Design, synthesis, and structure-activity relationship of novel CCR2 antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.12.050

A series of novel 1-aminocyclopentyl-3-carboxyamides incorporating substituted tetrahydropyran moieties have been synthesized and subsequently evaluated for their antagonistic activity against the human CCR2 receptor. Among them analog 59 was found to posses potent antagonistic activity.

Full text of DOI:10.1016/j.bmcl.2008.12.050

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1830–1834
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and structure–activity relationship of novel
CCR2 antagonists q
a
a
a
a
Shankaran Kothandaraman a, , Karla L. Donnely , Gabor Butora , Richard Jiao , Alexander Pasternak ,
Gregori J. Morriello a, Stephen D. Goble a, Changyou Zhou a, Sander G. Mills a, Malcolm MacCoss a,
Pasquale P. Vicario b, Julia M. Ayala b, Julie A. DeMartino b, Mary Struthers b,
*
Margaret A. Cascieri b, Lihu Yang a,
*
a Department of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
b Department of Immunology, Merck Research Laboratories, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 1-aminocyclopentyl-3-carboxyamides incorporating substituted tetrahydropyran moi-
eties have been synthesized and subsequently evaluated for their antagonistic activity against the human
CCR2 receptor. Among them analog 59 was found to posses potent antagonistic activity.
Ó 2009 Published by Elsevier Ltd.
Received 1 October 2008
Revised 8 December 2008
Accepted 10 December 2008
Available online 24 December 2008
Keywords:
CCR2 Antagonist
Chemotaxis
Aminocyclopentane carboxamide
Reductive aminations
Monocyte Chemotactic Protein-1 (MCP-1) is a member of the
chemokine family of pro-inflammatory proteins that are involved
in chemo trafficking and activation of leucocytes by the activation
of CCR2 receptors.1 MCP-1, is expressed not only from monocytes
but also in a host of other cells that includes T-cell, macrophages,
etc. There is a strong body of evidence to suggest that MCP-1 is
associated with inflammatory diseases such as rheumatoid arthri-
tis2 and atherosclerosis.3 Studies with CCR2 ꢀ/ꢀ4 and MCP-1ꢀ/ꢀ5
mice as well as with peptide based MCP-1 antagonists6 suggest
that blocking the interaction of CCR2 and MCP-1 may provide use-
ful therapy for the diseases indicated above.7,8
A host of CCR2 receptor antagonists have been identified, includ-
ing those detailed in the literature from this laboratory.9 Within the
scope of the present investigation two such CCR2 antagonists war-
rant attention ( Fig. 1). The nonselective quaternary salt 1 (TAK-
779) from Takeda10 was a potent CCR5 antagonist (IC50 = 1.4 nM)
in addition to being an equally impressive CCR2 antagonist
(IC50 = 28 nM). A second lead class is represented by the cyclopen-
tane based structure 2 that was recently disclosed.11 The cyclopen-
tane lead 2 showed a good potency in a functional assay to match its
excellent in vitro binding potency.
The present communication describes our preliminary efforts
towards the identification of a newer class of CCR2 receptor antag-
onists that displays high binding and functional potency in the hu-
man CCR2 receptor. In accomplishing these objectives, we have
incorporated features from structures such as 1 and 2 to generate
hybrid analogs represented by generic structure 5, shown below.
Our primary focus was the synthesis, SAR, and biological evalua-
tion for analogs of 5 (Fig. 2) that specifically altered the amino
end of the molecule. A cross species pharmacokinetic profile for
the best compound in this novel lead class is also discussed.
Analogs of 5 were synthesized by simply carrying out reductive
aminations of amine (4) and the pyranoketone (3) as shown below
(Fig. 3). The synthesis of substituted pyrans (3) and the chiral
cyclopentylamine (4) are discussed next.
O
CF3
N
N
H
H
N
O
CF3
2
1
N+
O
* Corresponding author. Tel.: +1 732 594 3979; fax: +1 732 594 2210.
