM-
Ig clone was subsequently adapted to CHO-SFM serum-free media (Gibco) and
maintained under selection for stable expression of VCAM-Ig. VCAM-Ig was
purified from crude culture supernatants by affinity chromatography on Protein
A/G
Sepharose (Pierce, Rockford, IL,) according to the manufacturer's instructions
and
desalted into 50 mM sodium phosphate buffer, pH 7.6, by ultrafiltration on a
YM-30
membrane (Amicon, Beverly, MA).
Step B Preparation of 1251-VCAM-Ig_.
VCAM-Ig was labeled to a specific radioactivity greater that 1000
Ci/mmole with 1251-Bolton Hunter reagent (New England Nuclear, Boston, MA; cat
# NEX120-0142) according to the manufacturer's instructions.The labeled
protein
- 113 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
was separated from unincorporated isotope by means of a calibrated HPLC gel
filtration column (G2000SW; 7.5 x 600 mm; Tosoh, Japan) using uv and
radiometric
detection.
Step C VCAM-Ig Bindin_ A~-ssay_.
Compounds of this invention were prepared in DMSO at 100x the
desired final assay concentration. Final concentrations were selected from a
range
between 0.001 nM-100 ~,M. Jurkat cells were centrifuged at 400xg for five mins
and
resuspended in binding buffer (25 mM HEPES, 150 mM NaCI, 3 mM KCI, 2 mM
, glucose, 0.1 % bovine serum albumin, pH 7.4). The cells were centrifuged
again and
resuspended in binding buffer supplemented with MnCl2 at a final concentration
of 1
mM. Compounds were assayed in Millipore MHVB multiscreen plates (cat#
MHVBN4550, Millipore Corp., MA) by making the following additions to duplicate
wells: (i) 200 ~.L of binding buffer containing 1 mM MnCl2; (ii) 20 ~,L of
1251-
VCAM-Ig in binding buffer containing 1 mM MnCl2 (final assay concentration ~
100
pM); (iii) 2.5 ~.L of compound solution or DMSO; (iv) and 0.5 x 106 cells in a
volume of 30 mL. The plates were incubated at rt for 30 mins, filtered on a
vacuum
box, and washed on the same apparatus by the addition of 100 ~,L of binding
buffer
containing 1 mM MnCl2. After insertion of the multiscreen plates into adapter
plates
(Packard, Meriden, CT, cat# 6005170, 100 ~L of Microscint-20 (Paclcard cat#
6013621) was added to each well. The plates were then sealed, placed on a
shaker for
seconds, and counted on a Topcount microplate scintillation counter (Packard).
Control wells containing DMSO alone were used to determine the level of VCAM-
Ig
binding corresponding to 0% inhibition. Contol wells in which cells were
omitted
25 were used to determine the level of binding corresponding to 100%
inhibition.
Binding of 1251-VCAM-Ig in the absence of cells was usually less than 5% of
that
observed using cells in the presence of vehicle. Percent inhibition was then
calculated for each test well and the IC50 was determined from a ten point
titration
using a validated four parameter fit algorithm.
EXAMPLE 72
Antagonism of a,~~i~ Dependent Binding to VCAM-Ig Fusion Protein.
Step A ot,~~,~ Cell line.
- 114 -
CA 02439952 2003-09-03
WO 02/074761 PCT/US02/08060
RPMI-8866 cells (a human B cell line oc~+(31-(3~+; a gift from Prof. John
Wilkins, University of Manitoba, Canada) were grown in RPMI/10% fetal calf
serum/
100 U penicillin/100 pg streptomycin/2 mM L-glutamine at 37°C, 5 %
carbon
dioxide. The cells were pelleted at 1000 rpm for 5 rains and then washed twice
and
resuspended in binding buffer (25 mM Hepes, 150 mM NaCI , 0.1 % BSA, 3 mM
KCI, 2 mM Glucose, pH 7.4).
Step B oc4~~ VCAM-Ig Binding Assay
Compounds of this invention were prepared in DMSO at 100x the
desired final assay concentration. Final concentrations were selected from a
range
between 0.001 nM-100 ~.M. Compounds were assayed in Millipore MHVB
multiscreen plates (Cat# MHVBN4550) by making the following sequential
additions
to duplicate wells: (i) 100 mL/well of binding buffer containing 1.5 mM MnCl2;
(ii)
10 mL/well lasl-VCAM-Ig in binding buffer (final assay concentration < 500
pM);
(iii) 1.5 mL/well test compound or DMSO alone; (iv) 38 mL/well RPMI-8866 cell
suspension (1.25 x lOG cells/well). The plates were incubated at rt for 45
rains on a
plate shaker at 200 rpm, filtered on a vacuum box, and washed on the same
apparatus
by the addition of 100 mL of binding buffer containing 1 mM MnCl2. After
insertion
of the multiscreen plates into adapter plates (Packard, Meriden, CT, cat#
6005178),
100 mL of Microscint-20 (Packard cat# 6013621) was added to each well. The
plates
were then sealed, placed on a shaker for 30 seconds, and counted on a Topcount
microplate scintillation counter (Packard). Control wells containing DMSO
alone
were used to determine the level of VCAM-Ig binding corresponding to 0%
inhibition. Wells in which cells were omitted were used to determine the level
of
binding corresponding to 100% inhibition. Percent inhibition was then
calculated for
each test well and the IC50 was determined from a ten point titration using a
validated
four parameter fit algorithm.
- 115 -
