Antagonists of the human CCR5 receptor as anti-HIV-1 agents. Part 1: Discovery and initial structure-activity relationships for 1-amino-2-phenyl-4-(piperidin-1-yl) butanes

Article abstract of DOI:10.1016/S0960-894X(00)00637-5

Screening of the Merck sample collection for compounds with CCR5 receptor binding afforded (2S)-2-(3,4-dichlorophenyl)-1-[N-(methyl)-N-(phenylsulfonyl)amino]-4-[spiro(2 ,3-dihydrobenzthiophene-3,4′-piperidin-1′-yl)]butane S-oxide (4) as a potent lead structure having an IC₅₀ binding affinity of 35 nM. Herein, we describe the discovery of this lead structure and our initial structure-activity relationship studies directed toward the requirement for and optimization of the 1-amino fragment.

Full text of DOI:10.1016/S0960-894X(00)00637-5

Bioorganic & Medicinal Chemistry Letters 11 (2001) 259±264
Antagonists of the Human CCR5 Receptor as Anti-HIV-1 Agents.
Part 1: Discovery and Initial Structure±Activity Relationships for
1-Amino-2-phenyl-4-(piperidin-1-yl)butanes
Conrad P. Dorn,a Paul E. Finke,a,* Bryan Oates,a Richard J. Budhu,a Sander G. Mills,a
Malcolm MacCoss,a Lorraine Malkowitz,b Martin S. Springer,b Bruce L. Daugherty,b
Sandra L. Gould,b Julie A. DeMartino,b Salvatore J. Siciliano,b Anthony Carella,c
Gwen Carver,c Karen Holmes,c Renee Danzeisen,c Daria Hazuda,c Joseph Kessler,c
Janet Lineberger,c Michael Miller,c William A. Schleifc and Emilio A. Eminic
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Immunology Research, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
cDepartment of Antiviral Research, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
Received 18 September 2000; accepted 9 November 2000
AbstractÐScreening of the Merck sample collection for compounds with CCR5 receptor binding a€orded (2S)-2-(3,4-dichloro-
phenyl)-1-[N-(methyl)-N-(phenylsulfonyl)amino]-4-[spiro(2,3-dihydrobenzthiophene-3,40-piperidin-10-yl)]butane S-oxide (4) as a
potent lead structure having an IC50 binding anity of 35 nM. Herein, we describe the discovery of this lead structure and our
initial structure±activity relationship studies directed toward the requirement for and optimization of the 1-amino fragment. # 2001
Published by Elsevier Science Ltd.
Human immunode®ciency virus type-1 (HIV-1) is an
enveloped virus that must fuse its envelope with the
plasma membrane of its host cell to gain cell entry.1
Early studies had identi®ed the T-lymphoid cell surface
protein CD4 as a primary receptor involved in the
necessary interaction with the gp120 viral envelope gly-
coprotein. However, while CD4 is required for HIV-1
entry into cells, CD4 alone was found not to be su-
cient.2 Thus, a search for a co-receptor was initiated.3 It
had been established that HIV-1 isolates can be divided
into two general classes depending on their cellular
tropism. M-tropic isolates can infect macrophages and
primary T-cells, but not T-lymphoid cell lines, and are
characterized as non-syncytium-inducing (NSI). T-tropic
isolates infect T-lymphoid cell lines, as well as primary T-
cells, but not macrophages, and are characterized as
syncytium-inducing (SI). In 1995, the chemotactic cyto-
kines (i.e., chemokines) MIP-1a, MIP-1b, and RANTES
were reported to be suppressors of HIV-1 cell entry for
the M-tropic variants, but not the T-tropic.4 Subse-
quently, the chemokine receptor CCR5, which binds
MIP-1a, MIP-1b, and RANTES, was identi®ed as a co-
receptor speci®c for the M-tropic HIV-1 isolates.5 9
Previously, CXCR4, also a chemokine receptor, had
been identi®ed as the primary co-receptor for the T-
tropic variants.10 Thus, these co-receptors, along with
CD4, interact with the viral gp120 to mediate the initial
steps of the virus' host cell infection.
