Human Toll-like Receptor (TLR) 8-Specific Agonistic Activity in Substituted Pyrimidine-2,4-diamines

Article abstract of DOI:10.1021/acs.jmedchem.6b00872

Activation of human toll-like receptor-8 (TLR8) evokes a distinct cytokine profile favoring the generation of Type 1 helper T cells. A multiplexed high-throughput screen had led to the identification of N⁴-butyl-5-iodo-6-methylpyrimidine-2,4-diamine as a pure TLR8 agonist, and a detailed structure-activity relationship study of this chemotype was undertaken. A butyl substituent at N⁴ was optimal, and replacement of the 5-iodo group with chloro, bromo, or fluoro groups led to losses in potency, as did the introduction of aromatic bulk. Drawing from our previous structure-based design, several 5-alkylamino derivatives were evaluated. Significant enhancement of potency was achieved in 5-(4-aminobutyl)-N⁴-butyl-6-methylpyrimidine-2,4-diamine. This compound potently induced Th1-biasing IFN-γ and IL-12 in human blood, but lower levels of the proinflammatory cytokines IL-1β, IL-6, and IL-8. These results suggest that the inflammatory and reactogenic propensities of this compound could be considerably more favorable than other TLR8 agonists under evaluation.

Full text of DOI:10.1021/acs.jmedchem.6b00872

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Article
Human Toll-like Receptor (TLR) 8-specific Agonistic
Activity in Substituted Pyrimidine-2,4-diamines
Mallesh Beesu, Alex C. D. Salyer, Kathryn L. Trautman, Justin K. Hill, and Sunil A. David
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00872 • Publication Date (Web): 11 Aug 2016
Downloaded from http://pubs.acs.org on August 13, 2016
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Human Toll-like Receptor (TLR) 8-specific Agonistic
Activity in Substituted Pyrimidine-2,4-diamines
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Mallesh Beesu, Alex C. D. Salyer, Kathryn L. Trautman,
Justin K. Hill, and Sunil A. David*
Department of Medicinal Chemistry,
University of Minnesota, Minneapolis, MN 55455, USA.
Running Title:
Keywords:
TLR-8 agonism in substituted pyrimidine-2,4-diamines
TLR8, TLR8 agonists, Pyrimidine-2,4-diamines, Vaccine adjuvants,
Innate immunity.
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Abstract
Activation of human tollꢀlike receptorꢀ8 (TLR8) evokes a distinct cytokine profile favoring the
generation of Type 1 helper T cells. A multiplexed highꢀthroughput screen had led to the
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identification of N ꢀbutylꢀ5ꢀiodoꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine as a pure TLR8 agonist, and a
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detailed SAR study of this chemotype was undertaken. A butyl substituent at N was optimal, and
replacement of the 5ꢀiodo group with chloro, bromo, or fluoro groups led to losses in potency, as
did the introduction of aromatic bulk. Drawing from our previous structureꢀbased design, several 5ꢀ
alkylamino derivatives were evaluated. Significant enhancement of potency was achieved in 5ꢀ(4ꢀ
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aminobutyl)ꢀN ꢀbutylꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine. This compound potently induced Th1ꢀ
biasing IFNꢀγ and ILꢀ12 in human blood, but lower levels of the proinflammatory cytokines ILꢀ1β,
ILꢀ6 and ILꢀ8. These results suggest that the inflammatory and reactogenic propensities of this
compound could be considerably more favorable than other TLR8 agonists under evaluation.
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Introduction
Tollꢀlike receptors (TLRs) constitute important sensors in the activation of innate immune
cells including monocytes, macrophages and dendritic cells. These cells function as sentinels
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against foreign antigens and pathogens, recognizing pathogenꢀassociated molecular patterns
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(
PAMPs) through patternꢀrecognition receptors (PRRs). Activation of PRRs initiate an array of
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ꢀ10
innate immune responses, which serve to augment subsequent adaptive immune responses.
