Evaluation of secondary amide replacements in a series of CCR5 antagonists as a means to increase intrinsic membrane permeability. Part 1: Optimization of gem-disubstituted azacycles

Article abstract of DOI:10.1016/j.bmcl.2009.11.072

Replacement of a secondary amide with an N-acyl or N-sulfonyl gem-disubstituted azacyle in a series of CCR5 antagonists led to the identification of compounds with excellent in vitro HIV antiviral activity and increased intrinsic membrane permeability.

Full text of DOI:10.1016/j.bmcl.2009.11.072

Bioorganic & Medicinal Chemistry Letters 20 (2010) 704–708
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Evaluation of secondary amide replacements in a series of CCR5 antagonists
as a means to increase intrinsic membrane permeability. Part 1:
Optimization of gem-disubstituted azacycles
a,
Rémy C. Lemoine a, , Ann C. Petersen , Lina Setti a, Jutta Wanner a, , Andreas Jekle b, Gabrielle Heilek b,
*
André deRosier b, Changhua Ji b, Pamela Berry c,à, David Rotstein a
a Department of Medicinal Chemistry, Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
b Department of Viral Diseases, Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
c DMPK, Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, CA 9430, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Replacement of a secondary amide with an N-acyl or N-sulfonyl gem-disubstituted azacyle in a series of
CCR5 antagonists led to the identification of compounds with excellent in vitro HIV antiviral activity and
increased intrinsic membrane permeability.
Received 1 September 2009
Revised 11 November 2009
Accepted 16 November 2009
Available online 7 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
CCR5
Azacycles
Intrinsic permeability
CCR5 is one of the many chemokine receptors involved in the
mediation of the inflammation process by activating and inducing
migration of several effector T cell subsets, such as antigen-pre-
senting leukocytes and Th1-polarized T cells. It is now recognized
that CCR5 is among the major regulators of Th1-cell recruitment in
autoimmune diseases.1 In addition to its role as a receptor on
inflammatory leucocytes, CCR5 is also the primary co-receptor
hydrophobes in the Western part of the molecule which will be re-
ferred to as the tail, and an aryl or heteroaryl in the Eastern part,
which will be referred to as the head).
Herein we describe part of our SAR focusing on the tail region.
Specifically, we describe the evaluation of alternative ways to pres-
ent hydrophobe 1 that would not involve the use of a secondary
amide. We hypothesized that by (re)moving the amide hydrogen
bond donor we would increase the intrinsic membrane permeabil-
ity of the series by reducing the desolvation energy cost (i.e., lead-
ing to a decrease in the membrane/solvent partition coefficient)
and/or by reducing interaction with the Pgp efflux pump.4 We
for macrophage-tropic HIV-1 virus. The
CCR5 allele causes the expression of a truncated, nonfunctional
CCR5 protein. Individuals homozygous for the CCR5 32 mutation
D32 mutation in the
D
are resistant to HIV-1 infection while heterozygosity prolongs
seroconversion time. While epidemiologic studies still have to
demonstrate more clearly whether or not CCR5 deficiency is asso-
ciated with overt or covert biologic adverse effects, CCR5 has pro-
ven to be an extraordinary target for the pharmaceutical industry,
with therapeutic applications in HIV-1/AIDS treatment2 as well as
potentials in various chronic or acute inflammatory diseases.
We recently disclosed a series of CCR5 antagonists (Fig. 1).3 This
series contains the four pharmacophores found in most of the
series reported in the literature (i.e., a tertiary basic amine, two
F
F
tail hydrophobe 1
4-, 5-, 6-membered
rings
compound A
N
O
H
HN
H
N
N
O
N
O
N
H
H
F
N
X = H or F
(hetero)aryl head
N
* Corresponding author. Tel.: +1 650 855 5774; fax: +1 650 855 5237.
E-mail address: remy.lemoine@roche.com (R.C. Lemoine).
Present address: Discovery Chemistry, Hoffmann-La Roche, 340 Kingsland Street,
Nutley, NJ 07110, USA.
Present address: DMPK, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ
07110, USA.
tail hydrophobe 2
Figure 1. A representative example of our series of CCR5 antagonists, A, showing
the four required pharmacophores and the proposed azacylic replacements of the
tail secondary amide.
