Exploration of piperidine-4-yl-aminopyrimidines as HIV-1 reverse transcriptase inhibitors. N-Phenyl derivatives with broad potency against resistant mutant viruses

Article abstract of DOI:10.1016/j.bmcl.2010.08.068

Further investigation of the recently reported piperidine-4-yl- aminopyrimidine class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) has been carried out. Thus, preparation of a series of N-phenyl piperidine analogs resulted in the identification of 3-carboxamides as a particularly active series. Analogs such as 28 and 40 are very potent versus wild-type HIV-1 and a broad range of NNRTI-resistant mutant viruses. Synthesis, structure-activity relationship (SAR), clearance data, and crystallographic evidence for the binding motif are discussed.

Full text of DOI:10.1016/j.bmcl.2010.08.068

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6020–6023
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Exploration of piperidine-4-yl-aminopyrimidines as HIV-1 reverse
transcriptase inhibitors. N-Phenyl derivatives with broad potency against
resistant mutant viruses
Guozhi Tang a, , Denis J. Kertesz b, , Minmin Yang a, , Xianfeng Lin a, Zhanguo Wang a, Wentao Li a,
⇑
⇑
Zongxing Qiu a, Junli Chen a, Jianghua Mei a, Li Chen a, Taraneh Mirzadegan b,à, Seth F. Harris b,§
,
Armando G. Villaseñor b, Jennifer Fretland b, William L. Fitch b, Julie Qi Hang b, Gabrielle Heilek b,
Klaus Klumpp b
a Roche R&D Center China, Shanghai 201203, China
b Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 June 2010
Revised 29 July 2010
Accepted 13 August 2010
Available online 19 August 2010
Further investigation of the recently reported piperidine-4-yl-aminopyrimidine class of non-nucleoside
reverse transcriptase inhibitors (NNRTIs) has been carried out. Thus, preparation of a series of N-phenyl
piperidine analogs resulted in the identification of 3-carboxamides as a particularly active series. Analogs
such as 28 and 40 are very potent versus wild-type HIV-1 and a broad range of NNRTI-resistant mutant
viruses. Synthesis, structure–activity relationship (SAR), clearance data, and crystallographic evidence for
the binding motif are discussed.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Non-nucleoside reverse transcriptase
Non-nucleoside reverse transcriptase
inhibitor (NNRTI)
HIV-1
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are an
important part of the successful combination therapy for HIV-1
known as HAART (highly active anti-retroviral therapy).1 The effi-
cacy of NNRTIs results from their binding to an allosteric site of
HIV reverse transcriptase (HIV-RT), which dramatically decreases
the ability of that enzyme to function in reproduction of the virus.2
Efavirenz and Nevirapine are two very effective first generation
NNRTIs, but a major problem with them has been the development
of viral resistance. This results from a low genetic barrier of the RT
enzyme to mutation and several viable mutant viruses can survive
treatment with these agents.3,4 Thus, there is a need for new NNR-
TIs with activity against the clinically significant mutant strains as
well as the wild-type HIV-1 virus.5
Etravirine has a broad range of activity against clinically important
mutant RT viruses but its development as a drug was hindered by
poor solubility.8 GW-678248 displays a good mutant profile along
with good pharmacokinetic properties (PK) and was developed as a
prodrug.9 We recently reported our efforts to combine aspects of
these two chemotypes, which resulted in the discovery of piperi-
dine-4-yl-aminopyrimidine structures such as 1–3 (Fig. 1). Our
H
Cl
O
H2N
Br
N
O
N
SO2NH2
O
O
N
H
N
CN
Etravirine (TMC125), which was recently approved for use in
treatment-experienced HIV patients,6 and the benzophenone
GW-6782487 are examples of second generation NNRTIs (Fig. 1).
Cl
CN
Etravirine(TMC125)
GW-678248
CN
H
H
SO2R2
N
O
N
N
N
⇑
Corresponding authors. Tel.: +86 21 38954910; fax: +86 21 50790291 (G.T.);
tel.: +1 650 961 4224 (D.J.K.).
