Synthesis of novel histamine H4 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.11.098

This letter describes the discovery and synthesis of a series of octahydropyrrolo[3,4-c]pyrrole based selective histamine hH4 receptor antagonists. The amidine compound 20 was found to be a potent and selective histamine H4 receptor antagonist with moderate clearance and a high volume of distribution.

Full text of DOI:10.1016/j.bmcl.2011.11.098

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1156–1159
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel histamine H4 receptor antagonists
⇑
Charlotte A.L. Lane, Duncan Hay, Charles E. Mowbray, Michael Paradowski, Matthew D. Selby ,
Nigel A. Swain, David H. Williams
Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Sandwich, Kent CT13 9NJ, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
This letter describes the discovery and synthesis of a series of octahydropyrrolo[3,4-c]pyrrole based
selective histamine hH4 receptor antagonists. The amidine compound 20 was found to be a potent and
selective histamine H4 receptor antagonist with moderate clearance and a high volume of distribution.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 4 September 2011
Revised 22 November 2011
Accepted 23 November 2011
Available online 1 December 2011
Keywords:
Histamine
Azaindole
Bicyclic
Amidine
Isostere
The fourth human histamine receptor H4 was disclosed in 2000
as a 390 amino acid transmembrane G-protein-coupled-receptor.1
The receptor is predominantly expressed in cells of the hematopoi-
etic lineage, including eosinophils, basophils and mast cells,
although recent reports in the literature show evidence for expres-
sion on non-hematopoietic cells2 and in the CNS.3
In this Letter we report on our efforts to identify a selective hH4
antagonist with good intrinsic pharmacokinetics. Given the high li-
gand efficiency of 1 (LE 0.58 kcal molÀ1 15
we perceived it to be an
)
attractive starting point for the program.16 Using the central amide
bond as the key disconnection, SAR of the ‘acid’ and ‘amine’ frag-
ments were investigated. A number of alternative 5,6- and 6,6-
fused carboxylic acid systems were coupled with N-methyl piper-
azine. Heteroatoms were introduced into the ring system in an at-
tempt to reduce lipophilicity and hence increase metabolic
stability; however, all of the analogues prepared showed reduced
activity relative to the original lead with representative examples
shown in Table 1.
The majority of the pharmacological research that has been
published in the literature has utilised compound
1 (JNJ-
7777120);4 however, more recently a number of other selective
tool compounds have been described including A-9439315 and
A-987306,6 along with numerous patent applications.7 Efficacy
for these small molecule tool compounds has now been demon-
strated in multiple pre-clinical models of inflammation and pain.5,8
These data in conjunction with data from H4 knockout mice9 sup-
port the position that there is potential that selective H4 receptor
antagonists represent a new class of therapy to treat a wide range
of diseases,10,11 including asthma and allergic rhinitis. Compound 1
was the first reported potent non-imidazole selective H4 antago-
nist and is reported to bind the human H4 receptor (hH4) with a
Ki of 4 nM and more than 1000-fold selectivity over the other his-
taminergic receptors. In our hands, the compound also shows high
affinity for the hH4 receptor (hH4 Binding Ki 8.0 nM,12 hH4 Func-
tional Ki 6.8 nM13). A limitation of compound 1 is its relatively high
intrinsic clearance (Cl 968 ml/min/kg) which results in a short T1/2
(0.7 h) in rat.14
An alternative system prepared with reasonable levels of po-
tency was the benzimidazole analogue 2 (JNJ 10191584).8 For
the imidazo-pyrazine and -pyridine analogues (3 and 4, respec-
tively) it is possible that the decrease in activity is driven in part
by removal of the H-bond donor, which has potential to bias the
conformation of the amide through an internal hydrogen bond.
The importance of this NH is supported by the fact that N-meth-
ylation has been shown to significantly reduce hH4 activity.4
However, it is not trivial to deconvolute whether this decrease
in activity is due to an intramolecular interaction of the methyl
(and subsequent conformational change), a negative interaction
of the methyl with the receptor, or disruption of an H-bond net-
work. Azaindoles, exemplified by 5, showed reduced activity rel-
ative to the indole 6 despite retaining the NH; however, the
ring’s
p system is less electron-rich than the indole which may
⇑
Corresponding author. Address: UCB, 216 Bath Road, Slough SL1 4EN, United
Kingdom. Tel.: +44 0 1753 534655; fax: +44 0 1753 536632.
negatively impact on any
might be making.