HIV-1 variants that exclusively use the CCR5 co-recep-
tor, now known as R5 variants, largely predominate
during the establishment and early stages of the infec-
tion in patients, although they appear to remain present
throughout the course of the disease. Viral variants that
utilize both the CCR5 and CXCR4 co-receptors, R5X4
variants, seem to arise during the course of infection
and may be associated with enhanced disease progres-
sion. Isolates that exclusively use CXCR4, X4 variants,
are rare in patients and arise through adaptation of viral
replication to T-lymphoid laboratory cell lines.
The importance of CCR5 for the establishment of the
initial infection in humans was demonstrated by human
genetic studies of high-risk individuals.11 Individuals
*Corresponding author. E-mail: paul_®nke@merck.com
0960-894X/01/$ - see front matter # 2001 Published by Elsevier Science Ltd.
PII: S0960-894X(00)00637-5

260
C. P. Dorn et al. / Bioorg. Med. Chem. Lett. 11 (2001) 259±264
homozygous for a 32-base pair deletion in the gene for
CCR5 do not express functional receptor on their cell
surfaces and have been identi®ed as being highly resistant
to HIV-1 infection,12 while infected individuals hetero-
zygous for the defective gene appear to exhibit delayed
disease progression.13 Given the importance of CCR5
for the establishment, and possible maintenance, of HIV-
1 infection in vivo, and the lack of an overt detrimental
phenotype in humans that do not express functional
CCR5, we initiated an e€ort to identify suitable CCR5
antagonists for use as potential anti-HIV-1 therapeutic
agents.14 16
CCR5 is a member of the seven-transmembrane G-protein
coupled receptor superfamily and is one of 18 currently
known chemokine receptors. There are over 50 reported
chemokine ligands that are classi®ed according to the pat-
tern of their ®rst two conserved cysteine residues. The two
main groups of chemokines are the CC (or b) chemo-
kines, having adjacent cysteines, and the CXC (or a) che-
mokines, having one intervening non-cysteine residue.17
phenyl)-4-(4-(substituted)piperidin-1-yl)butane deriva-
tives (2)26 were discovered as possible lead structures
(see Fig. 1). The best compound identi®ed was (2S)-2-
(3,4-dichlorophenyl)-1-[N-(methyl)-N-(phenylsulfonyl)-
amino]-4-[spiro(2,3-dihydrobenzthiophene-3,40-piperidin-
10-yl)]butane S-oxide (4, 1:1 mixture of sulfoxide dia-
stereomers) with an IC50=35 nM. The related sulfone 5
was also quite potent with an IC50=100 nM, while the
parent sul®de 3 was much less active (IC50=1000 nM).
The interaction of 4 with the CCR5 receptor was found
to be enantioselective at C-2, as evidenced by the much
lower anity of its (2R) diastereomers (IC50=870 nM).
The CCR5 binding enantioselectivity of the sulfoxide
isomers was not determined. Compounds 4 and 5 were
also found to be selective CCR5 antagonists in that
their IC50 values for CCR1, CCR2, and CCR3 were all
greater than 1000 nM (data not shown).
The identi®cation of CCR5 as a primary HIV-1 co-recep-
tor has initiated a ¯urry of activity to discover potent
antagonists in an e€ort to validate this mechanism of viral
entry as a viable therapeutic target. In addition, there
has been an explosion of research in the chemokine area
in general with several studies indicating that a potent
CCR5 antagonist might also be e€ective in the clinical
treatment of other disorders, such as rheumatoid arthri-
tis,18 asthma,19 and multiple sclerosis.20,21 To date, there
have been several reports of CCR5 antagonists in the
patent literature.1,22 The most potent structurally char-
acterized compound is Takeda's TAK-779 (1),23 which
is actually a dual CCR5 and CCR2b antagonist having
binding anities of 1.4 and 27 nM, respectively. Herein,
the discovery of a series of CCR5 selective 1-(N-(alkyl)-
N-(arylsulfonyl)amino)-2-(3,4-dichlorophenyl)-4-(4-
(substituted)piperidin-1-yl)butane structures (2) and our
initial SAR pertaining to the C-1 N-(alkyl)-N-(arylsul-
fonyl)amino moiety of 2 is described.24
A very important ®nding was that the analogous amides
were essentially inactive against CCR5 as illustrated with
the set of compounds 6±8.26 Thus, these initial binding
results seemed to indicate that the sulfonamide has a
distinct CCR5 receptor interaction and/or can uniquely
provide access to an important orientation for the phenyl
moiety which is capable of imparting the desired selec-
tive receptor binding interactions. Based on the activity
of the 2-thienyl derivatives 9 and 10 (IC50=65 and
120 nM, respectively), replacement of the phenyl with
di€erent heterocyles also appeared reasonable.