E-mail address: kothandaraman_shankaran@merck.com (S. Kothandaraman).
Figure 1. Takeda (1) and Merck (2) CCR2 antagonists.
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.12.050

S. Kothandaraman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1830–1834
1831
O
O
O
H
N
CF3
BocHN
O
BocHN
CF3
a, b
N
H
O
N
H
R
10
11
CF3
CF3
5, R = cycloalkyl and
heterocycloalkyl
CF3
c
H2N
N
H
. HCl
Figure 2. Proposed lead molecule.
CF3
4
O
O
Scheme 2. Reagents and conditions: (a) NaOH, MeOH/H2O, 60 °C; (b) EDC. HCl, 3,5-
bis-trifluoromethylbenzylamine hydrochloride, CH2Cl2, Hunig base, RT; (c) EtOAc/
HCl, 0 °C to RT.
CF
5
H N
3
2
N
H
+
[
]
n
CF
3
4
3
O
Figure 3. Reductive amination synthesis of 5.
O
OMe
OTMS
MeO
MeO
R
OH
f
CF3
18
CF3
As shown in Scheme 1, commercially available (1S,4R)-(+)-2-
azabicyclo-[2.2.1] hept-5-en-3-one (6) was transformed to the
ester 7 in a two step sequence of reactions involving catalytic
hydrogenation followed by the opening of the lactam ring. The
amino group in 7 was protected under O’Donnell conditions12 to
give the imine 8 that was carried forward crude to the next step.
The lithium enolate formed from the ester 8 using LDA was alkyl-
ated with 2-iodopropane to yield a mixture of diastereomers. To
facilitate the isolation of the desired product, the crude reaction
mixture was subjected to hydrolysis under acidic conditions (to
unravel the benzophenone imine), after which the regenerated
amine was subsequently protected as N-Boc. This produced a 1:1
mixture of 9 and the desired 10, which was readily separated. Pre-
vious results from this laboratory had disclosed that the cis-placed
amine/ester 10 led to the most potent analogs, while the com-
pounds derived from 9 were comparatively less active or inactive.13
The cis-ester 10 was saponified and then transformed to the
amide 11 under EDC conditions. Removal of the N-Boc protection
in 11 under acidic conditions produced the amine 4 as the hydro-
chloride salt (Scheme 2).
O
O
O
O
O
19
12, R = Me, Et,
n-Pr, F, OH, CF3
& Cyclopropyl
d, e
OMe
OH
OH
a
b
OMe
O
17
16
O
OMe
F
MeO
F
b
O
c
13
O
14
O
15
Scheme 3. Reagents and conditions: (a) m-CPBA/MeOH, 0 °C to RT; (b) MeOH/HCl;
(c) MeCN-MeOH/SlectflourTM
;
(d) TPAP oxidation; (e) CF3TMS, TBAF, THF; (f)
CF3COOH, CH2Cl2.
O
O
O
N
CF3
b, c
The syntheses of substituted pyran-4-ones (3) required a multi-
tude of approaches, as shown below. The synthesis of mono alkyl-
ated pyranoketones (12, R = Me, Et, n-Pr) was straight forward and
O
O
followed a literature procedure.14 The
a-fluoro and a-hydroxy ana-
logs were synthesized as shown in Scheme 3. Thus, 4-methoxy-3,
6-dihydro-2H-pyran 13 undergoes electrophilic fluorination15 with
selectflourTM in the presence of methanol to afford the acetal 14
that on treatment with acid gave 15. In an analogous fashion, 13
underwent smooth epoxidation followed by nucleophilic ring
opening of the epoxide with methanol to afford 16. Simple acidic
hydrolysis of 16 gave 17. The ketol 16 was further utilized to afford
19 as shown below (Scheme 3). A two step sequence that involved
TPAP oxidation16 of 16 followed by nucleophilic addition of the tri-
fluoromethyl anion17 to the intermediate ketone gave the silyl
ether 18. The ketol and the silyl protection in 18 were removed un-
der acidic conditions to give 19.
