The development of novel C-2, C-8, and N-9 trisubstituted purines as inhibitors of TNF-α production

Article abstract of DOI:10.1016/j.bmcl.2006.05.050

A series of C-2, C-8, and N-9 trisubstituted purine based inhibitors of TNF-α production are described. The most potent analogs showed low nanomolar activity against LPS-induced TNF-α production in a THP-1 cell based assay. The SAR of the series was optim

Full text of DOI:10.1016/j.bmcl.2006.05.050

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4360–4365
The development of novel C-2, C-8, and N-9 trisubstituted
purines as inhibitors of TNF-a production
Mark Sabat,a,* John C. VanRens,a Michael P. Clark,a Todd A. Brugel,a Jennifer Maier,a
Roger G. Bookland,a Matthew J. Laufersweiler,a Steven K. Laughlin,a
Adam Golebiowski,a Biswanath De,a Lily C. Hsieh,a Richard L. Walter,b
Marlene J. Mekelb and Michael J. Janusza
aProcter and Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Rd, Mason, OH 45040, USA
bP&G Analytical Discovery, Miami Valley Innovation Center, PO Box 538707, Cincinnati, OH 45253-8707, USA
Received 10 April 2006; revised 15 May 2006; accepted 16 May 2006
Available online 5 June 2006
Abstract—A series of C-2, C-8, and N-9 trisubstituted purine based inhibitors of TNF-a production are described. The most potent
analogs showed low nanomolar activity against LPS-induced TNF-a production in a THP-1 cell based assay. The SAR of the series
was optimized with the aid of X-ray co-crystal structures of these inhibitors bound with mutated p38 (mp38).
ꢀ 2006 Elsevier Ltd. All rights reserved.
F
In our efforts toward the discovery of therapeutics for the
treatment of inflammatory conditions we are developing
novel small molecule inhibitors of TNF-a (tumor necrosis
factor-a) via modulation of the MAPK (mitogen-activat-
ed protein kinase) cascade.1 The p38 protein is part of a
larger MAP kinase family involved in signal transduction
that also includes ERK (extracellular regulated kinase)
and JNK (c-Jun amino terminal kinase).1a,2 The p38
and JNK kinase families are activated in response to
infection, cellular stress, and cytokines.2 Additionally,
p38 is centrally involved in the regulation of many
proinflammatory cytokines most particularly TNF-a
(tumor necrosis factor-a) and IL-1b (interleukin-1b).2
The overexpression of these cytokines has been implicat-
ed in the pathogenesis of rheumatoid arthritis (RA),
osteoarthritis (OA), and Crohn’s disease.3,4 Thus, agents
that inhibit p38 MAPK can decrease levels of TNF-a and
the related IL-1b, and thereby reduce inflammation and
halt tissue destruction.4
F
S
N
N
N
O
S
N
F
O
N
H
Cl
Cl
N
SB203580
TNF-α IC50 = 72 nM
p38 kinase IC50 = 136 nM
VX745
TNF-α IC50 = 56 nM
p38 kinase IC50 = 10 nM
1
N 7
N
O
N
N
4
F
N9
Cl
H
F
IC50 = 4 nM (TNF-α)
6m
Figure 1. Inhibitors of p38 MAPK.
The prototypical p38 inhibitors (e.g. SB-203580, Fig. 1)5
contained a 4-aryl-5-pyridinyl motif and were found to
interact competitively with the p38 ATP binding site.
To date, numerous chemical scaffolds (e.g. imidazoles,
pyrroles, pyrimidines, pyridines, pyrimidones, indoles,
heteroindoles, ureas, and various fused bicyclic hetero-
cycles) containing a variety of functionality have been
reported to inhibit TNF-a production.2a,6 Of the non-
vicinal diaryl scaffolds VX745 has been shown to be a
potent and selective inhibitor of p38.2a,2d,5a,6a,7
Keywords: Purines; TNF-a; MAP kinase; p38; Mutated p38; THP-1;
THP-1 cell.