à
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.072

R. C. Lemoine et al. / Bioorg. Med. Chem. Lett. 20 (2010) 704–708
705
Boc
N
borohydride in the presence of cobalt(II) chloride. In this case,
the amine did not cyclize spontaneously onto the ester but re-
quired the use of methylmagnesium iodide as a base. The azetidi-
none was reduced with lithium aluminum hydride and the
azetidine nitrogen was protected with a Boc group. The benzyl
ether was then hydrogenolyzed and the primary alcohol was oxi-
dized to aldehyde 3 with TEMPO/sodium hypochlorite.
The CCR5 antagonists with varying head and tail substituents
(R1, R2, respectively) were prepared according to two general pro-
cedures (Scheme 3).
Boc
Boc
N
1
2
3
N
O
O
O
F
Figure 2. Structure of the three aldehydes required for the synthesis of compounds
in the piperidine-tail, pyrrolidine-tail, and azetidine-tail sub-series, respectively.
Boc
N
H
Boc
N
H
N
O
6
7
O
O
N
N
HN
4
N
(a)
(b)
H
Bn
H
H
O
N
O
O
O
O
N
(a)
Bn
H
(c)
X
Boc
Boc
X
H
N
2
5
H
N
N
(e)
(d)
9
HN
O
(a)
(b), (c)
N
O
OH
OH
R1
R2
Boc
8
Scheme 1. Synthesis of aldehyde 2. Reagents and conditions: (a) LHMDS, ethyl
bromoacetate, THF, À78 °C to rt, 24 h, 69%; (b) Raney-nickel, H2 (50 psi), MeOH,
48 h, 92%; (d) LAH, THF, 70 °C, overnight, 78%; (d) Boc2O, NaHCO3, THF/H2O, rt,
overnight, 80%; (e) Dess–Martin periodinane, CH2Cl2/tBuOH, rt, overnight, used
crude.
N
N
H
H
N
N
O
O
N
N
H
H
R1
R1
X
X
mostly focused on saturated gem-disubstituted 4-, 5-, and 6-azacy-
cles (Fig. 1).
(d), (e) or (f)
The synthesis of compounds in the piperidine-, pyrrolidine-,
and azetidine-tail sub-series required the synthesis of aldehydes
1, 2, and 3 (Fig. 2).
Scheme 3. General synthetic approach of analogs in the three sub-series. Reagents
and conditions: (a) NaBH(OAc)3, CH2Cl2, rt, 2 h; (b) H2 (1 atm), 20% Pd(OH)2, EtOH,
rt, 24 h, >95%; (c) R1COOH, EDCI, HOBt, Et3N (or DIPEA), CH2Cl2, rt, overnight, 40–
80%; (d) HCl 4 M in dioxane, CH2Cl2, rt, 2 h; (e) R2COOH, EDCI, HOBt, Et3N (or
DIPEA), CH2Cl2, rt, overnight or R2COCl, pyridine (or Et3N), CH2Cl2, 0 °C to rt,
overnight, or R2SO2Cl, Et3N, CH2Cl2, 0 °C to rt, overnight, 50–80%.
Aldehyde 1 was prepared according to literature procedures.5
Racemic aldehyde 2 was prepared according to the procedure de-
picted in Scheme 1.
Phenylacetonitrile was bis-alkylated with ethyl bromoacetate.
Reduction of the nitrile was carried out with hydrogen and Ra-
ney-nickel and the corresponding amine cyclized in situ with one
of the appended acetic esters to form lactam 4. Both the lactam
and the remaining ester were reduced with lithium aluminum hy-
dride to give aminoalcohol 5. The pyrrolidine nitrogen was pro-
tected with a Boc group and the alcohol was oxidized to the
aldehydes using Dess–Martin periodinane.
The preparation of aldehyde 3 was carried out by a similar se-
quence (Scheme 2). Ethyl phenylcyanoacetate was alkylated with
benzyl 2-bromoethyl ether. Selective reduction of the nitrile to
the corresponding primary amine was performed with sodium
OH
O
H
O
O
O
O
a
b
c
d
f
e
R2
=
N
g
R1 =
H
N
O
N
N
N
N
R1
F
N
Figure 3. Representative compounds 10–16 prepared in the piperidine-tail sub-
series.