Scheme 4. Reagents and conditions: (a) pyrrolidine, RT; (b) 5-(trifluoromethyl)dib-
enzothiophenium trifluoromethanesulfonate, DMAP, DMF, RT; (c) HCl.
OTBS
OH
O
OH
c, d, e
O
a, b
20
O
24
23
OH
f, g
h
O
25
O
26
O
O
Ph
Ph
c
a, b
Scheme 5. Reagents and conditions: (a) LDA, THF/HMPA, allylbromide; (b) O3/
CH2Cl2, ꢀ78 °C and then NaBH4: (c) O-NO2PhSeCN/n-Bu3P; (d) TBSCl, imidazole; (e)
H2O2/THF; (f) CH2N2, Pd(OAc)2, ether; (g) TBAF/THF; (h) Swern oxidation.
NH
H2N
N
O
+
O
6
O
7
8
O
O
BocHN
d, e
BocHN
O
More challenging trifluoromethyl and the cyclopropyl pyran-4-
ones (22 and 26), were obtained starting from 20 and are shown in
Schemes 4 and 5. Transformation of 20 to enamine 21 was accom-
plished with pyrrolidine at RT. The enamine 21 underwent smooth
trifluoromethylation18 that was subsequently followed by acidic
workup, as shown, to give 22.
O
10
9
Scheme 1. Reagents and conditions: (a) Pd/C, EtOAc, H2, RT; (b) MeOH, HCl; (c)
benzophenone imine, CH2Cl2; (d) LDA, 2-iodo-propane, THF, ꢀ78 to 0 °C; (e) 1 M
HCl and then NaHCO3/BOC2O, RT.

1832
S. Kothandaraman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1830–1834
Table 2
The preparation of cyclopropyl compound 26 began with the al-
Binding affinities and chemotaxis (CTX) in the 3,5-bis-trifluoromethylbenzylamide
kylation of 20 with allyl bromide to give the mono allylated com-
pound that in a subsequent step was ozonized followed by
reductive work up to give the diol 23. The diol was then trans-
formed to the olefin 24 in a three step sequence that involved con-
version of the diol 23 to the selenide, hydroxyl protection, followed
by selenoxide elimination19 to give 24. Cyclopropanation20 of the
olefin 24 followed by the removal of the TBS protecting group gave
25. Oxidation of 25 to give 26 was accomplished under Swern oxi-
dation conditions.21
seriesa,c
O
R1
H
R2
CF3
N
N
H
R3
O
CF3
R4
The synthesized pyran 4-ones (Schemes 3–5) and the commer-
cially available ketones were subjected to reductive aminations22,27
with 4 to furnish the desired products that were transformed to
their hydrochloride salts prior to being assayed. Mixtures of stereo-
isomer were initially submitted for the binding assay (Tables 1 and
2). If the initial results from the binding assay looked promising, the
racemic/diastereomeric amines were further separated by chiral
chromatography to yield individual diastereomers for further
follow up studies (Table 3).
Results and discussion: The compounds listed in Table 1, illus-
trates the effect on binding of systematic variation of the amine
end of the molecule. The binding assay23 involved a recombinant
version of the human CCR2 receptor expressed in CHO cells. Thus,
Entry
R1
R2
R3
R4
Binding IC50, nM hCCR2b
CTX (nM)
34
45
46
47
48
49
50
51
53
54
55
56
H
Me
Et
n-Pr
Cyclopropyl
OH
F
CF3
CF3
H
Me
Me
H
H
H
H
H
H
H
H
OH
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
Me
H
H
4.6
1.5
3.0
7.8
123
51
6.0
5.5
765
28
57
0.2
0.3
5.1
ND
ND
1.0
1.0
ND
ND
ND
ND
230
7
a
IC50 reported are averages of triplicate measurements whose standard errors
were normally <15% in a given assay. Assay to assay variability was with in
twofold based on the standard compound.
b
Displacement of [125I]-labeled of hMCP-1 from the CCR2 receptor expressed on
Table 1
CHO cells.