The
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0 functional TLRs in the human encode proteins with an extracellular domain having leucineꢀrich
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repeats (LRR) and a cytosolic domain called the Toll/ILꢀ1 receptor (TIR) domain. TLR1, ꢀ2, ꢀ4, ꢀ5,
and ꢀ6 recognize extracellular stimuli, while TLR3, ꢀ7, ꢀ8 and ꢀ9 function within the endolysosomal
compartment. The ligands for TLRs are highly conserved molecules such as lipopolysaccharides
(
LPS) and monophosphoryl lipid A (MPLA) (recognized by TLR4), lipopeptides (TLR2 in
combination with TLR1 or TLR6), flagellin (TLR5), single stranded RNA (TLR7 and TLR8),
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double stranded RNA (TLR3), and CpG motifꢀcontaining DNA (recognized by TLR9).
T lymphocytes bear antigen specific receptors on their cell surface to allow recognition of
foreign pathogens, and their effector functions are determined in part by the production of key
cytokines. The two main subsets of T lymphocytes are distinguished by cell surface markers Cluster
of Differentiation 4 (CD4) and CD8. CD4ꢀexpressing helper T lymphocytes are prolific cytokine
producers and can be further subdivided into Th1 and Th2 subsets, depending on specific cytokine
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signatures.
In the context of Th1ꢀbiased adaptive immune responses, TLR8 is of particular
significance. The engagement of TLR8, which is expressed predominantly in myeloid dendritic
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cells, monocytes, and monocyteꢀderived dendritic cells,
potently enhances the production of
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Th1ꢀpolarizing cytokines, tumor necrosis factorꢀα (TNFꢀα), interleukinꢀ12 (ILꢀ12), ILꢀ18, and
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interferonꢀγ (IFNꢀγ).
Our interest in Th1ꢀpolarizing small molecule agonists of TLR8 has led
to the exploration of a variety of pure TLR8 agonists with no detectable activity at TLR7, including
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the 2,3ꢀdiaminoꢀfuro[2,3ꢀc]pyridines (1a),
4ꢀaminoꢀfuro[2,3ꢀc]quinolones (1b),
3ꢀalkylꢀ
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quinolineꢀ2ꢀamines
(1c),
1ꢀalkylꢀ2ꢀaminobenzimidazoles
(1d),
2ꢀaminoꢀ3ꢀpentylꢀ5ꢀ
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alkylaminoquinolines (1e), and the 2ꢀaminoimidazoles (1f) (Fig. 1).
A recent multiplexed, reporter geneꢀbased highꢀthroughput screen led to the identification of
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N ꢀbutylꢀ5ꢀiodoꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine as a pure TLR8 agonist, and we report here a
structureꢀactivity relationship study of the 2,4ꢀdiaminopyrimidines. The protonatable C4ꢀamine
appears to serve as a Hꢀbond donor, since substitutions of the amine abrogated activity, and a butyl
substituent at this position was found to be optimal. A dramatic enhancement of potency (and
preservation of pure TLR8 agonistic activity) was achieved in substituting the iodo group at C5
with an aminobutyl group. This compound potently induced Th1ꢀbiasing IFNꢀγ and ILꢀ12 in
human blood, but lower levels of the proinflammatory cytokines ILꢀ1β, ILꢀ6 and ILꢀ8. These results
suggest that the inflammatory and reactogenic propensities of this compound could be considerably
more favorable than other TLR8 agonists under evaluation.
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Results and Discussion
The discovery and development of safe and effective vaccine adjuvants has been a central
research goal in our laboratory, which has led us to explore structureꢀactivity relationships (SAR) in
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a variety of innate immuneꢀstimulatory chemotypes.
We recently concluded a multiplexed,
reporter geneꢀbased highꢀthroughput screen with a view to identifying novel immunostimulatory
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molecular classes; 552 provisional hits were identified from among 123,943 compounds.
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Deconvolution and validation of hits led to the identification of N ꢀbutylꢀ5ꢀiodoꢀ6ꢀ
methylpyrimidineꢀ2,4ꢀdiamine (4b, Scheme 1, Table 1) as a pure TLR8 agonist.
