The effect of pKa on pyrimidine/pyridine-derived histamine H4 ligands

Article abstract of DOI:10.1016/j.bmcl.2014.10.013

During the course of our efforts toward the discovery of human histamine H₄ antagonists from a series of 2-aminiopyrimidines, it was noted that a 6-trifluoromethyl group dramatically reduced affinity of the series toward the histamine H₄ receptor. This observation was further investigated by synthesizing a series of ligands that varied in pK_a of the pyrimidine derived H₄ ligands by over five orders of magnitude and the effect on histamine H₄ affinity. This trend was then extended to the discovery of C-linked piperidinyl-2-amino pyridines as histamine H₄ receptor antagonists.

Full text of DOI:10.1016/j.bmcl.2014.10.013

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5489–5492
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The effect of pKa on pyrimidine/pyridine-derived histamine H4
ligands
⇑
Brad M. Savall , Steven P. Meduna, Jennifer Venable, Jianmei Wei, Russell C. Smith, Michael D. Hack,
Robin L. Thurmond, Patricia McGovern, James P. Edwards
Janssen Pharmaceutical Research & Development, LLC, 3210 Merryfield Row, San Diego, CA 92121, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
During the course of our efforts toward the discovery of human histamine H4 antagonists from a series of
2-aminiopyrimidines, it was noted that a 6-trifluoromethyl group dramatically reduced affinity of the
series toward the histamine H4 receptor. This observation was further investigated by synthesizing a ser-
ies of ligands that varied in pKa of the pyrimidine derived H4 ligands by over five orders of magnitude and
the effect on histamine H4 affinity. This trend was then extended to the discovery of C-linked piperidinyl-
2-amino pyridines as histamine H4 receptor antagonists.
Received 5 September 2014
Revised 26 September 2014
Accepted 1 October 2014
Available online 13 October 2014
Keywords:
Histamine H4
H4
Ó 2014 Elsevier Ltd. All rights reserved.
pKa
Pyrimidine
Pyridine
Aminopyrimidine
The histamine H4 receptor is a 390 amino acid G-protein coupled
receptor expressed on several types of immune system cells such as
eosinophils, mast cells, dendritic cells and other leukocytes.1–4
Antagonists of the histamine H4 receptor have demonstrated activ-
ity in animal models of pruritus, asthma, ulcerative colitis and rheu-
matoid arthritis.5,6 As such, we have had a long-standing interest in
the development of histamine H4 antagonists initially describing
JNJ 7777120 (1),7 the first potent and selective H4 antagonist.
Recently we reported on the SAR of a series of 6-alkyl-2,4-diamino-
pyrimidine histamine H4 antagonists which led to the identification
of the clinical compound JNJ 39758979 (2) (Fig. 1).8
During the course of this work we had also investigated a series
of pyrimidines lacking the primary amine at C-2, 4 (Fig. 2). Des-
aminopyrimidine analogues, such as 4, demonstrated less potency
for the histamine H4 receptor implicating a direct role for the pri-
mary amine. Recognizing that the aminopyrimidine likely has a
key interaction with one of the acid residues in the active site, it
was postulated that the pKa of that moiety could play a role in con-
trolling the activity, consistent with the analogs sharing a binding
motif with histamine.9,10 Calculated (ACD) and experimental
(potentiometric titration performed by pION) pKa measurements
revealed a drop in pKa, for the less potent analogue 4.
compound 5, which also resulted in a significant loss in affinity
for the histamine H4 receptor. These observations led us to further
define the requirements around the central core pyrimidine,
including the presence and location of heterocyclic and exocyclic
nitrogens. The importance of the overall basicity of the heterocycle
on histamine H4 receptor affinity was evaluated by modulating the
pKa by varying both the C-6 substituents and the properties of the
diamine at C-4 (Fig. 2 and Table 2).
In order to probe the pKa effect further, we systematically
altered the pKa of the 6-alkyl aminopyrimidines with electron
withdrawing substituents to determine the effect on histamine
H4 receptor affinity. The pKa measurements were performed by
Analiza; the first site of protonation at N-Me of the diamine
pKa(N-Me) with subsequent protonation at the heterocycle
pKa(Het) is consistent with the ACD predictions of basicity. We
NH2
Cl
O
N
N
N
Me
N
H
N
NH2
Me
N
Me
1
2
Additionally, a reduction in pKa was noted when electron with-
drawing groups were directly adjacent to the ring, that is, 6-CF3
JNJ 7777120
hH4 Ki = 5 nM
JNJ 39758979
hH4 Ki = 12 nM
Figure 1. Histamine H4 ligands.
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2014.10.013
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