Human binding affinities in the 3,5-bis-trifluoromethylbenzylamide seriesa
C
Analogs reported are mixtures of racemic/diastereomeric mixtures. ND, not
determined.
O
CF3
RHN
N
going from 4 to 7 member ring compounds (27–30), we notice that
the analog 29, with cyclohexyl ring, was two to three folds more
potent than the other analogs (27, 28, and 30). Significant enhance-
ment in the potency for 29 was accomplished by the placement of
heteroatom at the cyclohexane core. In particular, compound 34
with a distal oxygen atom on the tetrahydropyran ring registered
a 14-fold gain in potency over the cyclohexane analog 29. Interest-
ingly, the analog 36, with an additional methylene group in be-
H
CF3
Entry
R
IC50 (nM)b
193
Entry
R
IC50 (nM)b
362
27
36
O
28
29
30
31c
32
33
34
156
64
37
38
39
40
41
42
43
22
36
S
Table 3
Binding affinities and chemotaxis (CTX) of monosubstituted THP substituted analogs
in the 3,5-bis-trifluoromethylbenzylamide seriesa
S
O
R
H
175
469
85
631
CF3
N
O2S
N
H
O
21% 1 lM
CF3
HN
O
O
187
239
77
R
Entry
Binding IC50 (nM)b
CTX in nM
Mec
57
58
59
42
4
1.2
1
0.2
0.1
56
O
Etc
Fd
a
60
61
62
141
5.6
3.0
ND
0.3
0.3
4.6
O
63
64
65
259
14.3
8.6
ND
0.8
3.3
35
30
44
419
O
O
IC50 reported are averages of triplicate measurements whose standard errors
a
were normally <15% in a given assay. Assay to assay variability was with in
twofold based on the standard compound.
IC50 reported are averages of triplicate measurements whose standard errors
were normally <15% in a given assay. Assay to assay variability was with in
twofold based on the standard compound.
b
Displacement of [125I]-labeled of hMCP-1 from the CCR2 receptor expressed on
b
Displacement of [125I]-labeled of hMCP-1 from the CCR2 receptor expressed on
CHO cells.
c
Diastereomers separated by chiral OD column.
Diastereomers separated by chiral AD column. ND, not determined.
CHO cells.
d
c
The trifluoroaceate salt of the corresponding 31 was prepared.

S. Kothandaraman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1830–1834
1833
tween the amine and the pyran core, led to a significant loss in
binding compared to 34. The isomeric pyran analog 33 lost a con-
siderable potency in comparison to 34. The dramatic effect of the
oxygen atom in the ring that lead to the more potent analogs while
going from 29 to 34 was found to be general, as illustrated by other
compounds 31, 32, and 35 as shown, though all were significantly
less potent than 34. The effect was less profound when the ‘O’ atom
in 34 was replaced by more lipophilic ‘S’ as in 37. While the sulfone
39 lost significant activities compared to the parent (37) the more
binding and in chemotaxis compared to the C-3 methyl/ethyl ana-
logs. It is noteworthy that the most active analog (59 vs 57) in the
binding assays were also the most potent in the chemotaxis assays
as shown in Table 3.
While the contrasting potency between the monofluoro and
mono methyl/ethyl analogs could be easily explained by the differ-
ences in the basicity of nitrogen on the pyran core, the differences
in the potency between the stereoisomers 57 and 59 was hard to
elucidate. Corroborative work done for the analogs25,26 of 59 and
congeners suggest that the most potent analog 59 had a cis-dis-
posed substituents (b-oriented) on the pyran core.
basic piperidine analog 40 had only 20% activity at 1 lM. The bicy-
clic compounds (41–44) were all significantly less potent than 34
and these systems were not pursued further. The most potent ana-
log 34 (Table 1), was probed further to generate even more potent
analogs as shown below in Table 2.
The preparation of more potent analogs derived from 34 was
accomplished by the substitution of the pyran ring with alkyl, hy-
droxyl, and fluoro group at the either C-2 or C-3 position. The bind-
ing results for the newer analogs (Table 2) were compared with 34.