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It is to be noted that several aminopyrimidines have been reported in the patent literature
to possess mixed TLR7/8ꢀagonistic activity, and it was of particular interest to examine analogues
of 4b. SAR studies on 4b began with the reꢀsynthesis of the hit, as well as a verification of optimal
alkyl chain length which we previously have shown to be a crucial determinant of activity.
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Compound 4b (N ꢀbutyl), its homologs 4c (N ꢀpentyl), 4d (N ꢀhexyl), as well as a shorterꢀchain
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analogue 4a (N ꢀpropyl) were synthesized in a straightforward manner from commerciallyꢀavailable
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ꢀchloroꢀ6ꢀmethylpyrimidinꢀ2ꢀamine by iodination at C5 and aromatic nucleophilic substitution
with alkylamines (Scheme 1). Primary screens in reporter gene assays verified that 4b was a bona
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fide TLR8ꢀspecific agonist, and the N ꢀbutyl substituent in 4b was optimal (EC : 1.64 ꢀM, Table 1,
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Fig. 2). The C4ꢀamine appears to serve as a Hꢀbond donor, since substitutions of the amine (4e, 4f)
abrogated activity (Table 1). This was borne out in the bioisosteric 4ꢀbutoxy (4g) and 4ꢀthiobutyl
(
4h) analogues which were also inactive. However, we had previously observed in the case of the 3ꢀ
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alkylꢀquinolineꢀ2ꢀamines that the Cꢀalkyl compound, exemplified by 1c, was significantly more
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active than its heteroatomꢀlinked congeners, we sought to synthesize the Cꢀalkyl analogue with a
chain length comparable to that of 4b. The pentylꢀsubstituted pyrimidine analogue 4k was accessed
via Sonogashira coupling of 2 with 1ꢀpentyne, reduction of the alkyne, followed by iodination
(Scheme 1). Compound 4k as well as its precursors 4j and 4i were found to be inactive, indicating
that a Hꢀbond donor functionality was indispensable at C4.
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We then systematically examined the influence of various halogens at C5. The 5ꢀchloro and
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ꢀbromo analogues (6a and 6b, respectively) were obtained from 2 using conventional method
(Scheme 1). We found it expedient to synthesize the 5ꢀfluoro substituted 6c by standard
condensationꢀcyclization of 2ꢀfluoroethylacetoacetate with guandine via the 4ꢀhydroxyꢀ2ꢀaminoꢀ5ꢀ
fluoroꢀ6ꢀmethylpyrimidine intermediate (Scheme 1). Replacement of the 5ꢀiodo group with chloro
(6a), bromo (6b), or fluoro (6c) groups led to substantial losses in potency.
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We next explored the consequences of replacing the 6ꢀmethyl group of 4b, holding the N ꢀ
butyl group invariant. These target compounds (15ꢀ23) were readily accessed from commerciallyꢀ
available substituted 2ꢀaminoꢀ4ꢀchloropyrimidines (Scheme 2). The EC of the desꢀiodo analogue
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5 was 22 ꢀM (ca. thirteenꢀfold less active than 4b; Table 1, Fig. 2); the desꢀiodo, desꢀmethyl
compound 16 was feebly active (EC : 73.2 ꢀM); and the desꢀmethyl analogue 17 was entirely
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without TLR8ꢀagonistic activity (Table 1). The retention of activity in 15 prompted the exploration
of analogues with various electronꢀdonating and ꢀwithdrawing groups at C6, in the presence or
absence of iodo substitution at C5 (18ꢀ23); all of these compounds, however, were inactive,
pointing strongly to electronic, rather than steric effects of substitutions at C6. Crystallographic
studies are being planned to examine the structural basis of attenuation of activity in 15 and 16, and
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the complete loss of activity in 17.
Mindful of the prominent πꢀπ interactions that we had previously observed in the crystal
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structures of a variety of chemotypes with Phe405 in TLR8,
we asked if functionalizing
C5 with a bulky aromatic group would enhance potency. The C5ꢀbenzyl analogue 24, synthesized
from 4b via conventional Suzuki coupling (Scheme 3), resulted in diminution of activity, exhibiting
greatly attenuated areaꢀunderꢀthe curve in doseꢀresponse assays. We have, in the course of
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exploring several chemotypes,
observed that such compounds are inactive in secondary screens.