N
CO2Et
(a)
H
N
N
CO2Et
OBn
O
(b) (c)
OBn
(d)
Table 1
RANTES binding inhibition,6 cell fusion inhibition,7 and antiviral activity8 of
piperidine-tail compounds 10–16
Boc
N
Boc
H
N
Compounds Binding inhibition Cell fusion inhibition Antiviral activity
(g)
(e) (f)
N
R2 R1 IC50 (nM)
IC50 (nM)
IC50 (nM)
a
a
a
O
OH
OBn
10
11
13
14
15
16
a
b
c
d
e
c
f
f
f
f
f
g
>500
102
59
>2500
160
129
339
1000
37
ndb
>400
67
447
ndb
18
ndb
167
15
Scheme 2. Synthesis of aldehyde 3. Reagents and conditions: (a) NaH, benzyl 2-
bromoethyl ether, DMF, 0–60 °C, overnight, 60%; (b) NaBH4, CoCl2Á6H2O, MeOH, rt,
16 h, 40%; (c) MeMgI, Et2O/THF, 0 °C to rt, 3 h, 40%; (d) LAH, THF, 60 °C, 6 h; (e)
Boc2O, NaHCO3, THF/H2O, rt, 3 days, 49% (over two steps); (f) H2 (1 atm), 20%
Pd(OH)2, EtOH, rt, 24 h, >95%; (g) TEMPO, NaOCl, NaHCO3, CH2Cl2/H2O, 0 °C to rt,
4 h, used crude.
a
Values are means of at least two experiments.
Not determined.
b

706
R. C. Lemoine et al. / Bioorg. Med. Chem. Lett. 20 (2010) 704–708
Reductive N-alkylation of the aldehydes with template 63
OH
O
O
O
formed 7 as an orthogonally protected bis secondary amine inter-
mediate. The pyrrolidine N-benzyl group was hydrogenolyzed to
give an amine which was acylated using standard procedures to
give 8. Alternatively, the aldehydes were reacted with template
93 in which one of the pyrrolidine amines was already bearing
the head acyl group R1. The tail Boc group was then hydrolyzed
and the tail amine was acylated or sulfonylated.
From the first parallel synthesis array that was prepared (Fig. 3),
it became apparent that only hydrophobic bulky acyl groups R2
were tolerated (Table 1).
O
O
O
a
b
c
d
e
F
R2
N
O
S
F
O
H
O
g
f
N
O
F
N
H
O
R1
F
h
k
i
j
l
m
Introduction of some polarity such as in compound 15 abol-
ished the antiviral activity. The best compound prepared in the ar-
ray was 13 which showed an antiviral activity of 67 nM. During our
SAR studies (data not shown), we discovered that substituted het-
eroaryl R1 heads usually increased antiviral activity. We thus re-
placed the standard R1 = 5-(4,6-dimethylpyrimidine) head in
compound 13 with R1 = 5-(4,6-dimethyl-2-cyanopyridine)9 and
noticed a close to fourfold improvement in antiviral activity (com-
pound 16) (see Table 1).
We prepared a set of compounds varying the size of the pyrrol-
idine nitrogen substituent R2 (Fig. 4) and observed that in this sub-
series, the type of groups tolerated was even more limited than in
the piperidine-tail sub-series, with cyclopentyl being the best. In
this sub-series as well, R1 = 5-(4,6-dimethyl-2-cyanopyridine) in-
creased activity up to 15-fold compared to R1 = 5-(4,6-dimethyl-
pyrimidine) (compounds 17 and 18). We did not separate the
enantiomers of 17–20, the activity reported in Table 2 are for the
racemic mixtures (see Table 2).
Having learned from the piperidine- and pyrrolidine-tail sub-
series that CCR5 did not offer much space in the hydrophobe 1
binding region, a limitation that led to a mostly flat SAR, we antic-
ipated that as we reduced the size of the tail azacycle to a four-
membered ring we would be able to attach more diverse types of
substituents than in the previous sub-series (Fig. 5). This turned
out to be the case (see Table 3).
Unlike the piperidine- and pyrrolidine-tail sub-series, the azeti-
dine-tail sub-series accepted both amide and sulfonamide groups
(compounds 25 and 30). Also, while slightly polar groups were tol-
N
N
N
N
N
N
N
F
F
F
F
N
F
F
Figure 5. Representative compounds 25–41 prepared in the azetidine-tail series.