Additionally, the chemotaxis (CTX) data for the select compounds
are also displayed. It should be noted that the assay results for
the analogs displayed in Table 2 are for the racemic and diastereo-
meric mixtures.
O
Me
H
N
CF3
N
H
O
CF3
59
The pharmacokinetic profile (po dosing) for the most potent
analog (59) was measured in rat (Sprague–Dawley), dog (Beagle),
and rhesus and is shown below.
Table 2 reveals that in comparison to 34, the C-3 mono methyl
compound 45 gained a threefold improvement in binding and a
staggering 280-folds increase in the chemotaxis assay.24 The bind-
ing and chemotaxis data for the ethyl analog 46 in many respects
was similar to 45. The homologous n-propyl analog 47 was slightly
less potent in binding compared to either 45 or 46 and also had lost
significant potency (relative) in the chemotaxis assay. Interest-
ingly, all three compounds (45, 46, and 47) share a trend in that
they all had a similar binding (compared to 34) and also displayed
better potency during the chemotaxis assay. Going forward, the
bulkier cyclopropyl analog 48 lost considerable binding compared
to 34. This was also true for the aminol 49. The C-3 monofluoro and
trifluoromethyl analogs 50 and 51 displayed binding affinity simi-
lar to 34, but again showed superior chemotactic properties. Ana-
log 53 with geminal trifluoromethyl/hydroxyl at C-3, showed a
substantial loss of potency. The isomeric methyl analog compound
54 was considerably less potent in binding and stands in contrast
to either 45 or 46. The presence of an additional methyl leading
to the geminal dimethyl compound 55 resulted in significant loss
of affinity compared to 34. That the mono-substitution at C-3 of
THP ring (analog 45) led to an increase in potency and chemotaxis
was further underscored by the preparation of analog 56, with a
methyl substituents flanked at either side (C-3/C-30) of the pyran
ring leading to significant improvement in the binding potency
compared to 55.
Species Dose (mg/kg) AUC (
l
M h) Tmax (h) Cmax
(
l
M) F%
Rat
Dog
Rhesus
3
2
2
0.76
0.62
0.01
0.25
0.75
1.00
0.735
0.422
0.004
42
28
2
An oral dose of 3 mg/kg in rat gave a Cmax of 735 ng/ml with a Tmax
of 0.25 h and the bio-availability was 42%. The oral bio-availability
(at 2 mg/kg) for 59 in dogs was 28% and a dismal 2% in rhesus
monkeys.
In summary, we have successfully synthesized a new class of
CCR2 antagonists. The analog 59 displayed superb potency in both
the binding and chemotaxis assays. It also displayed reasonably
good PK in rats/dog, but poor oral absorption in rhesus. Efforts to
improve the deficiencies of 59 by modification of the cyclopentane
core and also at the amide end will be the subject of future
communications from these laboratories.
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1834
S. Kothandaraman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1830–1834
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ester group in 10 came via the X-ray of ‘wrong acid’ from the ester 9, where in
the amine and carboxyl group had a trans-relationship..
14. Smith, A. B.; Empfield, J. R.; Vaccaro, H. A. Tetrahedron Lett. 1989, 30, 7325.
15. Stavber, S.; Scotler-Pecan, T.; Zupan, M. Tetrahedron Lett. 1994, 35, 1105.
16. Griffith, W. P.; Ley, S. V.; Whitecombe, G. P.; White, A. D. J. Chem. Soc. Chem.
Commun. 1987, 1625.
dichloromethane (200 ml) was added benzophenone imine (10.2 g,
56.8 mmol) at RT and the resultant mixture was stirred for 24 h. The
reaction mixture was filtered and the filtrate was evaporated, to leave
behind an yellow oil that was triturated with ether (100 ml), filtered and
evaporated. This operation was repeated twice to ensure that the product was
free of ammonium chloride impurities. The resultant oil was thoroughly dried
under vacuo to yield the title compound (18.03 g, ꢂ100%) and required no
further purification. 1H NMR (CDCl3, 500 MHz): d 7.5–7.18 (m, 10H), 3.75 (m,
1H), 3.7 (s, 3H), 2.78 (m, 1H), 2.26–1.7 (m, 6H).