N
N
N
N
Br
Br
E-mail addresses: gordon.tang@roche.com (G. Tang), deniskertesz@comcast.net
R1
O
(D.J. Kertesz).
1: R1 = Cl, R2 = NH2
2: R1 = H, R2 = Me
Present address: Pharmablock R&D Co. Ltd, 10 Xuefu Rd., Pukou, Nanjing 210061,
PR China.
CN
3
CN
à
Present address: Johnson and Johnson R&D, LLC, San Diego, CA 92121, USA.
Present address: Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
§
Figure 1. Structures of Etravirine, GW-678248, and aminopyrimidine leads 1–3.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.068

G. Tang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6020–6023
6021
R4
R4
R3
Table 1
N
Cl
Enzyme inhibitory potency of N-phenyl piperidines 11–25
OH
O
a
N
R3
+
H
4: R3 = H
N
N
N
O
N
Cl
NC
5: R3 = Br
NC
R1
R2
6: R3 = others
Cl
N
R3
N
NHBoc
NHBoc
NHBoc
R2
R1
11-25
CN
R3
b or c
d
N
N
+
X
R2 = CN
ID #
R1
R2
H
WTa
IC50 (nM)
KYa
IC50 (nM)
Y188La
IC50 (nM)
N
H
X = F, Br, or I
NH2
R2
3
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
H
H
H
H
H
H
H
H
H
H
H
H
Br
H
Br
H
Br
Br
H
Br
Br
H
Br
H
Br
H
H
Br
—
192
24
19
15
32
19
7
24
42
11
103
51
16
9
112
>1000
72
622
967
30
16
>1000
100
238
352
772
>1000
192
265
334
>1000
105
194
—
70
34
—
831
—
—
—
—
—
—
874
108
R1
CONH2
R1
4-SO2NH2
3-SO2NH2
3-SO2NH2
2-CONH2
3-CONH2
3-CONH2
4-CONH2
3-SO2CH3
3-SO2CH3
3-CN
7
8
R2
R1
H
N
N
N
e
f
7, 8
N
N
R3
O
CN
R2
R1
R4
11-25 (R4 = Me)
9
3-CN
10: R2 = CONH2
32-40 (R4 = others)
H
3-CO2H
3-CONH2
3-CONH2
3-CONH2
—
4-Cl
5-Cl
6-Cl
—
Scheme 1. Reagents and conditions: (a) K2CO3, DMF, rt; (b) K2CO3, DMSO, 140 °C
(X = F); (c) K2CO3, CuI, proline, DMSO, 90 °C (X = Br, or I); (d) NaOH, H2O2, EtOH,
40 °C; (e) TFA, CH2Cl2, rt; (f) 4 or 5, DIPEA, NMP, 100 °C.
28
76
26
>1000
19
Etravirine
a
Values are the mean of at least two independent heteropolymeric enzyme
inhibition assays (see Ref. 12).
publication describing that work focused on optimization of the
N-benzyl piperidines, which led to compounds 1 and 2 as highly
active examples of the series.10 In the present effort we have ex-
plored a series of N-phenyl analogs for which compound 3 was
the lead structure. As will be described below, that effort has
yielded compounds with potency profiles well surpassing those
of the initial derivatives 1–3.
Table 2
Enzyme inhibitory potency of 50-substituted analogs
H
N
O
N
The first phase of our new explorations involved determining
the best substitution pattern for the N-phenyl ‘head’ ring. For this
purpose we kept the 2,6-dimethyl-4-cyanophenyl ‘tail’ ring con-
stant and used either Br or H as a 50-substituent on the pyrimidine
ring. The syntheses of analogs 12–25 required the initial prepara-
tion of the intermediates 4, 5, 7, and 8 as shown in Scheme 1. Thus,
the treatment of 2,4-dichloropyrimidines with 3,5-dimethyl- 4-cy-
ano-phenol gave phenyl ethers 4 and 5 in excellent yields. Sepa-
rately, the use of 4-Boc-aminopiperidine to displace the fluorine
atom from an appropriately substituted aryl ring gave the N-phen-
ylpiperidine intermediates 7. In an alternative procedure, the inter-
mediates 7 could also be made by copper catalyzed couplings of
aryl iodides or bromides with the same aminopiperidine reagent.