*
Corresponding author. Tel.: +1 513 622 1272; fax: +1 515 622
3681; e-mail: sabat.mp@pg.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.050

M. Sabat et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4360–4365
4361
The purine scaffold has also been exploited for designing
X
X
biologically relevant molecules including inhibitors of
kinase signaling.8 Utilizing structure based drug design
in conjunction with X-ray co-crystallography we
achieved the synthesis of a series of novel inhibitors of
TNF-a containing a C-2, C-8, and N-9 trisubstituted
purine scaffold. This communication details the synthe-
sis, biological activity, and the mode of binding for this
class of compounds in mutated p38.
N
N
N
N
R1
N
N
c
a, b
F
4
O
O
N
F
OH
O
N
F
R1
F
8a,c-d
7
Scheme 3. General procedure for preparation of 7 and 8a, c, d.
Reagents and conditions: (a) Pd/C, H2, EtOH, 45 ꢁC, 2 h, 79%; (b) 2-
(2-halo-phenyl)-2-hydroxy-acetimidic acid ethyl ester, EtOH, reflux,
18 h, 24–38%; (c) MnO2, DCM, 1 h, 74–85%.
The trisubstituted purines were prepared from readily
available 2,4-dichloro-5-nitro-pyrimidine 19 using gener-
al procedures depicted in Schemes 1–3. Early lead mol-
ecules which contain C-2 anilino- or alkylamino-groups
were accessed via a 4-step sequence (Scheme 1). Initial
displacement of the 4-chloro of 1 with various alkyl
amines was performed at 0 ꢁC in THF. Subsequent
substitution of the 2-chloro with various amines
occurred readily to give 2.10 These compounds were then
hydrogenated with Pd on carbon and the resultant
amines were treated with a series of isothiocyanates to
provide purines 3a–n (Table 1).11
TNF-a production in LPS (lipopolysaccharide) stimu-
lated human monocytic cells (THP-1).13–15 The initially
synthesized compound 3a (Table 1) was a weak inhibitor
of TNF-a release (IC50 = 6.6 lM). Exchange of the 4-
aminotetrahydropyran moiety in analog 3a for an alkyl
group slightly improved potency (3b; IC50 = 3.2 lM).
Maintaining R2 and insertion of a methylene or ethyl-
ene spacer group into the chlorophenyl group (3c and
3d) resulted in a loss of activity (IC50 > 10 lM). Similar
results were obtained for compound 3e which contained
a 2-chlorobenzamide group at position C-8. Installation
of a 2,6-difluoroaniline group at C-2 gave the most
potent analog 3f (IC50 ꢀ 1.5 lM). We then examined
the effect of incorporating various alkyl groups at posi-
tion N-9 (3g–k). Purines with an ethyl (3g;
IC50 = 542 nM) or isopropyl (3h; IC50 = 630 nM) group
at N-9 displayed increased activity, whereas larger func-
tionality and the cyclopropyl group at this position
attenuated potency (3i–k). Having explored N-9 substi-
tution we next attempted to vary the ring substituents on
R2. Replacement of the 2,6-difluoroaniline group pres-
ent in 3f and 3g with a 2-fluoro-, 4-fluoro- or 2, 4-diflu-
oroaniline group at C-2 gave analogs with poor activity
(3l–n).
Dichloropyrimidine 1 was also used to generate pur-
ines containing a C-2 phenoxy group (Schemes 2
and 3). While displacement of amines at the 2-chloro
position occurred readily at rt, substitution with phe-
nols required reflux for 3–6 h in DIPEA. Catalytic
reduction of 4 followed by reaction with 1,10-carbon-
yldiimidazole and treatment with POCl3afforded 5.
The 8-chloro of compound 5 was then displaced with
various anilines, 2-chlorophenol and 2-chlorobenze-
nethiol, to give 6a–m. Compound 4 was also treated
with 2-(2-halo-phenyl)-2-hydroxy-acetimidic acid ethyl
ester.12 The resulting benzylic alcohol 7 was subse-
quently oxidized with MnO2 in DCM to furnish 8a,
c, d (Scheme 3).
To facilitate the SAR and delineate the binding mode of
these molecules an X-ray crystallographic structure16–18
of 3g with mutated p38 (mp38) was obtained (Fig. 2).