In our previous efforts, crystallographic observations in two regioisomeric, dual TLR7/8ꢀ
active imidazoquinolines had suggested that the formation of a strong ionic Hꢀbond (salt bridge)
with the side chain carboxylate of Asp545 not only resulted in enhancement of agonistic activity in
primary screens, but also in higher proinflammatory cytokine induction in whole human blood
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assays; we were successful in testing this hypothesis whereby strategically grafting an aminoalkyl
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group on an aminoquinoline scaffold resulted in a twentyꢀfold enhancement of potency. Our
attention therefore turned to 5ꢀalkylamino derivatives of 15. Sonogashira coupling of 4b with
aminoꢀsubstituted alkynes (Scheme 3) afforded access to compounds 27aꢀc (propylꢀ, butylꢀ, pentylꢀ
amino derivatives). The potency of the propylamino derivative 27a increased nearly tenꢀfold (2.31
ꢀM) relative to that of 4b (22 ꢀM); the potency of the butylamino compound 27b was ca. 73ꢀfold
higher (0.3 ꢀM; Table 1, Fig. 2) and appeared to plateau with the pentylamine homologue, 27c (0.3
ꢀM). We were gratified that this approach resulted in enhanced potency.
In our earlier work on the exploration of SAR in the 2ꢀaminoimidazoles, we had observed
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that the introduction of a phenyl group at C4 allowed for aromatic stacking with nearꢀperfect
coplanarity between the C4ꢀphenyl ring of Phe405 of TLR8; the C4ꢀphenyl group was accomodated
in a pocket tightly circumscribed and delimited by the hydrophobic residues Tyr353, Val378,
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Val520 and Gln519. We were unsuccessful in enhancing potency in the C5ꢀderivatized analogue
4, and it remained for us to explore the functionalization of C6. The C6ꢀphenyl analogue 28,
synthesized via Suzuki coupling (Scheme 4) resulted in a TLR8ꢀselective agonist with an EC of
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.7 ꢀM (Table 1, Fig. 2). We were, however, surprised to find that its 5ꢀiodo derivative 29 was
inactive. Compound 29 served as a convenient substrate for the syntheses of the 5ꢀalkylamino
derivatives 32aꢀc (Scheme 4). Activity in TLR8ꢀspecific primary screen was reinstated in the 5ꢀ
butylamino (32b; EC : 2.7 ꢀM) and 5ꢀpentylamino (32c; EC : 2.35 ꢀM) analogues, similar to
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what we had observed in the 27aꢀc series of compounds.
We screened the hit compound 4b, alongside the 6ꢀmethyl analogue 15, the 5ꢀbutylamino
analogue 27b, the 6ꢀphenyl substituted 28, and its 5ꢀbutylamine derivative 32b, for cytokine and
chemokine induction in human PBMCs using a 41ꢀanalyte multiplexed immunoassay platform.
TLR8 agonists upregulate the production of proinflammatory cytokines such as TNFꢀα, ILꢀ1β, ILꢀ
8
, as well as a number of chemokines; in addition, TLR8 agonists appear particularly potent in
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inducing the Th1ꢀbiasing cytokines IFNꢀγ and ILꢀ12.