Compounds wherein R2 was to be carboxamide were made by
hydrolysis of the corresponding nitriles 7 to give intermediates 8.
Deprotections of 7 and 8 gave amine products 9 and 10, and their
further couplings with the 2-chloropyrimidine reagents 4 and 5
gave the desired compounds of structures 11–25.11 The inhibitory
activities of these products were measured against recombinant
wild-type and mutant RT enzymes, and results for the wild-type
(WT), the K103N/Y181C double mutant (KY) and the Y188L mutant
are given in Table 1.12
The preferred substitution pattern for the N-benzyl piperidine
series represented by 1 and 2 had been a polar substituent at the
4-position of the head group together with a small lipophilic
2-substituent.10 It was appreciated that the best pattern for the
N-phenyl piperidines would likely vary from the N-benzyl series
due to differences in geometry and available vectors. As shown
in Table 1, a polar substituent on the N-phenyl head group was in-
deed needed for the best activity in this series as well, but the
3-position was the preferred location. Thus, the 3-sulfonamide 12
and the 3-carboxamides 15 and 16 were among the most potent
N
R3
N
CONH2
CN
ID #
R3
WTa
KYa
Y188La
IC50 (nM)
IC50 (nM)
15
16
26
27
28
29
30
31
Br
H
Cl
F
CH3
CF3
19
7
30
16
13
14
15
41
17
64
70
34
203
35
—
—
118
—
24
14
12
86
19
109
NH2
NO2
a
Values are the mean of at least two independent heteropolymeric enzyme
inhibition assays (see Ref. 12).
inhibitors of the WT, KY, and Y188L enzymes. In fact, the carbox-
amide was clearly the superior 3-substituent over the sulfonamide,
cyano, sulfone, and carboxylic acid groups exemplified in deriva-
tives 12–13 and 18–22. Addition of a second head-ring substituent
in compounds 23–25 did not add activity in this series. The supe-
rior potencies associated with the 3-carboxamide in this data set
were sufficiently striking that this substituent was kept constant
in our further structural modifications.
The next variable we examined was the 50-substituent on the
pyrimidine core ring. Our crystal structures and modeling studies
had suggested that such substituents could occupy a small hydro-
phobic pocket formed by G138, V179 and Y181, and modulate the
potency of these molecules. Thus, several analogs were made

6022
G. Tang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6020–6023
Table 3
according to the chemistry of Scheme 1 by employing intermedi-
ates 6 where R3 is other than H or Br, and activities of the products
26–31 are summarized in Table 2. Although no single substituent
was clearly superior, it did appear that small hydrophobic groups
(R3 = H, F or CH3) are preferable at this position.
The two final SAR variables we investigated were substituents
on the tail ring and the nature of the linker to that ring. The 2,6-
bis-substitution pattern of the tail ring appears necessary to main-
tain the orthogonal relationship of that ring with the core, which
seems to be a characteristic of Etravirine-like structures.10 The
flanking methyl groups are suspected as a metabolic liability, but
other substituents can be tolerated in at least one of the two posi-
tions.13 Exchange of the ether linkage for an amino (NH) might be
expected to affect activity via effects on the conformation and
polarity of the molecule.