Once CCR5 was identi®ed as a primary HIV-1 co-recep-
tor, an extensive screening of the Merck sample collection
was initiated. To accomplish this, a high-throughput
binding assay of [125I]-MIP-1a to stably expressed human
CCR5 receptors in Chinese hamster ovary (CHO) cells
was developed.25 From this screening e€ort, a number
of 1-(N-alkyl-N-phenylsulfonylamino)-2-(3,4-dichloro-
Further characterization of these initial leads was also
obtained from HIV-1 viral replication inhibition studies in
isolated peripheral blood mononuclear cells (PBMCs).27
In this assay, compounds 4 and 9 showed consistent
inhibition with IC95 values of 6±12mM while weaker
Figure 1. Structures and CCR5 activities of lead compounds.

C. P. Dorn et al. / Bioorg. Med. Chem. Lett. 11 (2001) 259±264
261
CCR5 antagonists gave lower or no inhibition. Consistent
with selective CCR5 inhibition, 4 was ine€ective when an
X4-tropic HIV-1 strain was used (data not shown). These
inhibition studies provided encouragement to pursue
these newly identi®ed lead structures.
1,2-dichloroethane30 a€orded 17. Removal of the Boc
protecting group with HCl in methanol and sulfonyla-
tion a€orded the sul®de derivatives 19. Selective mono-
oxidation of the sul®des with 1 equiv of Oxone131 at
20 ꢀC for 2±5 min in methanol a€orded the ®nal sulf-
oxide compounds 20 as shown in Figure 2 and Table 1.
The sulfoxides were all isolated as 1:1 inseparable mix-
tures of the R and S stereoisomers at sulfur. Alternatively,
use of excess Oxone1 for extended time at room tem-
perature gave the corresponding spiro-sulfones 21.
The synthesis of these and related compounds26,28 (see
Table 1) started from the chiral acid 11 and utilized a
synthetic sequence previously reported from these
laboratories (Scheme 1).29 Conversion to an appropriate
amide 12 (R1=H, Me, Et) allowed for introduction of
the desired ®nal C-1 N-alkyl group. Reduction of these
amides a€orded the amines 13, which were protected as
the Boc derivatives 14 while the left hand piperidine was
incorporated. Thus, the two-step oxidation of the allyl
group with catalytic osmium tetroxide and N-methyl-
morpholine N-oxide (NMO) followed by periodate clea-
vage of the intermediate diols was carried out to a€ord the
aldehyde 15. Reductive alkylation of the spiro-piperidine
16 with aldehydes 15 using either sodium cyanoboro-
hydride in methanol or sodium triacetoxyborohydride in
Other nitrogen-based derivatives were also prepared
(Scheme 2). Oxidation of the Boc intermediate 17 (R1=
Me) gave the sulfoxide 22, which on deprotection with
TFA a€orded the N-methylamine derivative 23. Reduc-
tive alkylation of 18 (R1=Me) with benzaldehyde led to
the benzylic amine 24, which on oxidation as above
a€orded the sulfoxide 25 and sulfone 26. Alternatively,
acylation of 18 with a variety of acid chlorides, chloro-
formates, isocyanates and carbamoyl chlorides yielded
several carbonyl derivatives which on oxidation as
above a€orded the sulfoxides 27±30.
Table 1. Structure and CCR5 binding activities for the sulfonyl
derivatives 4, 5, 20, and 21
The preparation of some oxygen-based derivatives also
started with acid 11 (Scheme 3). Reduction with lithium
aluminum hydride a€orded the alcohol 31, which could
be alkylated to give the ether 32 or acylated to the ester
33. These were then carried through the same sequence
of reactions as above to give the sulfoxides 34 and 35,
respectively.