As depicted in Fig. 3, 27b induces a
cytokine/chemokine signature prototypical of TLR8 agonists in ex vivo human blood stimulation
experiments, the potency (EC : 3 ꢀg/mL; 9.26 ꢀM for TNFꢀα) of which exceeds that of the
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benzimidazole 1d (used as a comparator/reference compound). Compound 27b was also found to be
particularly potent in inducing Th1ꢀbiasing IFNꢀγ and ILꢀ12, while the levels of the
proinflammatory cytokines ILꢀ1β, ILꢀ6 and ILꢀ8 appear considerably lower than those observed for
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d (Fig. 3). We have hitherto not observed a cytokine profile characterized by an apparent
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dissociation between strong IFNꢀγ, ILꢀ12 and chemokine responses on the one hand, and
considerably weaker ILꢀ1β, ILꢀ6 and ILꢀ8 levels on the other. These results suggest that the
inflammatory and reactogenic propensities of 27b could be considerably more favorable than other
TLR8 agonists that we are currently examining in detail in preclinical studies. Notably, the C6ꢀ
phenyl substituted analogues 28 and 32b are significantly weaker than both the hit compound 4b, as
well as the bestꢀinꢀclass aminopyrimidine 27b. We had previously observed an apparent
dissociation of activity in primary cellꢀbased reporter assays and secondary screens in human
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PBMCs,
and the basis for why the C6ꢀphenyl substituted analogues 28 and 32b are active in
primary screens, and become highly attenuated in human PBMCs remains to be elucidated.
In an effort to understand the mechanistic basis of amplified adaptive immune functions
resulting from TLR8 engagement, we had previously used multiꢀcolor flow cytometry to interrogate
+
activation markers (CD40, CD80) in major cellular subsets (granulocytes, monocytes (CD14 ), T
+
+
ꢀ
+
cells (CD3 ), B cells (CD19 ), NK cells (CD3 CD56 ) and cytokineꢀinduced killer cells
+
+
(
CD3 CD56 ) in human whole blood; we had shown that TLR8 stimulation strongly induces CD80
expression in the monocytes, signifying a possible specific role for TLR8 agonists in enhancing
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antigen presentation. We therefore also examined CD80 responses by the aminopyrimidine
compounds (4b, 15, 27b, 28, 32b; Fig. 4). As depicted in Fig. 4, the hit compound 4b, itself, was
more active (1.5 ꢀg/mL; 5 ꢀM) than 1d (3 ꢀg/mL; 14 ꢀM in upregulating CD80, and 27b was very
potent (0.5 ꢀg/mL; 1.5 ꢀM).
The activation of TLRs by cognate ligands culminates in the recruitment of cytosolic
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3
3
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4
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5
5
5
5
5
5
5
5
5
6
Toll/interleukinꢀ1 receptor (TIR) domainꢀcontaining adaptors coupling TLRs to downstream
effector proteins. Five adaptors have thus far been identified in signaling: Myeloid differentiation
primary response gene 88 (MyD88), MyD88ꢀadapterꢀlike (MAL), TIRꢀdomainꢀcontaining adapterꢀ
inducing interferonꢀβ (TRIF), TRIFꢀrelated adaptor molecule (TRAM) and Sterileꢀalpha and
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9
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3
5, 36
Armadillo motif containing protein (SARM).
These adaptors are thought to converge on either
MyD88ꢀ or TRIFꢀdependent pathways leading to the production of inflammatory cytokines
3
5, 36
(Myd88ꢀdependent), or IFNα/β (TRIFꢀdependent).
The structural features in smallꢀmolecule ligands that govern the recruitment of downstream
signaling pathways is not understood as yet, and our results indicating strong IFNꢀγ, ILꢀ12, and
chemokine responses and weaker proinflammatory responses by 27b suggest that selective targeting
of these signaling pathways may be a tractable goal in this chemotype, amenable to classic
medicinal chemistry approaches. These finding have been instructive, and efforts are currently
underway to further explore substitutions at C5 and C6.
Experimental Section
Chemistry. All of the solvents and reagents used were obtained commercially and used as such
unless noted otherwise. Moistureꢀ or airꢀsensitive reactions were conducted under nitrogen
atmosphere in ovenꢀdried (120 ºC) glass apparatus. Solvents were removed under reduced pressure
using standard rotary evaporators. Flash column chromatography was carried out using RediSep Rf
‘Gold’ high performance silica columns on CombiFlash Rf instruments unless otherwise
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mentioned; thinꢀlayer chromatography was carried out on silica gel CCM preꢀcoated aluminum
sheets. Purity for all final compounds was confirmed to be greater than 98% by LCꢀMS using a
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Zorbax Eclipse Plus 4.6 mm x 150 mm, 5 ꢀm analytical reverse phase C18 column with H Oꢀ
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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9
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2
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4
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8
9
0
CH CN and H OꢀMeOH gradients and an Agilent 6520 ESIꢀQTOF Accurate Mass spectrometer
3
2
(mass accuracy of 5 ppm) operating in the positive ion acquisition mode.