Enzyme inhibitory potency of compounds with tail ring and linker modifications
H
N
N
N
N
R3
CONH2
Y
R4
CN
ID #
R3
R4
Y
WTa
IC50 (nM)
KYa
IC50 (nM)
Y188La
IC50 (nM)
16
28
32
33
34
35
36
37
38
39
40
43
44
45
46
H
CH3
H
Br
Cl
CH3
H
Br
Cl
CH3
Br
H
Br
H
Br
CH3
CH3
Cl
Cl
Cl
O
O
O
O
O
O
O
O
O
O
O
NH
NH
NH
NH
7
12
8
28
20
6
18
56
19
21
6
23
19
36
15
16
15
6
34
—
—
413
48
—
64
—
101
66
29
—
75
—
17
13
20
54
28
50
11
5
18
16
17
10
Phenyl ethers 32–40 where tail ring substituent R4 is other than
methyl were prepared from the appropriate intermediates 4–6 by
the chemistry of Scheme 1. A different method was used for syn-
thesis of the NH-linked compounds in order to achieve regioselec-
tivity in the coupling reactions. Thus, as shown in Scheme 2, the
treatment of 4-chloro-2-methylsulfanyl pyrimidine with 2,6-
disubstituted-4-cyanoanilines followed by an MCPBA oxidation
gave methylsulfones 41. The 50-Br intermediates 42 were prepared
by bromination of 41 with NBS. Displacement of the methylsulfone
groups of 41 and 42 with amine 10 then afforded the desired NH-
linked products 43–46.11
Enzyme inhibitory activities are presented in Table 3 for repre-
sentative products containing the R4-groups Cl, OCH3, CH3, and F,
the linkers O and NH, and various 50-substituents. Most of these
combinations seemed well tolerated. Indeed, several compounds
had quite excellent enzyme potencies, with 32 and 40 demonstrat-
ing single digit nanomolar activities versus wild-type and KY mu-
tant enzymes.
At this point antiviral data was collected for several of the most
promising compounds. Thus, inhibitory potencies were measured
for the WT, KY, and Y188L viruses, as well as for two other fre-
quently encountered mutant strains L101I/K103N and V106A.14
Their protein shifted potencies against the WT virus were also
determined.15 This data along with human liver microsome clear-
ance data (HLM) for selected analogs and reference NNRTIs is pre-
sented in Table 4. It is evident from the table that activity profiles
significantly superior to the reference compounds have been
achieved with the present scaffold. The most potent compounds
are 28, 32, 35, and 46, and the profile of 28 is particularly impres-
sive when the low protein shift is noted. Unfortunately, these most
potent compounds were also cleared rapidly in HLM assays. The
facile microsomal clearance is not totally unexpected given the
lipophilicity of the molecules and the known susceptibility of aro-
Cl
OCH3
OCH3
OCH3
OCH3
F
CH3
CH3
Cl
Cl
42
a
Values are the mean of at least two independent heteropolymeric enzyme
inhibition assays (see Ref. 12).
matic methyl groups to oxidative metabolism. Indeed, metabolite
ID studies confirmed the oxidation of the tail ring methyl groups,
and somewhat unexpectedly, they also found products of degrada-
tion via N-dephenylation.16
A second set of analogs of interest includes 40, 43, and 44,
which all show reasonable clearance as well as good antiviral activ-
ity. Compound 44 in particular has a mutant profile very similar to
that of Etravirine, and 40 has even better potential when the pro-
tein shift is considered. Although it is difficult to generalize about
the antiviral SAR of the N-phenyl piperidine scaffold, it does appear
that the small, lipophilic 50-substituents (H, Br or CH3) on the core
ring are most consistent with good potency, and a variety of R4-
groups are tolerated in place of methyl as a tail ring flanking sub-
stituents. Also, a source of extra polarity in the molecule seems
helpful in reducing HLM clearance. This is seen with the 2-fluoro
tail ring compound 40 and the NH-linked analogs 43 and 44,
although not with 46.
Co-crystals of 3-substituted analogs with wild-type HIV-1 en-
zyme could not be prepared in good enough quality for X-ray dif-
fraction work. However, it was possible to determine a crystal
structure for the 4-sulfamoylphenyl piperidine 11,17 and an over-
lay with our previously reported structure of the 4-methanesulfo-
nyl N-benzyl piperidine 210 is shown in Figure 2. It is clear that a 3-
substituent on the N-phenyl group would occupy essentially the
same space as the 4-substituent of the N-benzyl ring, and this is
evidently the preferred locus as implied by the SAR of both series.
It places the polar head group near the solvent–protein interface
and it is also well positioned for interaction with the V106 back-
bone NH. Further noteworthy interactions include the edge-to-face
approach of the tail ring to W229, H-bonding of the pyrimidinyl
NH with the K101 backbone carbonyl, and the stacking of the
head-ring with P236. Also of interest is the nearly identical place-
ment of the piperidine ring nitrogens of 11 and 2. The latter had
been shown to make a hydrogen bond with the backbone NH of
K103 via a water molecule,10 and a similar interaction is implied
for 11. The diffraction density for the bound water is not as conclu-
sive in the case of 11, but crystal structure distances and modeling
support its presence in essentially the same position as noted for 2.