Table 3
RANTES binding inhibition,6 cell fusion inhibition,7 and antiviral activity8 of the
azetidine-tail compounds 25–41
Compounds Binding inhibition Cell fusion inhibition Antiviral activity
R2 R1 IC50 (nM)
IC50 (nM)
IC50 (nM)
a
a
a
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
a
b
c
d
e
f
g
h
c
g
h
g
h
g
h
g
h
i
i
i
i
i
i
i
i
j
j
j
k
k
l
l
m
m
67
28
55
27
56
27
28
30
38
30
66
38
60
17
24
21
22
65
136
559
186
310
458
10
93
92
5
168
15
53
<0.3
1
60
53
204
77
130
40
21
39
59
4
23
12
55
2
4
2
3
61.2
4
a
Values are means of at least two experiments.
a
b
O
O
erated (compounds 27 and 29), better antiviral activity was seen
with hydrophobic substituents. In general, the more hydrophobic
the substituent, the more active the compound (25, 26, 28, 31,
32), with 4,4-difluorocyclohexyl being the best. The activity of
the series was further improved by introducing R1 = 5-(4,6-di-
methyl-2-cyanopyridine) as the head substituent (compounds 31
and 38). When the azetidine nitrogen R2 acyl substituent was
3,3-difluorocyclobutyl, electron-withdrawing groups such as tri-
fluoromethyl at C2 of the pyrimidine improved potency close to
twofold (compounds 35 compared to 32). When the pyridine head
was used instead of the pyrimidine head, C2-substitution improved
potency close to 10-fold (compounds 39 and 41 compared to 37).
The boost in potency was the same for both substituents (cyano
or trifluoromethyl). Interestingly, when the azetidine nitrogen R1
acyl substituent was the optimal 4,4-difluorocyclohexyl, the com-
bined effect of a better tail hydrophobe 1 and an optimal substi-
tuted pyridine head seemed to be more than additive. Indeed,
compound 40 (substituent = trifluoromethyl) was greater than
10-fold more potent (activity below detection level) than the
unsubstituted equivalent, 36. Compound 38 (substituent = cyano)
on the other hand was only sixfold more potent than 36, which
indicated to us that the trifluoromethyl substituent was better
than the cyano substituent at increasing the activity. In other
words, the right combination of tail and head substituents led to
=
R2
N
H
c
d
N
O
N
H
R1
=
N
N
N
N
Figure 4. Representative compounds 17–20 prepared in the pyrrolidine-tail sub-
series.
Table 2
RANTES binding inhibition,6 cell fusion inhibition,7 and antiviral activity8 of
pyrrolidine-tail compounds 17-20
Compounds Binding inhibition Cell fusion inhibition Antiviral activity
R2 R1 IC50 (nM)
IC50 (nM)
IC50 (nM)
a
a
a
17
18
19
20
a
a
b
b
c
d
c
116
46
242
69
361
85
351
24
1943
360
ndb
nd
d
a
Values are means of at least two experiments.
Not determined.
b

R. C. Lemoine et al. / Bioorg. Med. Chem. Lett. 20 (2010) 704–708
707
Table 4
CO2H
O
O
OMe
O
(b) (c)
(a)
Apparent fluxes in 21-day Caco-2 assays with or without elacridar and apparent
efflux ratio
N
F
N
F
CO2Me
CO2Me
Compounds Papp: ABa
21-day Caco-2
Papp: AB
21-day Caco-2 with 21-day Caco-2
Efflux ratio BAb/AB
43
42
F
without elacridar
2
lM elacridar
without elacridar
CO2H
COCl
CO2Et
A
0.22
2.98
2.73
2.55
0.53
0.44
1.72
1.59
5.41
55.38
6.39
6.78
8.25
32.26
40
(e)
(d)
13
16
18
38
39
40
41
14.9
17.5
14.3
10.2
10.7
14.6
13.2
NH
O
N
Cl
N
45
48
46
Cl
(f)
CO2H
CO2Et
CO2Et
10.35
10.69
N
N
N
Apparent apical to basolateral fluxes (cm/s  10À6).
a
(c)
(g) (h)
44
F
47
Apparent basolateral to apical fluxes (cm/s  10À6), data not shown.
b
I
F
F
F
F
F
compartments were performed and were shown to be within the
set target values in all cases. Finally, the good solubility in the as-
say buffer of the compounds tested did not lead us to believe that
the observed results could be biased either way. We describe here
the results of only a small set of compounds (see Table 4), but the
trend was shown to be general.
Scheme 4. Syntheses of acids 42 and 44. Reagents and conditions: (a) Cs2CO3,
MeOTf, (b) trifluoroacetamide, acetone/MeOH, 0 °C to rt overnight, 44%; (c) KOH,
EtOH/H2O, 40 °C, overnight, acidic work-up, 66% for acid 42, 75% for acid 44; (d)
POCl3, 80 °C; (e) EtOH, 0 °C then rt, 30 min (60% over two steps); (f) NaI, TMSCl,
75 °C, 4 h,%; (g) CuI, KF, TMSCF3, NMP, 70 °C, overnight; (h) H2 (40 psi), 10% Pd/C,
AcOH, MeOH, rt, 48 h, 45% over three steps.