4
-Chloro-5-iodo-6-methylpyrimidin-2-amine (3). To a solution of compound 2 (143.6 mg, 1
mmol) in anhydrous DMF (5 mL) was added NIS (225 mg, 1 mmol), and the reaction mixture was
stirred for 12 h. The reaction mixture was diluted with water and extracted with EtOAc (3 x 30 mL).
The combined organic layer was dried over Na SO , concentrated under reduced pressure, and the
2
4
crude material was purified by flash chromatography (40% EtOAc/hexanes) to obtain the
1
13
compound 3 as a white solid (210 mg, 78 %). H NMR (500 MHz, MeOD) δ 2.55 (s, 3H). C NMR
+
(
126 MHz, MeOD) δ 173.86, 165.51, 163.76, 79.58, 30.16. MS (ESIꢀTOF) for C H ClIN [M + H]
5 5 3
calculated 269.9289, found 269.9350.
4
N -Butyl-5-iodo-6-methylpyrimidine-2,4-diamine (4b). To a solution of compound 3 (53.8 mg,
0
.2 mmol) in MeOH (3 mL), was added Et N (56 µL, 0.4 mmol) and butylamine (39.5 µL, 0.4
3
mmol). The reaction mixture was stirred for 12 h at 70 ºC. The reaction mixture was cooled to room
temperature and the solvent was removed under reduced pressure. The reaction mixture was diluted
with water and extracted with EtOAc (3 x 20 mL). The combined organic layer was dried over
Na SO and concentrated under reduced pressure, and the crude material was purified by flash
2
4
chromatography (70% EtOAc/hexanes) to afford the compound 4b as a white solid (46 mg, 75%).
1
H NMR (500 MHz, CDCl ) δ 5.24 (s, 1H), 4.76 (s, 2H), 3.42 – 3.34 (m, 2H), 2.40 (s, 3H), 1.63 –
3
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
1
3
1
.53 (m, 2H), 1.44 – 1.34 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 166.52,
3
+
1
62.16, 161.04, 68.50, 41.59, 31.65, 28.84, 20.26, 13.99. MS (ESIꢀTOF) for C H IN [M + H]
9
15
4
calculated 307.0414, found 307.0408.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
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9
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0
Compounds 4a and 4c-f were synthesized similarly as compound 4b.
4
5
-Iodo-6-methyl-N -propylpyrimidine-2,4-diamine (4a). Compound 3 (53.8 mg, 0.2 mmol), Et N
3
(56 µL, 0.4 mmol) and propylamine (32.8 ꢀL, 0.4 mmol) were used as reagents. White solid (40
1
mg, 68%). H NMR (500 MHz, CDCl ) δ 5.27 (s, 1H), 4.78 (s, 2H), 3.39 – 3.31 (m, 2H), 2.40 (s,
3
1
3
3H), 1.67 – 1.56 (m, 2H), 0.97 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 166.55, 162.18,
3
+
1
2
61.05, 68.47, 43.57, 28.84, 22.77, 11.56. MS (ESIꢀTOF) for C H IN [M + H] calculated
8
13
4
93.0258, found 293.0249.