The above compounds were evaluated for CCR5 bind-
ing activity in the described [125I]-MIP-1a assay.25 Initial
modi®cation of the N-alkyl indicated that methyl was
optimal since the N-H derivatives 19a and 20a were con-
siderably less potent and extension to the N-ethyl analo-
gues 19b, 20b, and 21b was also detrimental as shown in
Figure 2. While all three sulfur oxidation states were
usually screened, in almost all cases the sulfoxide deri-
vative was preferred and these derivatives will be
emphasized in the following discussion. Since the sulf-
oxide isomers were not separated, any possible sulfoxide
stereochemical preference for CCR5 interaction was not
determined. The corresponding sulfones were usually
equipotent or slightly less active while the sul®des were
much poorer inhibitors, or inactive.
Structure
R2
CCR5a
Compound
n
IC50 (nM)b
3
4
5
9
10
0
1
2
1
2
1
1
1
1
1
1
1
1
1
2
2
1
1
1
1
1
1
1
1
1
1
Phenyl
Phenyl
Phenyl
2-Thienyl
2-Thienyl
NMe2
Benzyl
Methyl
n-Octyl
Cyclopentyl
Cyclohexyl
2-Cl-Phenyl
3-Cl-Phenyl
4-Cl-Phenyl
3-NO2-Phenyl
4-NO2-Phenyl
4-MeO-Phenyl
4-Ph-Phenyl
Naphth-1-yl
Naphth-2-yl
Indan-5-yl
3-Thienyl
1000
35
100
60
120
340
590
850
1400
100
100
80
70
40
150
60
40
40
360
60
20c
20d
20e
20f
20g
20h
20i
20j
20k
21l
21m
20n
20o
20p
20q
20r
20s
20t
20u
20v
20w
As alluded to above, the sulfonamide appeared to be a
critical factor for activity since the related amides 6±8 (and
all substituted benzamide analogues as well,26 data not
shown), were essentially inactive. However, there was a
hint of activity with the phenylacetamide 27 and the car-
bamate 28 (CCR5 IC50=640 and 780 nM, respectively).
The other nitrogen based derivatives that were prepared
in Scheme 2 were also found to be poor inhibitors, such
as the t-butyl carbamate 22 (35% I @ 1000 nM) and
ureas 29 and 30 (69 and 73% I @ 10,000 nM). Deletion
of the entire phenylsulfonamide as in the N-methyl
amine 23 resulted in complete loss of activity. Removal
of just the sulfonyl moiety as in the benzylamines 25
(40% I @ 1000 nM) and 26 (IC50=1000 nM) also led to
greatly diminished activity, indicating that a basic moi-
ety was not tolerated or that the sulfonamide was a key
70
>4000c
100
90
Pyridin-3-yl
Quinolin-8-yl
Quinolin-3-yl
1-Me-Imidazol-4-yl
120
340
aSee ref 25 for the procedure.
bThe IC50 results are an average of three independent titrations with
calculated standard errors of less than 15%. The assay-to-assay varia-
tion was generally Æ2-fold based on the results of a standard compound.
cCompound 20s gave 20% inhibition at 4000 nM, the highest con-
centration tested.

262
C. P. Dorn et al. / Bioorg. Med. Chem. Lett. 11 (2001) 259±264
Scheme 1. Reagents: (a) (COCl)2, DMF (cat), DCM, rt; (b) R1NH2 (aq, 5 equiv), THF, rt; (c) NH4OH, THF, rt; (d) LAH, THF, rt; (e) DIBAL-H,
THF, rt; (f) (Boc)2O, DIPEA, DCM, rt; (g) OsO4 (cat), NMO, 2:1:1 v/v/v acetone/t-butanol/water, rt; (h) NaIO4, 4:1 v/v THF/water, rt; (i) 16-HCl,
NaBH(OAc)3, DIPEA, DCE, rt; (j) 16-HCl, NaCNBH3, DIPEA, MeOH, rt; (k) HCl, MeOH, rt; (l) R2SO2Cl, DIPEA, DCM, rt; (m) Oxone1 (1.2
equiv), MeOH, 20 ꢀC, 2±5 min; (n) Oxone1 (3 equiv), MeOH, rt.
Figure 2. CCR5 activities of N-alkyl derivatives.
pharmacophore for this sca€old. In agreement with the
latter, the corresponding oxygen derivatives 34 and 35
were also found to have poor or no anity (41 and 4% I
@ 1000 nM, respectively).