O
O
N
S
R4
N
SCH3
NH2
N
R3
a, b
+
N
HN
R4
NC
N
Cl
CN
41: R3 = H
42: R3 = Br
c
H
N
d
N
R3
N
CONH2
HN
R4
43-46
CN
Scheme 2. Reagents and conditions: (a) neat, 160 °C; (b) MCPBA, CH2Cl2, 20 °C; (c)
NBS, CH2Cl2, 20 °C; (d) 10, DIPEA, DMSO, 150 °C.

G. Tang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6020–6023
6023
Table 4
Antiviral potencies of several aminopyrimidine derivatives compared to reference compounds
Analog
R3a
R4a
Ya
WTb
WT, 40% HuSc
Protein shiftc
K103N/Y181Cb
Y188Lb
K103N/L100Ib
V106Ab
HLMd
28
32
35
40
43
44
46
1
CH3
H
CH3
Br
H
Br
Br
—
CH3
Cl
Cl
O
O
O
O
NH
NH
NH
—
—
—
1.1
0.4
1.6
7.3
0.5
1.5
1.2
6.1
2.1
1.8
4.5
7.5
4.0
17.4
8.6
2.5
27.1
9.8
8.6
8.0
12.2
10.1
3.8
5.3
5.5
28.0
15.2
11.7
4.9
7.3
9.2
82.8
<1.2
1.6
1.4
5.5
3.7
2.7
<1.2
2.8
3.1
>100
6.7
42.0
7.4
18.4
>100
10.8
2.9
5.9
9.5
>100
0.6
0.4
0.7
3.9
0.8
0.9
0.5
2.0
2.0
5.7
730
274
580
82
31
42
418
151
29
14.2
18.2
13.9
14.2
9.9
48.7
25.9
18.1
F
CH3
CH3
Cl
—
—
Etravirine
Efavirenz
—
—
—
—
a
b
c
Substituents R3, R4, and Y are as defined in Table 3.
Cell based antiviral assay in the presence of 10% FBS, IC50’s in nM (see Ref. 14).
WT activity in the presence of 40% human serum (HuS) indicates protein shifted activity, also given as ratio (see Ref. 15).
d
Human liver microsome clearance in ll/min/mg.
Acknowledgments
The authors thank Eric Sjogren and Robert Wilhelm for many
helpful discussions. We also are grateful to Yongguo Li, and Wen-
zhi Chen for their analytical expertise, and thank Yu Li, Eugene
Chow and Jun Mei Yan for their bioassay diligence.
References and notes
1. De Clercq, E. Chem. Biodivers. 2004, 1, 44.
2. Balzarini, J. Curr. Top. Med. Chem. 2004, 4, 921.
3. Wainberg, M. A. J. Acquir. Immune Defic. Syndr. 2003, 34, S2.
4. Delaugerre, C.; Rohban, R.; Simon, A.; Mouroux, M.; Tricot, C.; Agher, R.;
Huraux, J. M.; Katlama, C.; Calvez, V. J. Med. Virol. 2001, 65, 445.
5. Sweeney, Z. K.; Klumpp, K. Curr. Opin. Drug Discov. Devel. 2008, 11, 458.
6. Madruga, J. V.; Cahn, P.; Grinsztejn, B.; Haubrich, R.; Lalezari, J.; Mills, A.;
Pialoux, G.; Wilkin, T.; Peeters, M.; Vingerhoets, J.; de Smedt, G., et al Lancet
2007, 370, 29.
7. Ren, J.; Chamberlain, P. P.; Stamp, A.; Short, S. A.; Weaver, K. L.; Romines, K. R.;
Hazen, R.; Freeman, A.; Ferris, R. G.; Andrews, C. W.; Boone, L.; Chan, J. H.;
Stammers, D. K. J. Med. Chem. 2008, 51, 5000.