4
5
-Iodo-6-methyl-N -pentylpyrimidine-2,4-diamine (4c). Compound 3 (53.8 mg, 0.2 mmol), Et N
3
(
56 µL, 0.4 mmol) and amylamine (46.4 ꢀL, 0.4 mmol) were used as reagents. White solid (55 mg,
1
8
1
6%). H NMR (500 MHz, CDCl ) δ 5.24 (s, 1H), 4.67 (s, 2H), 3.42 – 3.34 (m, 2H), 2.41 (s, 3H),
3
1
3
.65 – 1.55 (m, 2H), 1.41 – 1.31 (m, 4H), 0.92 (t, J = 6.7 Hz, 3H). C NMR (126 MHz, CDCl ) δ
3
166.66, 162.21, 161.05, 68.58, 41.87, 29.26, 29.25, 28.91, 22.56, 14.18. MS (ESIꢀTOF) for
+
C H IN [M + H] calculated 321.0571, found 321.0566.
10
17
4
4
N -Hexyl-5-iodo-6-methylpyrimidine-2,4-diamine (4d). Compound 3 (53.8 mg, 0.2 mmol), Et N
3
(
56 µL, 0.4 mmol) and hexylamine (52.6 ꢀL, 0.4 mmol) were used as reagents. White solid (56 mg,
1
8
4%). H NMR (500 MHz, CDCl ) δ 5.25 (s, 1H), 4.76 (s, 2H), 3.41 – 3.33 (m, 2H), 2.41 (s, 3H),
3
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3
1
.63 – 1.54 (m, 2H), 1.40 – 1.29 (m, 6H), 0.89 (t, J = 6.6 Hz, 3H). C NMR (126 MHz, CDCl ) δ
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
1
66.51, 162.16, 161.03, 68.51, 41.89, 31.65, 29.49, 28.84, 26.74, 22.72, 14.18. MS (ESIꢀTOF) for
+
C H IN [M + H] calculated 335.0727, found 335.0895.
11
19
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
4
N -Butyl-5-iodo-N ,6-dimethylpyrimidine-2,4-diamine (4e). Compound 3 (53.8 mg, 0.2 mmol),
Et N (56 µL, 0.4 mmol) and Nꢀmethylꢀ1ꢀbutanamine (47.4 ꢀL, 0.4 mmol) were used as reagents.
3
1
White solid (46 mg, 72%). H NMR (500 MHz, CDCl ) δ 4.73 (s, 2H), 3.38 (t, J = 7.5 Hz, 2H),
3
13
2
.98 (s, 3H), 2.50 (s, 3H), 1.68 – 1.58 (m, 2H), 1.37 – 1.25 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H). C
NMR (126 MHz, CDCl ) δ 170.11, 167.82, 161.10, 68.63, 53.08, 39.65, 30.36, 29.78, 20.23, 14.09.
3
+
MS (ESIꢀTOF) for C H IN [M + H] calculated 321.0571, found 321.0593.
1
0
17
4
4
4
N -Benzyl-N -butyl-5-iodo-6-methylpyrimidine-2,4-diamine (4f). Compound 3 (53.8 mg, 0.2
mmol), Et N (56 µL, 0.4 mmol) and Nꢀbenzylꢀnꢀbutylamine (71.7 ꢀL, 0.4 mmol) were used as
3
1
reagents. White solid (62 mg, 78%). H NMR (500 MHz, CDCl ) δ 7.32 – 7.29 (m, 4H), 7.26 – 7.21
3
(m, 1H), 4.75 (s, 2H), 4.63 (s, 2H), 3.34 (t, J = 7.5 Hz, 2H), 2.53 (s, 3H), 1.62 – 1.51 (m, 2H), 1.30
13
–
1
1.19 (m, 2H), 0.87 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 170.42, 167.72, 161.14,
3
38.75, 128.40, 127.99, 127.03, 70.82, 54.22, 50.38, 30.38, 29.77, 20.27, 14.06. MS (ESIꢀTOF) for
+
C H IN [M + H] calculated 397.0884, found 397.0888.