p-stacking interactions the ligand
E-mail address: matt.selby@ucb.com (M.D. Selby).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.098

C. A. L. Lane et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1156–1159
1157
Table 1
Table 2
Structure and activity for compounds 1–6
Structure and hH4/hH3 activity for compounds 7–16
R1
Compd Structure
hH4 bind hH4 func. hH3 bind clogP
R2
R3
Ki (nM)
Ki (nM)
Ki (nM)
O
N
N
Cl
O
X
O
X
N
H
1
8.0
6.8
15,300
2.5
1.9
N
H
N
Cl
R4
N
2
47.7
39.6
20,000
N
H
Compd
R1
R2
R3
R4
hH4 bind
hH4 func.
hH3 bind
Ki (nM)
Ki (nM)
Ki (nM)
O
X
N
7a
8
9
10
11
12
13
14
15
16
—
H
Me
F
H
H
H
H
H
H
—
Cl
F
F
F
H
F
Cl
Cl
Cl
—
H
H
H
H
H
F
H
H
H
—
16
129
52
74
83
102
128
1960
2670
5080
27
218
97
134
145
189
236
3300
4490
8180
8750
nd
nd
nd
15,500
nd
17,900
nd
nd
3
20,100
3170
>20,000
>11,400
nd
À0.1
N
N
Me
Me
Me
Me
Me
Me
Et
Cl
O
X
N
4
5
28,600
1.7
N
O
3100
118
>29,900
46
36,600
0.7
1.7
N
X
O
X
N
H
iPr
H
nd
64
>20,000
(nd = not determined).
N
H
a
Compound 7 is indole shown previously.
Where X = N-methyl piperazine.
(nd = not determined).
however, there was a approximately 10-fold decrease in hH4
activity.
Previous literature data4,17 suggested that the pendant basic
centre is a key pharmacophoric element. As a consequence, our pri-
mary focus was to look for alternative diamine fragments to re-
place the piperazine. Substructure searching within the Pfizer file
and literature identified about 100 diverse low MWT (<200) dia-
mine fragments which had potential to mimic the piperazine of
1. A library was prepared using 5-chloroindole-2-carboxylic acid
as the coupling partner. One of the most promising leads to emerge
from this library work was compound 7.
We were keen to explore the SAR around 8; however, simple
amide formation from the benzimidazole acids was precluded be-
cause of the facile decarboxylation of the corresponding parent
carboxylic acids. Instead the required compounds were accessed
from the nitro aniline precursors as shown in Scheme 1.18,12a
Unfortunately, despite preparing over 90 analogues varying the
substitution on the benzimidazole, we were never able to match
the potency that had been seen with the indoles. SAR of the termi-
nal substituent on the amino group was also very limited, with
sharp decrease in activity seen for both the desmethyl compound
16 and homologues 14 and 15. The highest potency achieved
was for compound 9; however this compound showed reduced
metabolic stability in vitro and so was not pursued further (see Ta-
ble 5).
Cl
O
N
N
H
7
Three key azaindole analogues 17, 18 and 19 were also prepared
(Table 3). The 5-chloro analogue 18, gave a 10-fold potency in-
crease relative to the 5-H compound 17, whereas the 6-chloro ana-
logue 19 gave a threefold decrease in activity. These compounds
provided some of the strongest evidence that the C-5 substituent
of the 6,5-system was making a specific interaction with the recep-
tor, as opposed to binding through a non-specific lipophilic interac-
tion. Further variation of the substitution was limited by the
synthetic access to the template.
N
Replacing the piperazine with the bicyclic octahydropyrrol-
o[3,4-c]pyrrole moiety gave a compound 7 which retained selec-
tive hH4 activity (Table 2) and showed increased metabolic
stability in both rat and human in vitro (Table 5). Molecular mod-
elling clearly shows the potential of the fused system to mimic
piperazine, particularly if the bicyclic system adopts a ‘boat-like’
conformation as shown in Figure 1. The fused system demon-
strated a significant increase in pKa relative to the piperazine
(8.28 vs 6.86), which resulted in a log unit drop in LogD for direct
analogues.
A key risk for the indole analogues was the fact that the com-
pounds consistently gave positive results in a reactive metabolite
screen which is used as an early indicator for potential of idiosyn-
cratic toxicity.18 The exact nature of the reactive metabolite was
not elucidated; however, indoles have the potential to form reac-
tive metabolites via epoxidation of the C2–C3 bond, or to generate
quino-methide type intermediates following metabolism of the C-
5 position. We were thus keen to re-evaluate alternative acid frag-
ments. Based on the precedence from 2 we prepared the benzimid-
azole analogue 8 (Table 2). Compound 8 was negative in the
reactive metabolite assay and retained good metabolic stability;
During the purification of the 5,6-difluoro benzimidazole ana-
logue 13, a low yield of the amidine analogue 20 was also isolated.