Preparation of (10): To a solution of LDA (prepared from disopropylamine
(7.7 g, 76.1 mmol) and n-BuLi (30.4 ml, 2.5 M solution in hexane, 76.1 mmol) in
THF (120 ml) at ꢀ78 °C was slowly added intermediate 8 (18.0 g, 58.6 mmol).
The resultant burgundy colored solution was stirred for 20 min after which it
was quenched with 2-iodopropane (14.9 g, 88 mmol). The resultant solution
was gradually warmed over 3 h to 0 °C and held there for an additional 3 h.
Reaction mixture was quenched with water and extracted with EtOAc. The
solvent layer was washed with water, brine, dried (anhydrous magnesium
sulfate) and concentrated to yield an oil. The removal of Schiff base protection
at the amine group was brought about by taking the crude oil (20.0 g) in THF
(100 ml) and treating it with HCl (5.0 ml, 12 M) and was allowed to stir at RT
for 3 h. After the removal of all volatiles, the hydrochloride salt was taken in
dichloromethane (250 ml), saturated solution of sodium bicarbonate solution
(250 ml) and BOC2O (26.0 g, 1.4 equiv). The resultant mixture was vigorously
stirred overnight at RT. The solvent layer was separated and washed with
water, brine, dried (anhydrous magnesium sulfate) and concentrated to yield
oil. Purification by flash column chromatography and elution with hexane:
EtOAc (19:1) afforded the title compound (4.91 g, 30%). 1H NMR (CDCl3,
500 MHz): d 4.79 (br, 1H), 4.01 (m, 1H), 3.71 (s, 3H), 2.18–1.60 (m, 6H), 1.44 (s,
9H), 0.87 (d, J = 6.9 Hz, 3H), 0.86 (d, J = 6.9 Hz, 3H).
17. Krishnamurti, R.; Bellew, D. R.; Prakash, G. K. S. J. Org. Chem. 1991, 56, 984.
18. Umemoto, T.; Ishihara, S.; Adachi, K. J. Fluorine Chem. 1995, 74, 77.
19. Grieco, P. A.; Gilman, S.; Nishizawa, M. J. Org. Chem. 1976, 41, 1485.
20. Cossy, J.; Belotti, D.; Pete, J. P. Tetrahedron 1990, 46, 1859.
Preparation of (11): This was a two step preparation that involved saponification
of ester 10 to afford the intermediate acid followed by amidation.
21. Omura, K.; Swern, D. J. Org. Chem. 1978, 34, 1651.
22. Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah, R. D. J.
Org. Chem. 1996, 61, 3849.
Step A: To ester 10 (4.91 g, 17.2 mmol) in MeOH (100 ml) was added a solution
of LiOH (3.6 g, 85 mmol) dissolved in water (20 ml) and THF (10 ml). The
resultant mixture was heated at 80 °C until the reaction was complete (18 h).
MeOH was removed under vacuo and the crude was taken in Water/EtOAc
(200 ml, 1:4) and cooled to 0 °C. The pH of the reaction mixture was carefully
adjusted to 6 and the EtOAc layer was separated and washed with water, brine,
dried (anhydrous magnesium sulfate) and concentrated to yield oil. Purification
by flash column chromatography and elution with hexane: EtOAc (1:1) + 2%
AcOH gave acid (3.9 g, 84%) that was carried further.
Step B: To acid (10) from Step A (2.09 g, 7.71 mmol), 3,5-bis-trifluoromethyl-
benzylamine hydrochloride (2.26 g, 8.1 mmol) in DCM (50.0 ml) was added 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.96 g, 15.4 mmol) and
followed by diisopropylethylamine (2.1 g, 16.2 mmol). The resultant mixture
was stirred at RT for 18 h. The reaction mixture was diluted DCM and twice
washed with 1 N HCl, once with aqueous sodium carbonate and once with
brine, dried (anhydrous magnesium sulfate), concentrated, purified by flash
column chromatography. Eluting with hexane/EtOAc (2:3) gave 11 (2.23 g,
64%).