5490
B. M. Savall et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5489–5492
withdrawing fluorine 14 provides an additional 0.5 drop in the
NH2
NH2
N
1
pKa and similar reduction in affinity (Ki = 126 nM). At this point,
additional fluorine substitution lowers the pKa below 5.0 dramati-
cally reducing the affinity of the ligand to the H4 receptor (15, 5
>10,000 nM). However, when the trifluoromethyl group is not
directly attached to the pyrimidine ring (16, 17), the pKa remains
above 5.0 with subsequent restoration of affinity.
N
N
Me
N
N
Me
3
N
F3C
Me
5
N
N
N
Me
Me
N
N
N
3
4
5
In addition to the halogen substitution on the 6-alkyl-2-amino
pyrimidine series, we targeted a range of calculated pKa’s via a
combination of other groups on pyrimidine and pyridine cores
(Table 1). It is interesting that the ACD calculated pKa ranges from
6.5 to 9.2 for compounds 19–24, while the measured values show
little change (6.3–6.6). In the case of the 1,5-pyrimidines (19–22)
pKa and affinity did not display a similar trend seen for the
1,3-pyrimidines. This is most readily seen by comparing the
1,3-pyrimidine 3 to the 1,5-pyrimidine 19, where there is no
change in the pKa, but the affinity goes form 21 nm to >300 nM
demonstrating pKa is not the only requirement of the receptor.
The des-amino derivative 20 has a similar pKa as 19 and is slightly
less potent (460 nM). In the 1,5-pyrimidine series the addition of
an N-linked substituent at the 6-position dramatically increases
the affinity in both the amino 21 and des-amino 22 cases. But in
this series too, the des-amino derivative 22 is of similar potency
to 21 and has a similar pKa as 22. Finally, the C-6 N-linked pyri-
dine/aminopyridine series shows the 2-amino derivative 23 being
more potent than the 2-des-amino analog 24.
21 nM
1.5
456 nM
1.8
hH4 Ki:
clogP:
> 10,000 nM
1.6
ACD calc pKaa
Expt pKab
8.5
7.5
4.5
3.1
7.1
5.9
Figure 2. Initial Observations of Role of pKa. aACD/pKa DB, version 12.01, Advanced
Chemistry Development, Inc., Toronto, ON, Canada, www.acdlabs.com, 2013. bThe
initial pKa determinations were performed by pION Inc., using the potentiometric
method.11
observed a trend of decreasing affinity with decreasing pKa(Het)
(Fig. 3). Protonation of the N-Me of the pyrrolidine were roughly
equivalent across all of the compounds with pKa(N-Me) = 8.3–8.7
[pKa(N-Me) data not shown in tables]. Starting with the ethyl
derivative 3, replacement of the terminal methyl with the iso-
steric chlorine 13 lowers the pKa by 0.3 resulting in a reduction
in affinity from 21 to 92 nM. Changing to the more electron
NH2
N
N
Me
R
N
NH
entry
R =
3
13
Cl
14
15
5
16
F3C
17
F
F
Me
F3C
F3C
F
ACD pKaa
Analiza pKab
8.4
6.7
6.0
6.6
5.5
5.3
4.5
6.8
5.4
7.8
7.8
6.5
4.4
3.1
hH4 Kic
21 nM
92 nM 126 nM >10,000 nM >10,000 nM 11 nM
16 nM
Figure 3. pKa versus Affinity of 6-Alkyl-2,4-dimaminopyrimidines. aACD/pKa DB, version 12.01, Advanced Chemistry Development, Inc., Toronto, ON, Canada,
www.acdlabs.com, 2013. bAll data was obtained using a pKa PRO Analyzer (AATI, Ames, IA). An electrophoretic separation is performed in parallel across 24 different pH
values, providing a direct measure of overall compound charge versus pH with the compounds are detected by UV at 228 nm. The total number of pKa values are predicted by
relating mobility and compound molecular weight using pKa EstimatorÒ software (AATI, Ames, IA). The compounds were diluted 100 fold with 10 mM NaOH for a final
compound concentration of 300 lM and 1% DMSO. The compounds were transferred into 24 consecutive wells of a 96 well skirted PCR plate for analysis with the aqueous
method. All compounds gave high quality data in the aqueous method. cDisplacement of [3H]histamine from the recombinant histamine H4 receptor. Ki values were run in
triplicate and calculated according to Cheng and Prusoff.14
Table 1
pKa of N-methylpyrrolidine compounds 18–24
R1
1
3
N
X
Me
NH
R2
Y
5
N
Compound #
X
Y
R1
R2
hH4Ki (nM)
Calcd pKa (Het)
Expt pKa (Het)
3
19
20
21
22
23
24
N
CH
N
N
N
N
NH2
NH2
H
NH2
H
Et
i-Pr
i-Pr
21
8.4
6.5
6.8
7.2
7.6
9.2
9.3
6.3
6.3
6.5
6.3
6.4
6.5
6.6
CH
CH
CH
CH
CH
CH
320
460
6.0
6.0
3.1
19
Isopropyl amine
Isopropyl amine
Isopropyl amine
Isopropyl amine
CH
CH
NH2
H