The apical to basolateral (AB) apparent fluxes (with elacridar) of
compounds 13, 16, 18, 38, 39, 40, 41 were all at least twofold great-
er than that of the representative primary-amide-tail compound A
(Fig. 1). Since no correlation could be made between the AB appar-
ent fluxes (without elacridar) and clog P, log D, or polar surface
area, we concluded that this was due to an overall increase in
intrinsic permeability of the gem-disubstituted azacyclic series
versus our original series. The increase was even noticeable in
the AB apparent fluxes (without elacridar) of compounds 13, 16,
18, 40, and 41 in which the possible efflux influence of Pgp seemed
to be reduced and/or compensated. Interestingly, despite the in-
crease in intrinsic permeability, the AB apparent fluxes (without
elacridar) of compounds 38 and 39 were fairly low compared to
other compounds. To us, this indicated that Pgp might transport
those compounds more efficiently and that the efflux might no
longer be compensated by the increase in permeability.
While we were able to improve the intrinsic permeability of our
lead series in vitro, high metabolic clearance precluded us from
observing an effect in vivo (data not shown). However, it will be
addressed on a parallel series in part 2.
In this Letter, we described the synthesis and evaluation of
three new sub-series of saturated gem-disubstituted azacylic
CCR5 antagonists. Careful tuning of head and tail substituents al-
lowed the identification of derivatives with excellent in vitro
HIV-1 antiviral activity. Moreover, compounds within the three
sub-series showed increased intrinsic permeability compared to
that of the original primary amide tail series, as measured in the
Caco-2 permeability assay.
the identification of compounds with substantially better than ex-
pected antiviral potencies. The mechanistic explanation of such a
cooperative effect is still not clear, but we hypothesized that it
could be related to some dynamic effect of such combination lead-
ing to a more efficient allosteric rearrangement10 of the CCR5
receptor which is required for antiviral activity after binding of
the antagonist.
The preparation of compounds 33–35 and 40, 41 required the
introduction of heteroaryl heads derived from acid 42 (R1COOH
where R1 = j) and acid 44 (R1COOH where R1 = m). They were syn-
thesized according to the sequences depicted in Scheme 4. Acid 42
was synthesized in three steps from the commercially available 3-
methyl-pentane-2,4-dione using a modified literature procedure.11
The enolate of 2-acetyl-3-oxo-butyric acid methyl ester was
trapped with methyl triflate to form intermediate 43 which was
then treated with trifluoroacetamidine under neutral conditions
at room temperature to give the ethyl ester of 42 in 44% yield.12
The ester was then hydrolyzed to the acid with potassium hydrox-
ide. Evaporation of the reaction mixture and acidification of an
aqueous solution of the recovered residue led to the crystallization
of the acid 42 which was obtained in 66% yield (see Scheme 4).
The synthesis of acid 44 was carried out in six steps from the
commercially available 2,4-dimethyl-6-oxo-1,6-dihydro-pyridine-
3-carboxylic acid. The starting material was treated with phospho-
rus oxychloride to give acyl chloride 45 which was then treated
with ethanol to give ester 46. The chloride was exchanged with
an iodide and 47 was then treated with trifluoromethyl carbene
generated in situ from trimethylsilyltrifluoromethane and potas-
sium fluoride. Subsequent hydrogenolysis allowed the purification
of the trifluoropyridine 48 from the unsubstituted pyridine gener-
ated by reduction of chloride 46 carried over from the iodination
reaction or reduction of the unreacted iodide 47. Saponification
of the ester led to the isolation of the acid 44 in 75% yield.
Representative compounds from the three sub-series were
tested in a 21-day Caco-2 permeability assay.13 In order to address
the potential influence of Pgp in the apparent permeability, the as-
say (in both directions) was run in the presence or absence of the
Pgp inhibitor elacridar.14 We believe that the results of the assay in
the presence of elacridar gave us a good estimate of the intrinsic
permeability versus the apparent permeability observed in the as-
say without elacridar, by eliminating the influence of Pgp on po-
tential substrates. Also, in order to alleviate the possibility of
over- or under-estimating the flux values, mass recovery in both
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