23. Initially, the binding affinities were evaluated in the CHO assay; later the human
monocyte based assay was used. Radioligand competition binding assays:
human monocytes (2 ꢁ 105), or CHO cells expressing human CCR2b (5 ꢁ 104)
were incubated with 125I-hMCP-1 (20–50 pM) and various concentrations of
unlabeled chemokines in binding buffer for 60 min at room temperature. The
binding buffer contains 50 mM Hepes, 5 mM MgCl2, and 1 mM CaCl2, pH 7.4. 125I-
hMCP-1 was purchased from Perkin Elmer Life Sciences, Inc., with a specific
activity of 2200 Ci/mmol. The assay was terminated by filtration of the reaction
mixture through GF/B filter plates (presoaked in 0.1% polyethyleneimine) using a
Packard Cell Harvester, filter plates were washed with 25 mM Hepes, pH 7.5,
containing 500 mM NaCl and dried in an incubator for at 37 °C for 30 min. The
plates were loaded with Microscint 0 (Packard) and counted in a Topcount NXT
(Packard). The software program Prism (GraphPad) was used for all..
24. Ayala, J. M.; Goyal, S.; Liverton, N. J.; Claremon, D. A.; O’Keefe, S. J.; Hanlon, W.
A. J. Leukoc. Biol. 2000, 67, 869.
25. Butora, G. Unpublished experimental observations. Stereochemical
relationship was established by comprehensive analysis of the high field
NMR studies of the analogs, that were independently synthesized by a route,
unlike the one described herein..
26. We believe that the trans-diastereomer (both the methyl and amine in diaxial
disposition) probably was formed during this reaction and but eluded our
detection/separation by chiral columns..
Preparation of (4): To a solution of 2.23 g (11) was added a saturated HCl (4 N
solution in dioxane, 25.0 ml) and the mixture stirred at RT. After 1.5 h at RT, the
volatiles were removed under reduced pressure to leave behind an oil that was
triturated with ether followed by filtration to afford hydrochloride salt of 4
(1.79 g, 100%) as a white solid. LC–MS for C18H22F6N2O [M+H+] calculated 396.4;
found 397.2 (M+H).
27. General procedures
Typical reductive amination of (4) with ketones: The reductive amination of (4)
with ketones are similar to the Ref. 22. Thus to a stirred solution 4 (33 mg,
0.076 mmol) in 2 ml of DCM at RT was added 16 lL (0.095 mmol) of
diisopropylethylamine and ketone (0.076 mmol). To the reaction mixture was
added 4 beads of molecular sieves (4A) followed by 24 mg of Na(OAc)3BH. After
stirring overnight the reaction mixture was evaporated, the DCM layer was
washed with brine, dried (anhydrous MgSO4), and evaporated to give the crude
product. Further purification to afford the pure product was effected by Gilson
reverse phase separation. This procedure was employed to prepare the analogs
27–56.
Preparation of (7): A mixture of (1S,4R)-(+)-2-azabicyclo[2.2.1]-hept-5-en-3-
one (10.3 g, 94.4 mmol) in EtOAc (200 ml) and 10% Pd/C (0.5 g), was
hydrogenated at RT. After 24 h the reaction mixture was filtered and
evaporated leaving behind 10. Four grams (100%) of the product that was
taken up in 250 ml methanol and HCl (12 M, 6 ml). The resultant mixture was
stirred at RT, until the reaction was complete (72 h). Evaporation of methanol
followed by drying under high vacuo, yielded title compound as an off white
solid (16.0 g, 96%). 1H NMR (D2O, 500 MHz): d 3.70 (s, 3 H), 3.01 (m, 1H), 2.38
(m, 1H), 2.16–1.73 (m, 6H).