The phenyl was also determined to be important for
potent CCR5 binding anity in that the N,N-dimethyl-
sulfonylurea 20c, the benzylsulfonamide 20d and the
alkyl sulfonamides 20e±h were all less active (Table 1),
although the cycloalkyl derivatives 20g and 20h dis-
played moderate CCR5 anity. Substitution on the
phenyl ring of the lead compound 4 was then investi-
gated. From this work it was clear that ortho and meta
substitution was not desirable (i.e., compounds 20i, 20j,
and 21l), but para substituents with a broad range of
structural properties were tolerated (i.e., compounds
20k, 21m, 20n, and 20o). In accord with this SAR, the b-
naphthyl derivative 20q was better than the a-naphthyl
20p. Also, 3,4-alkyl disubstitution as with indane 20r
a€orded slightly diminished binding, presumably due to
the meta e€ect.
Scheme 2. Reagents: (a) Oxone1 (1.2 equiv), MeOH, 20 ꢀC, 2±
5 min; (b) TFA, rt; (c) benzaldehyde, NaHB(OAc)3, DIPEA, DCE, rt;
(d) Oxone1 (3 equiv), MeOH, rt; (e) PhCH2COCl, DIPEA, DCM, rt;
(f) PhOCOCl, DIPEA, DCM, rt; (g) PhNCO, DIPEA, DCM, rt; (h)
PhN(Me)COCl, DIPEA, DCM, rt.
3-thienyl isomer 20s indicated a dramatic loss in binding
(20% I @ 4000 nM). A variety of heterocycles, for
example pyridine 20t, quinolines 20u and 20v and imi-
dazole 20w, were also prepared and were shown to have
only moderate activity. Thus, the simple N-(methyl)-N-
(phenylsulfonyl)amino moiety of the original lead 4
remained the optimal functionality at C-1 of this butane
sca€old to achieve potent CCR5 binding.
Based on the initial screening result for the 2-thienyl
derivative 9 (IC50=60 nM), it was hoped that the phenyl
could be replaced with a variety of other aromatic het-
erocycles. However, as also shown in Table 1, even the
The initial lead structures were assayed in a modi®ed
PBMC-based viral replication assay as previously

C. P. Dorn et al. / Bioorg. Med. Chem. Lett. 11 (2001) 259±264
263
4. Cocchi, F.; DeVico, A. L.; Garzino Demo, A.; Arya, S. K.;
Gallo, R. C.; Lusso, P. Science 1995, 270, 1811.
5. Choe, H.; Farzan, M.; Sun, Y.; Sullivan, N.; Rollins, B.;
Ponath, P. D.; Wu, L.; Mackay, C. R.; LaRosa, G.; Newman,
W.; Gerard, N.; Gerard, C.; Sodroski, J. Cell 1996, 85, 1135.
6. Doranz, B. J.; Rucker, J.; Yi, Y.; Symth, R. J.; Samson,
M.; Peiper, S. C.; Parmentier, M.; Collman, R. G.; Doms,
R. W. Cell 1996, 85, 1149.
7. Dragic, T.; Litwin, V.; Allaway, G. P.; Martin, S. R.;
Huang, Y.; Nagashima, K. A.; Cayanan, C.; Maddon, P. J.;
Koup, R. A.; Moore, J. P.; Paxton, W. A. Nature 1996, 381,
667.
8. Deng, H.; Liu, R.; Ellmeier, W.; Choe, S.; Unutmaz, D.;
Burkhart, M.; Di Marzio, P.; Marmon, S.; Sutton, R. E.; Hill,
C. M.; Davis, C. B.; Peiper, S. C.; Schall, T. J.; Littman, D. R.;
Landau, N. R. Nature 1996, 381, 661.
9. Alkhatib, G.; Combadiere, C.; Broder, C. C.; Feng, Y.;
Kennedy, P. E.; Murphy, P. M.; Berger, E. A. Science 1996,
272, 1955.
Scheme 3. Reagents: (a) LAH, THF, rt; (b) BnBr, NaH, DMF, rt; (c)
BzCl, DIPEA, DCM, rt; (d) see Scheme 1, steps g±m.
10. Feng, Y.; Broder, C. C.; Kennedy, P. E.; Berger, E. A.
Science 1996, 272, 872.
11. Paxton, W. A.; Martin, S. R.; Tse, D.; O'Brien, T. R.;
Skurnick, J.; VanDevanter, N. L.; Padian, N.; Braun, J. F.;
Kotler, D. P.; Wolinsky, S. M.; Koup, R. A. Nature Med.