The mutated p38a herein described was a double mutant
(S180A, Y182F) of murine p38a.18 Inhibitor 3g orients
itself in the mp38 enzyme (PDB accession code
2GTM) such that the 2-chloroaniline ring (at C-8) lies
in the hydrophobic (specificity) pocket adjacent to
Thr106. There was a hydrogen bond between N-1 of
the pyrimidine ring and the Met109 N–H. Additionally,
the hydroxyl of Thr106 formed a hydrogen bond with
N-7 of the inhibitor. A water molecule (removed for
clarity in Fig. 2) was observed to bridge Lys53 and the
N–H of position C-8 on the purine ring. The N-9 methyl
Tables 1 and 2 summarize the screening results for a
variety of trisubstituted purines on the inhibition of
R3
NH
NO2
N
N
R1
NO2
Cl
N
N
N
a, b
c, d
HN
R2
N
HN
R2
N
NH
R1
Cl
N
1
2
3a-n
Scheme 1. General procedure for the preparation of compounds 3a–n.
Reagents and conditions: (a) alkylamine, THF, 0 ꢁC, 10 min, 42–94%;
(b) alkylamine or aniline, rt, 3–6 h, 24–72%; (c) Pd/C, H2, EtOH, rt,
1.5 h, 81–98%; (d) various isothiocyanates, DCM, DCC, DIPEA, rt,
15 min then reflux 2 h, 7–64%.
N
N
f
NO2
N
a, b
c, d, e
F
N
N
R2
Cl
1
N
O
N
F
N
NH
R1
O
N
F
O
N
F
R1
R1
6a-m
F
F
5
4
Scheme 2. General procedure for preparation of 6a–m. Reagents and conditions: (a) ethylamine or isopropylamine, THF, 0 ꢁC, 10 min, 79%
(R1 = ethyl or isopropyl); (b) 2,6 difluorophenol, DIPEA, reflux, 3–6 h, 32–48%; (c) Pd/C, H2, EtOH, 45 ꢁC, 2 h, 87–98%; (d) 1,10-
carbonyldiimidazole, THF, 21 ꢁC, 1 h, 94–97%; (e) POCl3, DCM, reflux, 24 h, 29–40%; (f) various phenols and thiophenols, DIPEA, 120 ꢁC, 6 h,
4–32%.

4362
M. Sabat et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4360–4365
Table 1. IC50 values for purine derivatives 3a–n
N
N
R1
R3
NH
N
HN
R2
N
R1
R2
R3
TNF-a IC50 (nM)
a
Compound
Cl
3a
Me-
6579
3171
O
Cl
Cl
Cl
3b
3c
3d
3e
3f
Me-
HO
HO
HO
Me-
>10,000
>10,000
>10,000
1541
Me-
Cl
Me-
O
F
O
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Me-
F
F
F
F
F
F
F
F
F
F
F
F
3g
3h
3i
Et-
542
Isopropyl-
630
>1000
798
3j
n-Butyl-
t-Butyl-
Et-
3k
3l
>1000
2110
3m
3n
Me-
10,000
6556
F
F
Me-
F
a IC50 of LPS-stimulated TNF-a production in human monocytic cells (THP-1).
group was positioned toward Leu171, however this sub-
stituent appeared too short for maximal interaction with
Using insights from the co-crystal structure of mp38
with 3g we synthesized compound 6a (Table 2) incorpo-
rating a 2,6-difluorophenoxy group at C-2 and an ethyl
group at N-9. These changes corrected the deficiencies
observed in the X-ray co-crystal structure (O at C-2
H-bonds the amide N–H of Gly110 and the ethyl at N-
9 is better positioned toward Leu171) thus providing a
significant increase in potency (IC50 = 69 nM) relative
to 3g. The use of an O linker (6b) resulted in a moderate
decrease in activity (IC50 = 86 nM). The corresponding
S linked analog 6c was better tolerated (IC50 = 14 nM).
Leu171
.
In the Met109-Gly110 region of the protein a peptide flip
was observed (the carboxyl of Met109 turned away from
the inhibitor and the amide N–H of Gly110 turned in to-
ward the inhibitor) (Fig. 2). This orientation induced a
repulsive interaction between the N–H of the 2,6-diflu-
orophenylanalino-hydrogen at C-2 and the N–H of
Gly110
.