B. M. Savall et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5489–5492
5491
Table 2
pKa of piperazine/piperidine compounds
NH2
1
3
N
X
R1
Y
5
NH
Compound #
X
Y
R1
hH4Ki (nM)
Calcd pKa (Het)
Expt pKa (Het)
25
26
27
28
29
30
N
N
N
CH
CH
CH
N
Cyclopentane
Cyclopentane
Cyclopentyl amine
Cyclopentyl amine
Cyclopentyl amine
Cyclopentyloxy
1
7.2
4.1
7.2
6.4
8.3
4.8
7.9
4.5
6.1
6.1
6.5
4.7
CH
CH
CH
N
1547
>1000
50
2.0
>10,000
CH
1) Meldrums acid
pyridine, CH2Cl2
Though the 1,5 pyrimidines lacked a trend with pKa, we wanted
to further elaborate on the trends observed in the 1,3-pyrimidine
series. A unique set of molecules was produced when the heterocy-
cle pKa was modulated by altering the diamine portion of the mol-
ecule. The 6-alkyl-2-aminopyrimidine 25 contains a piperazine as
the aliphatic basic amine with Ki = 1 nM. Replacement of the prox-
imal piperazine nitrogen to give the piperidine derivative 26
results in a 1000-fold loss in potency for H4. This can be attributed
to the large drop in pKa, almost 4 units, which was anticipated by
the experimental calculation with pKa (ACD) = 4.1. Based on our
results with the C-4 N-linked derivatives (vide supra), we thought
it would be possible to regain affinity by modulating the pKa of the
ring nitrogen by replacing the 6-alkyl group with an aminoalkyl
substituent. While the pKa(Het) was increased to 6.2 (experimen-
tal), the cyclohexylamine compound 27 was inactive (Ki
>1000 nM). However, switching to the aminopyridine core (28)
increased the H4 affinity (Ki = 50 nM) with a similar pKa(Het) = 6.1.
The piperazine equivalent of 28 (compound 29) is also potent at
the H4 receptor. The cyclohexylalkoxy derivative 30 was inactive
at the histamine H4 receptor; consistent with a low pKa = 4.7.
O
O
O
F C
3
F C
OMe
3
Cl
33
2) MeOH, 65 °C
24-50%
32
NH
NH
K2CO3,
MeOH, 23 °C
HCl
2
H N
2
NH
NH2
N
H N
N
N
O
O
2
F C
2
F
OMe
OMe
35
34
NH2
N
N
O
HO
OMe
36
Scheme 1. Attempted synthesis of compound 16.
NH2
b
a
O
O
N
N
Cl
HO
OMe
OH
37
NH2
38
NH2
c
d
N
N
N
N
Cl
BOC
Cl
N
Cl
N
Me
39
40
Me
Me
N
Me
Me
e
N
N
N
f
N
N
N
Cl
BOC
F3C
BOC
N
N
N
Me
NH2
N
41
42
Me
N
F3C
N
NH
Me
16
a) guanidine HCl, KOtBu, MeOH, 23 °C (46%); b) POCl3(96%); c) (R)-BOC-3-
methylaminopyrrolidine, Et3N, IPA (78%); d) acetylacetone, 120 °C (26%); e) methyl 2-
chloro-2,2-difluoroacetate, KF, CuI, DMF (29%); f) CH3CN, 6.0 N HCl(aq) (52%)
Scheme 2. Modified synthesis for compound 16.