8. Davies, S. L.; Castaner, J.; Silvestre, J. S.; Bayes, M. Drugs Future 2005, 30, 462.
9. Sahlberg, C.; Zhou, X. Anti-infect. Agents Med. Chem. 2008, 7, 101.
10. Kertesz, D. J.; Brotherton-Pleiss, C.; Yang, M.; Wang, Z.; Lin, X.; Qui, Z.;
Hirschfeld, D. R.; Gleason, S.; Mirzadegan, T.; Dunten, P. W.; Harris, S. F.;
Villaseñor, A.; Hang, J. Q.; Heilek, G. M.; Klumpp, K. Bioorg. Med. Chem. Lett.
2010, 20, 4025.
11. For
a more detailed description of these syntheses see: Kertesz, D. J.;
Figure 2. Overlay of crystal structures of N-phenyl piperidine 11 (bronze) and the
N-benzyl piperidine 2 (ivory).
Brotherton-Pleiss, C.; Yang, M. US 2008/0146595.
12. Enzymatic assay: IC50 values were obtained from a SPA heteropolymeric assay.
RNA-dependent DNA polymerase activity was measured using a biotinylated
primer oligonucleotide and tritiated dNTP substrate. Newly synthesized DNA
was quantified by capturing the biotinylated primer molecules on streptavidin
coated Scintillation Proximity Assay (SPA) beads (Amersham). Each value
represents the average of at least two independent assays.
13. Ludovici, D. W.; De Corte, B. L.; Kukla, M. J.; Ye, H.; Chih, Y. H.; Lichtenstein, M.
A.; Kavash, R. W.; Koen, A.; de Bethune, M. P.; Azijn, H.; Oauwels, R.; Lewi, P. J.;
Heeres, J.; Koymans, L. M. H.; de Jonge, M. R.; Van Aken, K. J. A.; Daeyaert, F. F.
D.; Das, K.; Arnold, E.; Janssen, P. A. J. Bioorg. Med. Chem. Lett. 2001, 11, 2235.
14. Replication assay: EC50 values were obtained following incubation of compound
with infected MT4 cells in the presence of 10% fetal bovine serum. The assay
assesses the reduction in the cytopathic effect of HIV-1 on MT4 cells in the
presence of HIV-RT inhibitors. Each value represents the average of at least two
independent assays. For a detailed description of the antiviral assay see:
Pauwels, R.; De, C. E.; Desmyter, J.; Balzarini, J.; Goubau, P.; Herdewijn, P.;
Vanderhaeghe, H.; Vandeputte, M. J. Virol. Methods, 1987, 16, 171.
In summary, we have developed a new N-phenyl piperidinyl
aminopyrimidine scaffold combining facets of the K101 and K103
binding classes of NNRTIs. Representative compounds 28 and 32
have achieved remarkable potency and mutant profiles, but they
are readily cleared metabolically. Other compounds like 40 and
44 have reasonable clearance and possess lesser but still very good
antiviral potencies. It appears that having the major interactions
made with conserved residues and the protein backbone, com-
bined with the opportunity for extra interactions in the P236/
V106 pocket and with K103, have afforded these molecules a
remarkable immunity to RT mutant variability. It also seems the
potential of very high potency with adequate metabolic stability
may yet be realized from the N-substituted piperidinyl aminopy-
rimidine motif. Continued efforts towards these goals have in-
cluded modifying the overall polarity of the molecules and
further investigations of the SAR, and the results of those studies
will be the subject of future reports.
15. Determination of potency versus wild type virus was repeated in the presence
of 40% human serum (HS) for these compounds. The resulting ‘protein shifted
potency’ as compared to the result with 10% FBS (see Ref. 14) is considered an
indicator of potential for in vivo activity.
16. Wang, J.; DeMaio, W.; Chandrasekaran, A.; Shen, L.; Bach, A., II; Scatina, J.;
Talaat, R. Curr. Drug Discovery Technol. 2006, 3, 101.
17. Atomic coordinates for the structure of 11 bound to HIV-RT were deposited
with the RCSB Protein Data Bank (PDB) under the access code 3 NBP.