M. Sabat et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4360–4365
Table 2. IC50 values of the amine derivatives of 6a–m, 7, and 8a–d
4363
F
N
N
R1
N
R2
X
O
N
F
R1
X
R2
TNF-a IC50 (nM)
a
Compound
6a
6b
6c
6d
6e
6f
Et-
Et-
NH
O
2-Chloro
2-Chloro
69
86
Et-
Et-
S
2-Chloro
2-Methyl
2-Fluoro
14
48
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
Et-
Et-
35
2-Methoxy
2-Hydroxy
2-Amino
2050
906
473
237
273
300
100
4
6g
6h
6i
Et-
Et-
Et-
Et-
2-Chloro-4-fluoro
2-Chloro-4-methyl
2,3-Dichloro
2,6-Dichloro
2-Fluoro
6j
6k
6l
Et-
Et-
6m
7
Isopropyl-
Et-
Et-
CHOH
C@O
S@O
C@O
C@O
2-Chloro
2-Chloro
169
4
8a
8bb
8c
8d
Et-
Et-
2-Chloro
2-Fluoro
2-Chloro
>1000
207
650
Isopropyl-
a IC50 of LPS-stimulated TNF-a production in human monocytic cells (THP-1).
b Analog 8b was synthesized from 6c with 1 eq. Oxoneꢂ.
2,3-dichloro (6k; IC50 = 300 nM) or the 2,6-dichloro
(6l; IC50 = 100 nM) reduced potency. Finally,
replacement of the N-9 ethyl on compound 6a with an
isopropyl group gave the most potent purine 6m in this
series (IC50 = 4 nM) (Table 2 and Fig. 3). This inhibitor
showed all expected binding interactions with mp38
(PDB accession code 2GTN) including the torsional flip
at Met109-Gly110 reported to confer p38 selectivity.7b
Additional SAR included replacement of the linker het-
eroatom at C-8 with an alcohol moiety to afford 7
Figure 2. Key hydrogen bonds between 3g and mp38 with key
mismatch at Gly110 backbone N–H.
This was attributed to a more favorable orientation of
the 2-chloro phenyl ring in the hydrophobic pocket.
Replacement of the 2-chloro with a methyl 6d or fluo-
rine 6e increased activity (IC50 = 48 nM; IC50 = 35 nM).
Further changes at the 2 position of the 8-phenoxy ring:
methoxy-, hydroxy-, and amino- (6f–h) led to a drop in
activity.
Attempts to improve activity by disubstitution (6i–l) on
the 8-phenoxy ring with: 2-chloro-4-fluoro (6i;
IC50 = 237 nM), 2-chloro-5-methyl (6j; IC50 = 273 nM),
Figure 3. Key hydrogen bonds between 6m and mp38 including the
torsional flip at Met109-Gly110.

4364
M. Sabat et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4360–4365
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(IC50 = 169 nM), oxidation of this group with MnO2
affords the potent keto analog 8a (IC50 = 4 nM). Ex-
change of the ketone linker in 8a with a sulfoxide group
muted activity (8b; IC50 > 1000 nM). Substitution of the
2-chloro atom with 2-fluoro 8c or the exchange of the N-
9 ethyl for isopropyl 8d was also deleterious and under-
scores the sensitivity of the binding pocket to these
inhibitors.
The most potent analogs (6c–e, 6m, and 8a) were
screened for in vitro metabolism.20 Compounds 6d, 6e,
and 6m were moderately metabolized, while analogs 6c
and 8a suffered high loss (56, 65, 61, 75, and 82% loss,
respectively). Inhibitors with the greatest metabolic sta-
bility (6d and 6m) were subsequently evaluated in a rat
PK study to determine bioavailability and half-life. Both
molecules displayed low BA (ꢀ9%) and possessed a
short T1/2 (1.5 h).
In summary we have described a series of novel
trisubstituted purines which inhibit the production of
TNF-a. The design, synthesis, and mode of binding
(to mutated p38) of these compounds was detailed. We
hope to report further progress in development of this
series in the near future.
7. (a) Ferraccioli, G. F. Curr. Opin. Anti-Inflamm. Immunod-
ulatory Invest. Drugs 2000, 2, 74; (b) Fitzgerald, C. E.;
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Narula, S. S.; Dalgarno, D. C.; Haraldson, C.; Keats, J.;
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Acknowledgments
We are grateful to A. L. Roe, C. A. Cruze, W. E.
Schwecke, C. R. Dietsch for pharmacokinetic studies,
M. Buchalova for chemical stability and solubility stud-
ies, and M. Mekel for X-ray co-crystallization studies.