1996, 2, 412.
12. Liu, R.; Paxton, W. A.; Choe, S.; Ceradini, D.; Martin,
S. R.; Horuk, R.; MacDonald, M. E.; Stuhlmann, H.; Koup,
R. A.; Landau, N. R. Cell 1996, 86, 367.
13. Michael, N. L.; Chang, G.; Louie, L. G.; Mascola, J. R.;
Dondero, D.; Birx, D. L.; Sheppard, H. W. Nature Med. 1997,
3, 338.
described.27 Using the R5-tropic HIV-1 YU-2 strain,
compounds 4 and 9 yielded IC95 values in this assay of
6±12 mM, which initially indicated that the desired inhi-
bition of viral entry is possible with a small molecule
antagonist. As indicative of the weaker CCR5 binding
data for most of the subsequent derivatives, poorer
results were generally seen in this screen. However, the 4-
substituted derivatives 20k, 21m, 20n, and 20o appeared
equipotent to 4 and 9.
14. Bates, P. Cell 1996, 86, 1.
15. Moore, J. P. Science 1997, 276, 51.
16. Cohen, O. J.; Kinter, A.; Fauci, A. S. Immunol. Rev. 1997,
159, 31.
In summary, screening of the Merck sample collection
identi®ed several potent lead structures for the inhibition
of [125I]-MIP-1a binding to CCR5. The pivotal nature of
the C-1 N-(methyl)-N-(phenylsulfonyl)amino moiety of
the original lead structure 4 was con®rmed in an extensive
study of other nitrogen- as well as oxygen-based deriva-
tives. Alkyl substituents for the phenyl were also found to
be less active. Further elaboration of the phenyl indi-
cated that para substitution was possible with a variety
of functionality and without loss in binding activity,
which might be important for future modi®cations to
achieve proper pharmacokinetic or biological proper-
ties. While several heterocyclic replacements for the
phenyl were prepared, their binding activity was not
improved over 4. Viral growth inhibition was also
demonstrated with 4 and 9, as well as several analogous,
equipotent compounds. Thus, the N-(methyl)-N-(phe-
nylsulfonyl)amino moiety was identi®ed as an impor-
tant subunit for selective CCR5 binding with this
sca€old and its further utilization will be discussed in
several subsequent publications related to the modi®ca-
tion of the 2-phenyl and 4-piperidine moieties.32
17. For a recent chemokine review, see Baggiolini, M.;
Dewald, B.; Moser, B. Annu. Rev. Immunol. 1997, 15, 675.
18. Pipitone, N.; Pitzalis, C. Curr. Opin. Anti-in¯ammat.
Immunomodulat. Investigat. Drugs 2000, 2, 9.
19. Hall, I. P.; Wheatley, A.; Christie, G.; McDougall, C.;
Hubbard, R.; Helms, P. J. Res. Lett. 1999, 354, 9186.
20. Barcellos, L. F.; Schito, A. M.; Rimmler, J. B.; Vittingh-
o€, E.; Shih, A.; Lincoln, R.; Callier, S.; Elkins, M. K.;
Goodkin, D. E.; Haines, J. L.; Pericak-Vance, M. A.; Hauser,
S. L.; Oksenberg, J. R. Immunogenetics 2000, 51, 281.
21. Sellebjerg, F.; Madsen, H. O.; Jensen, C. V.; Jensen, J.;
Garred, P. J. Neuroimmunology 2000, 102, 98.
22. Horuk, R.; Ng, H. P. Med. Res. Rev. 2000, 2, 155.
23. Shiraishi, M.; Aramaki, Y.; Seto, M.; Imoto, H.; Nishi-
kawa, Y.; Kanzaki, N.; Okamoto, M.; Sawada, H.; Nishi-
mura, O.; Baba, M.; Fujino, M. J. Med. Chem. 2000, 43, 2049.
24. Dorn, C. P.; Finke, P. E.; Oates, B.; Budhu, R. J.; Cald-
well, C. G.; MacCoss, M.; Mills, S. G.; Malkowitz, L.;
Springer, M. S.; Schleif, W. A.; Carella, A.; Carver, G.;
Holmes, K.; Emini, E. A. Abstracts of Papers, Part 2, 219th
National Meeting of the American Chemical Society, San
Francisco, CA, March 26±30, 2000; American Chemical
Society: Washington, DC, 2000; MEDI #117.