The exact mechanism for formation of the amidine is not known;
however, we hypothesise that it was formed during the chromato-
graphic purification of the compounds with an eluent system con-
taining ammonia. Surprisingly, the amidine 2019 showed
a
significant potency advantage over the amides, retaining good
metabolic stability. Given the potency of the amidines, an opti-
mised route was developed making use of the 2-cyano benzimi-
dazoles as the key intermediate (Scheme 2).12a,20
The SAR for aromatic substitution of the amidines mirrored that
of the corresponding amides (Table 4), with all compounds show-
ing at least a fivefold potency increase relative to the amide ana-
logue. In-silico pKa calculations suggested that the amidines were
likely to sit as the zwitterionic species, protonated on the amidine
and deprotonated on the benzimidazole. Support for this was later

1158
C. A. L. Lane et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1156–1159
Figure 1. Molecular modelling showing ‘boat’ conformation of piperazine and bicyclic system. Distances are marked in angstroms.
R1
R1
Table 4
R2
R3
NH2
NO2
R2
R3
NH2
NH2
i
ii
Structure and hH4 Binding activity for compounds 20–26
Compd
R1
R2
R3
hH4 bind
Ki (nM)
hH4 func.
Ki (nM)
hH3 Bind
Ki (nM)
R1
20
21
22
23
24
25
26
H
H
Me
F
H
F
F
Cl
F
F
F
F
9.5
26
4.6
49
4.6
70
6.5
17.2
56
9.3
24
9.1
20
12.4
3090
nd
nd
R2
R3
R1
O
N
N
H
H
H
H
F
iii
R2
R3
N
N
nd
CCl3
H
1190
>20,000
3810
N
H
F
H
N
H
H
R4
(nd = not determined).
Scheme 1. Synthesis of benzimidazole amides (8–16). Reagents and conditions: (i)
Fe/HCl, EtOH, H2O, reflux; (ii) Cl3CC(NH)OMe, AcOH, rt; (iii) K2CO3, MeCN/H2O,
amine, rt.
regarding the role that the NH plays in driving activity. However,
the single crystal structure does not directly support this hypothe-
sis since the crystallised molecule was a methanol solvate, which
may have had significant impact on conformation in the solid state.
Whilst solid samples of the amidines were stable, further profil-
ing of the compounds indicated some level of chemical instability
in certain solvents (e.g., methanol). Stability in biological assay
media was unaffected; however, we were aware that the instabil-
ity would hamper further development of the compound. Cyclic
amidines were hypothesised to offer increased chemical stability
and a model compound 27 was prepared. No evidence of chemical
instability was observed; however, as anticipated, the compound
was devoid of hH4 activity. Based on this we designed compound
28. Chemical stability was retained, but unfortunately the com-
pound was inactive against hH4.
Table 3
hH4 binding data for compounds 17–19
R2
O
N
R3
N
N
H
N
Compd
R2
R3
hH4 bind
Ki (nM)
17
18
19
H
Cl
H
H
H
Cl
973
83
2090
R1
R1
R2
R3
R2
R3
N
N
i
CN
CCl3
N
H
N
H
R1
R2
R3
NH
N
N
ii
N
H
N
Scheme 2. Synthesis of benzimidazole amidines (20–26). Reagents and conditions:
(i) NH3(aq), EtOH, H2O, À5 °C to rt; (ii) N-acetyl- -cysteine, MeOH, amine, reflux.
L
provided by a single crystal X-ray structure of 25 shown in Figure
2.21 We believe that a potential consequence of the zwiterionic
nature would be an increased tendency for the molecule to sit in
a planar conformation, which ties in with previous hypotheses
Figure 2. Single-crystal X-ray structure of 25.

C. A. L. Lane et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1156–1159
1159
Table 5
Herrity, N. C.; Vawter, L.; Sarau, H. M.; Ames, R. S.; Davenport, C. M.; Hieble, J.
P.; Wilsom, S.; Bergsma, D. J.; Fitzgerald, L. R. Mol. Pharmacol. 2001, 59, 434.