We would like to acknowledge that X-ray data were col-
lected at beamline 17-BM in the facilities of the Industri-
al Macromolecular Crystallography Association
Collaborative Access Team (IMCA-CAT) at the Ad-
vanced Photon Source, Argonne National Laboratory.
Supporting institutions may be found at www.ser-
cat.org/members.html. Use of the Advanced Photon
Source was supported by the US Department of Energy,
Office of Science, Office of Basic Energy Sciences, under
Contract No. W-31-109-Eng-38.
9. Whittaker, N.; Jones, T. S. G. J. Chem. Soc. Abstracts
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10. Under the described conditions initial substitution occurs
predominately at the 4-chloro position of pyrimidine 1.
However, a small amount of the unwanted 2-chloro
material was detected (1H NMR) see: Taylor, E. C.;
Thompson, M. J. J. Org. Chem. 1961, 26, 5224, The
unwanted regioisomer was removed after the second
desired displacement (intermediates 2 and 4).
11. (a) Seth, P. P.; Robinson, D. E.; Jefferson, E. A.; Swayze,
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References and notes
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Robinson, F.; Silvestro, L.; Pearson, G.; Xu, B.-e.;
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2002, 298, 1911, For representations of the MAPK
signaling cascade, see Refs. 2a,d,e.
13. Duplicate cultures of human monocytic cells (THP-1)14
cells (2.0 · 105/well) were incubated for 15 min in the
presence or absence of various concentrations of inhibitor
before the stimulation of cytokine release by the addition
of lipopolysaccharide (LPS, 2 lg/ml). The amount of
TNF-a released was measured 4 h later using an ELISA
(R&D Systems, Minneapolis, MN). The viability of the
cells after the 4 h incubation was measured using MTS
assay15 (Promega Co., Madison, WI).
14. Mohler, K. M.; Sleath, P. R.; Fitzner, J. N.; Cerretti, D.
P.; Alderson, M.; Kerwar, S. S.; Torrance, D. S.; Otten-
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20. (a) Measured as percent loss at 4 h in rat hepatocytes;
(b) In vitro metabolism assay procedure: In vitro meta-
bolic stability of analogs in plated rat hepatocytes
(Sprague–Dawley) obtained from Cedra Corporation.
Metabolic activity was determined in triplicate using a
total volume of 0.2 mL containing 0.25 lM NCE incu-
bated in rat hepatocyte and matrigel blank microtiter
plates. The plates were maintained at 37 ꢁC throughout
the study. Samples were removed from wells at 0, 2, and
4 h, and NCE samples were analyzed by HP-LC/MS/MS
with reverse-phase chromatography. To improve analyt-
ical efficiency, compounds were grouped together (post-
incubation) into a multi-compound assay. Samples from
like time-points containing the different compounds were
combined and an internal standard (1.1 ng/mL stock) was
added. Results for each compound were expressed as the
ratio of the compound response area over the internal
standard response area. Percent loss was calculated by
dividing the 2 and 4 h ratios by the 0 h ratio.
16. The mutant enzyme cannot be phosphorylated and,
therefore, it is homologous to the inactive form of
murine p38a. Protein expression and purification were
carried out as previously described for the murine
enzyme.17 For crystallization, mutated p38a was incu-
bated overnight (12–16 h) with 1 mM compound.
Cocrystals were grown by hanging drop vapor diffu-
sion using PEG as a precipitating agent and overall
protocols similar to those previously described for the
human enzyme.18 Crystals typically diffracted to better
˚
than 2.0 A resolution and were of the previously
˚
reported space group: P212121; a = 65.2 A, b = 74.6 A,
˚
19
˚
c = 78.1 A.
X-ray data were collected at beamline
17-BM in the facilities of the Industrial Macromole-
cular Crystallography Association Collaborative Access
Team (IMCA-CAT) at the Advanced Photon Source.
These facilities are supported by the companies of the
Industrial Macromolecular Crystallography Association
through a contract with Illinois Institute of Technol-
ogy (IIT), executed through IIT’s Center for Synchro-
tron Radiation Research and Instrumentation. Use of
the Advanced Photon Source was supported by the
US Department of Energy, Basic Energy Sciences,
Office of Science, under Contract No. W-31-109-
Eng-38.