5492
B. M. Savall et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5489–5492
NH
NH2
2
NH2
NH2
a
b
N
N
N
N
a
c
N
N
NH
2
N
N
HN
HN
HN
Cl
NH
NH
Cl
Cl
NH
NH
N
N
25
NH
47
30
46
Cl
2
43
H2N
N
N
(Pin) B
2
d
N
44
BOC
26
NH
NH
2
29
2
b
N
N
N
N
NH2
NH2
N
Cl
(Pin) B
2
H
N
45
d
N
N
H
b
27
NH
N
44
46
BOC
O
O
Cl
a) 1. BOC-piperazine, Et3N, EtOH, 90 °C 2. HCl/formic acid (13%) b) 1.
48
NH
31
cat. RuPhos Pd(II) precat, cat. RuPhos, Na2CO3 or K3PO4, dioxane/H2O,
100 °C 2. Pd/C, H2, MeOH 3. HCl/dioxane (19-28%)
a) cyclopentylamine, 160 °C (42%) b) cyclopentanol, NaH, THF, 130 °C
(27%) c) piperazine, Yb(OTf)2 H2O, IPA, 160 °C (80%) d) 1. 34, cat.
RuPhos Pd(II) precat, cat. RuPhos, Na2CO3, dioxane/H2O 2. Pd/C, H2,
MeOH 3. HCl/formic acid or 5 N HCl/IPA (13-55%)
Scheme 3. Synthesis of 2-aminopyrimidine piperidine analogues.
Chemistry: The majority of the pyrimidine derivatives in this
communication were prepared as previously reported.8 In the case
of the trifluoroethyl pyrimidine derivative (5), the standard route
was not successful (Scheme 1). In short, guanidine is condensed
with a b-ketoester and the resulting pyrimidinone is transformed
into a chloropyrimidine which is elaborated to the final derivatives.
When this synthetic protocol was applied to the requisite CF3
b-ketoester 33, the conversion to the desired pyrimidine was not
successful. The reaction was not clean, and the only identifiable
product was the aminopyrimidinone ester 36, which could arise
by a sequence where the addition of the guanidine occurs with
subsequent elimination/Michael addition followed by hydrolysis
at the fluorine containing carbon (33 ? 34 ? 35).
Scheme 4. Synthesis of 2-aminopyridine piperidine analogues.
similar disposition as histamine. Indeed, the 6-alkyl-2,4-diamino
pyrimidines showed that ligands with a pKa <5.0 had reduced affin-
ity for the histamine H4 receptor. And generally, compounds with
too low of pKa had reduced H4 affinity. Though the pKa trend did
not appear to directly transfer to the 1,5-pyrimidine series, all com-
pounds in that series had the ring nitrogen pKa greater than 6.5 and
at this point it is unknown how lowering the pKa in that series would
impact affinity. Potency could also be maintained even when replac-
ing the typical diamine portion of the 2-aminopyrimidine series
with a piperidine. However, this could only be accomplished with
the 2-aminopyridine core with the ring nitrogen at the N-1 position.
It is our hope that this data will help to refine the published models
of the histamine H4 receptor.
Scheme 2 outlines the modified syntheses used to make deriv-
ative 16. The c-chloro-b-ketoester 37 is condensed with guanidine
with subsequent hydrolysis of the alkyl chlorine to the alcohol 38.
Treatment with POCl3 provides the bis-chloride 39, and addition of
the BOC-protected aminopyrolidine selectively adds to the hetero-
aryl chloride providing the alkyl chloro intermediate 40. The free
NH2 is protected as a dimethylpyrrole12 41 and the CF3 is installed
using copper mediated trifluoro-methylation13 to provide interme-
diate 42. Treatment with aqueous HCl provides the fully deprotec-
ted product 16.
Acknowledgments
The authors would like to thank Aimee Kestranek and Justin
Longenberger of Analiza for the robustness of their pKa
determinations.
Synthesis of the C-linked analogues are shown in Schemes 3 and
4. Starting from known mono-chloro pyrimidine 43, addition of
Boc-piperazine under basic conditions followed by BOC cleavage
afforded 25. The corresponding piperidine analogues were accessi-
ble by taking pyrimidyl chlorides 43 and 45 and subjecting to
cross-coupling with 44 using the RuPhos Pd-precatalyst affording
the carbon-linked scaffolds. Saturation of the olefins followed by
acidic cleavage of the Boc protecting group afforded the desired ana-
logues 26 and 27. The pyridine analogues were obtained in a similar
fashion by initial nucleophilic displacement of the pyridyl chloride
providing a separable mixture of constitutional isomers. Subsequent
amine addition or the cross-coupling sequence yielded the pyridine
compounds with analogous substitution patterns (Scheme 4).
During the course of our histamine H4 drug discovery program,
we determined that the heterocyclic ring nitrogen provided an
affinity boost relative to the des-nitrogen counterparts. Initial
observations led us to suspect that pKa may be playing an important
role in affinity of these pyrimidine derived ligands for the histamine
receptor coupled with the ability of these compounds to bind in a
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