2. Sander, L. E.; Lorentz, A.; Sellge, G.; Coeffier, M.; Neipp, M.; Veres, T.; Frieling,
T.; Meier, P. N.; Manns, M. P.; Bischoff, S. C. Gut 2006, 55, 498.
3. Connelly, W. M.; Shenton, F. C.; Lethbridge, N.; Leurs, R.; Waldvogel, H. J.; Faull,
R. L. M.; Lees, G.; Chazot, P. L. Br. J. Pharmacol. 2009, 157, 44.
4. Jablonowski, J. A.; Grice, C. A.; Chai, W.; Dvorak, C. A.; Venable, J. D.; Kwok, A.
K.; Ly, K. S.; Wei, J.; Baker, S. M.; Desai, P. J.; Jiang, W.; Wilson, S. J.; Thurmond,
R. L.; Karlsson, L.; Edwards, J. P.; Lovenberg, T. W.; Carruthers, N. I. J. Med. Chem.
2003, 46, 3957.
5. Cowart, M. D.; Altenbach, R. J.; Liu, H.; Hsieh, G. C.; Drizin, I.; Milicic, I.; Mileer,
T. R.; Witte, D. G.; Wishart, N.; Fix-Stenzel, S. R.; McPherson, M. J.; Adair, R. M.;
Wetter, J. M.; Bettencourt, B. M.; Marsh, K. C.; Sullivan, J. P.; Honore, P.;
Esbensahe, T. A.; Brioni, J. D. J. Med. Chem. 2008, 51, 6547.
The in vitro and in vivo rat PK parameters for key compounds
Compd HLM (
ll/min/
RLM (
mg)
ll/min/
Cl (ml/min/
mg)
T½
(h)
Vd (l/
kg)
mg)
1
2
7
8
9
11
13
20
28
19
<7
<7
<7
<7
<7
<7
61
nd
36
36
214
40
968a
700b
nd
0.2a
0.7b
nd
2.3a
23b
nd
nd
nd
nd
nd
nd
nd
161c
58c
31c
0.7hc
0.8hc
11hc
6.1c
4.1c
48c
40
10
6. Liu, H.; Alternbach, R. J.; Carr, T. L.; Chandran, P.; Hsieh, G. C.; Lweis, L. G.;
Manelli, A.; Milicic, I.; Marsh, K. C.; Miller, T. R.; Strakhova, M. I.; Vortherms, T.
A.; Wakefield, B. D.; Wetter, J. M.; Witte, D. G.; Honore, P.; Esbenshade, T. A.;
Brioni, J. D.; Cowart, M. D. J. Med. Chem. 2008, 51, 7094.
7. Kiss, R.; Keseru, G. M. Expert Opin. Ther. Patents 2009, 19, 119; Smits, R. A.;
Leurs, R.; de Esch, I. J. P. Drug Discovery Today 2009, 14, 745.
(nd = not determined).
a
3 mg/kg subcutaneous.
10 mg/kg subcutaneous.
b
c
1 mg/kg intravenous.
8. Venable, J. D.; Cai, H.; Chai, W.; Dvorak, C. A.; Grice, C. A.; Jablonowski, J. A.;
Shah, C. R.; Kwok, A. K.; Kiev, S. L.; Pio, B.; Wei, J.; Desai, P. J.; Jiang, W.; Nguyen,
S.; Ling, P.; Wilson, S. J.; Dunford, P. J.; Thurmond, R. L.; Lovenberg, T. W.;
Karlsson, L.; Carruthers, N. I.; Edwards, J. P. J. Med. Chem. 2005, 48, 8289.
9. Dunford, P. J.; Williams, K. N.; Desai, P. J.; Karlsson, L.; McQueen, D.; Thurmond,
R. L. J. Allergy Clin. Immunol. 2007, 119(1), 176.
10. Jablonowski, J. A.; Carruthers, N. I.; Thurmond, R. L. Mini-Rev. Med. Chem. 2004,
4, 993.
11. (a) Dunford, P. J.; Thurmond, R. L. Prog. Respir. Res. 2010, 39, 187; (b) Bhatt, H.
G.; Agrawal, Y. K.; Raval, H. G.; Manna, K.; Desai, P. R. Mini-Rev. Med. Chem.