25. The CCR5 binding assay was a modi®cation of that
described in ref 33. In summary, the CCR5 binding assay uti-
lized [125I]-MIP-1a as the ligand and was carried out by mixing
200 mL of 50 mM Hepes bu€er, pH 7.4, containing 5 mM
MgCl2, 1 mM CaCl2, 0.5% BSA, 10 mg/mL each of the pro-
tease inhibitors aprotinin, chymostatin and leupeptin, 0.1 mM
PMSF, and 0.01 mM phosphoramidon with 5 mL of com-
pound at increasing concentrations or DMSO for the back-
ground, 20 mL of [125I]-MIP-1a (2Â104 cpm, 2200 Ci/mmol)
and 10 mL of bu€er or unlabeled MIP-1a (100 nM ®nal con-
centration). Assays were initiated by adding intact CHO cells
References and Notes
1. For a review of HIV-1 entry mechanisms and current entry
inhibitors, see Blair, W. S.; Lin, P.-F.; Meanwell, N. A.; Wal-
lace, O. B. Drug Discovery Today 2000, 5, 183.
2. Maddon, P. J.; Dalgleish, A. G.; McDougal, J. S.; Clap-
ham, P. R.; Weiss, R. A.; Axal, R. Cell 1986, 47, 333.
3. For a review of this co-receptor search, see Fauci, A. S.
Nature 1996, 384, 529.

264
C. P. Dorn et al. / Bioorg. Med. Chem. Lett. 11 (2001) 259±264
(3Â104) stably expressing 106 CCR5 receptors per cell and
28. Mills, S. G.; Springer, M. S.; MacCoss, M. WO Patent 98/
25,605, 1998; Chem. Abstr. 1998, 129, 81760e.
29. Hale, J. J.; Finke, P. E.; MacCoss, M. Bioorg. Med. Chem.
Lett. 1993, 3, 319.
incubated for 1 h at 24 ꢀC. Separation of free from bound
[
125I]-MIP-1a was carried out on a Packard Filtermate 196
using GF/C ®lters pre-soaked in 0.33% PEI. IC50 values were
calculated using standard methods from the binding results
using ®nal concentrations of test compound in the range from
0.13 to 10,000 nM. The titrations were usually done in tripli-
cate with the average IC50 value being reported. The calcu-
lated standard errors were normally less than 15% in a given
assay. Assay-to-assay variability was within Æ2-fold based on
a standard compound.32
30. Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Mar-
yano€, C. A.; Shah, R. D. J. Org. Chem. 1996, 61, 3849.
31. Trost, B. M.; Curran, D. P. Tetrahedron Lett. 1981, 22,
1287.
32. Finke, P. E.; Meurer, L. C.; Oates, B.; Mills, S. G.; Mac-
Coss, M.; Malkowitz, L.; Springer, M. S.; Daugherty, B. L.;
Gould, S. L.; DeMartino, J. A.; Siciliano, S. J.; Carella, A.;
Carver, G.; Holmes, K.; Danzeisen, R.; Hazuda, D.; Kessler, J.;
Lineberger, J.; Miller, M.; Schleif, W. A.; Emini, E. A. Bioorg.
Med. Chem. Lett. 2001, 11, 265.
26. MacCoss, M.; Mills, S. G.; Shah, S. K.; Chiang, Y.-C. P.;
Dunn, P. T.; Koyama, H. US Patent 6,013,652, 2000; Chem.
Abstr. 2000, 132, 78470s.
27. Condra, J. H.; Schleif, W. A.; Blahy, O. M.; Gabryelski, L. J.;
Graham, D. J.; Quintero, J. C.; Rhodes, A.; Robbins, H. L.;
Roth, E.; Shivaprakash, M.; Titus, D.; Yang, T.; Teppler, H.;
Squires, K. E.; Deutsch, P. J.; Emini, E. A. Nature 1995, 374, 569.
33. Siciliano, S. J.; Kuhmann, S. E.; Weng, Y.; Madani, N.;
Springer, M. S.; Lineberger, J. E.; Danzeisen, R.; Miller,
M. D.; Kavanaugh, M. P.; DeMartino, J. A.; Kabat, D. J.
Biol. Chem. 1999, 274, 1905.