2010, 10, 1293; (c) Engelhardt, H.; Smits, R. A.; Leurs, R.; Haasksma, E.; de Esch,
I. J. P. Curr. Opin. Drug Discov. Devel. 2009, 12, 628.
F
Cl
N
N
N
N
N
H
H
N
H
N
H
N
F
H
N
H
27
28
12. Binding affinity to the H4 receptor (hH4 Bind) was assessed using an in vitro
radioligand ([3H]-histamine) filtration binding assay in a stable CHO cell line
expressing recombinant human H4 receptor with the Ga15 protein. Detailed
Given the excellent levels of potency and metabolic stability,
compound 20 was selected for further progression. Rat pharmaco-
kinetics indicated moderate clearance (31 mL/min/kg) and high
volume of distribution (48 L/kg) with 20% oral bioavailability (Ta-
ble 5). The high volume observed for 20 was not unexpected given
the dibasic nature of the compound.
In conclusion we identified a metabolically robust piperazine
isostere that provided compounds which retained H4 potency
and selectivity. The amidines exemplified by 20 represent selective
compounds which were suitable for further evaluation. Prelimin-
ary results for the in vivo pharmacological characterisation of 20
have been communicated.22
experimental
conditions
are
provided
in
(a)
‘Preparation
of
octahydropyrrolo[3,4-c]pyrrole derivatives as histamine H4 receptor ligands’
Lane, C. A. L.; Price, D. A. U.S. Patent Application US2006111416.
13. Functional Ki values for antagonists were determined using HEK-293 cells
expressing the full-length human H4R and a CRE-b-lactamase reporter gene.
Antagonists reversed histamine inhibition of forskolin-stimulated cAMP.
Detailed experimental conditions are provided in reference (a) ‘Preparation
of substituted 2,4-diaminopyrimidines ad histamine H4 receptor ligands’ Bell,
A. S.; Lane, C. A. L.; Mowbray, C. E.; Selby, M. D.; Swain, N. A.; Williams, D. H.
World Patent Application WO 2007072163.
14. Zhang, M.; Thurmond, R. L.; Dunford, P. J. Pharmacol. Ther. 2007, 113, 594.
15. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430.
16. A high throughput screen was run in parallel, results of which have been
disclosed in (a) Masood, M. A.; Selby, M. D.; Bell, A. S.; Mansfield, A. C.; Gardner,
M.; Smith, G. F.; Lane, C.; Kenyon-Edwards, H.; Osborne, R.; Jones, R. M.; Liu, W.
L.; Brown, C. D.; Clarke, N.; Perrucio, F.; Mowbray, C. E. Bioorg. Med. Chem. Lett.
2011, 21, 6591.
Acknowledgments
17. Terzioglu, N.; van Rijn, R. M.; Bakker, R. A.; DeEsch, I. J. P.; Leurs, R. Bioorg. Med.
Chem. Lett. 2004, 14, 5251.
18. Soglia, J. R.; Harriman, S. P.; Zhao, S.; Bareria, J.; Cole, M. J.; Boyd, J. G.; Contillo,
L. G. J. Pharm. Biomed. Anal. 2004, 36, 105.
19. Paradowski, M.; Lane, C. A. L.; Peakman, T. Synlett 2011, 1543.
20. Lange, U. W. E.; Schafer, B.; Baucke, D.; Buschmann, E.; Mack, H. Tetrahedron
Lett. 1999, 40, 7067.
21. CCDC 850451 contains the supplementary crystallographic data for this paper.
These data can be obtained free of charge via www.ccdc.cam.ac.uk/
data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by
contacting The Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK, fax: +44 1223 336033.
22. This work was first presented at the Gordon Research Conference on Medicinal
Chemistry at Colby-Sawyer College, New London, NH, USA, August 12, 2010 in
a presentation by Charles E. Mowbray titled: ‘The Discovery and Evaluation of
PF-3893787: A Novel Histamine H4 Receptor Antagonist’. The compounds
described in this letter have been disclosed in Ref. 12a and the findings of
in vivo toxicological investigations have been reported in detail by Mowbray, C.
E.; Bell, A. S.; Clarke, N. P.; Collins, M; Jones, R. M.; Lane, C. A. L.; Liu, W. L.;
Newman, S. D.; Paradowski, M.; Schenck, E. J.; Selby, M. D.; Swain, N. A.;
Williams, D. H. Bioorg. Med. Chem. Lett. 2011, 21, 6596.
The authors wish to acknowledge the contributions to this pro-
ject by numerous colleagues at Pfizer in the departments of Biol-
ogy, Chemistry and Pharmacokinetic, Dynamics and Metabolism
and Pharmaceutical Sciences and in particular Cheryl Doherty of
Pharmaceutical Sciences.
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