16
21
4
4
-Butoxy-5-iodo-6-methylpyrimidin-2-amine (4g). To a solution of compound 3 (53.8 mg, 0.2
mmol) in 1ꢀbutanol (3 mL), was added K CO (138 mg, 1 mmol). The reaction mixture was stirred
2
3
for 24 h at 85 ºC. The reaction mixture was cooled to room temperature and the solvent was
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9
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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6
removed under reduced pressure. The reaction mixture was diluted with water and extracted with
EtOAc (3 x 20 mL). The combined organic layer was dried over Na SO and concentrated under
2
4
reduced pressure, and the crude material was purified by flash chromatography (30%
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EtOAc/hexanes) to afford the compound 4g as a white solid (40 mg, 65%). H NMR (500 MHz,
CDCl ) δ 4.92 (s, 2H), 4.28 (t, J = 6.6 Hz, 2H), 2.49 (s, 3H), 1.79 – 1.70 (m, 2H), 1.54 – 1.43 (m,
3
1
3
2
H), 0.97 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 169.68, 168.10, 162.01, 67.60, 67.54,
3
+
30.88, 28.29, 19.35, 13.96. MS (ESIꢀTOF) for C H IN O [M + H] calculated 308.0254, found
9
14
3
3
08.0250.
4
-(Butylthio)-5-iodo-6-methylpyrimidin-2-amine (4h). To a solution of compound 3 (53.8 mg,
0
.2 mmol) in MeOH (3 mL), was added Et N (56 µL, 0.4 mmol) and 1ꢀbutanethiol (43 µL, 0.4
3
mmol). The reaction mixture was stirred for 12 h at 70 ºC. The reaction mixture was cooled to room
temperature and the solvent was removed under reduced pressure. The reaction mixture was diluted
with water and extracted with EtOAc (3 x 20 mL). The combined organic layer was dried over
Na SO and concentrated under reduced pressure, and the crude material was purified by flash
2
4
chromatography (30% EtOAc/hexanes) to afford the compound 4h as a white solid (50 mg, 77%).
1
H NMR (500 MHz, CDCl ) δ 4.92 (s, 2H), 3.05 (t, J = 7.4 Hz, 2H), 2.49 (s, 3H), 1.72 – 1.63 (m,
3
1
3
2H), 1.52 – 1.40 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 173.52, 167.19,
3
+
1
3
60.88, 81.55, 32.27, 30.95, 28.65, 22.26, 13.86. MS (ESIꢀTOF) for C H IN S [M + H] calculated
9
14
3
24.0026, found 324.0021.
4
-Methyl-6-(pent-1-yn-1-yl)pyrimidin-2-amine (4i). To a solution of compound 2 (72 mg, 0.5
mmol) in 2:1 mixture of CH CN (6 mL) and Et N (3 mL) were added Pd(PPh ) (57.8 mg, 0.05
3
3
3 4
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mmol) and CuI (19 mg, 0.1 mmol). The reaction mixture was degassed with dry nitrogen for 5 min.,
then 1ꢀpentyne (98.5 µL, 1 mmol) was added. The resulting reaction mixture was stirred for 12 h
under nitrogen atmosphere. After completion of the reaction, the reaction mixture was diluted with
water and extracted with ethylacetate (3 x 30 mL). The combined organic layer was dried over
Na SO , concentrated under reduced pressure, and the crude material was purified by flash
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
chromatography (70% EtOAc/hexanes) to obtain the compound 4i as a pale yellow solid (65 mg,
1
74%). H NMR (500 MHz, CDCl ) δ 6.57 (s, 1H), 5.06 (s, 2H), 2.40 (t, J = 7.1 Hz, 2H), 2.31 (s,
3
13
3
H), 1.70 – 1.58 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 168.43, 162.92,
3
+
1
51.71, 113.30, 94.00, 79.32, 23.88, 21.77, 21.41, 13.70. MS (ESIꢀTOF) for C H N [M + H]
10 13 3
calculated 176.1182, found 176.1172.
4
-Methyl-6-pentylpyrimidin-2-amine (4j). To a solution of compound 4i (35.4 mg, 0.2 mmol) in
anhydrous EtOAc (20 mL) was added a catalytic amount of Pd/C, and the reaction mixture was
subjected to hydrogenation at 50 psi for 5 h. The reaction mixture was filtered, and the filtrate
concentrated under reduced pressure. The crude material was purified using silica gel column
1
chromatography (60 % EtOAc/hexanes) to obtain 4j as white solid (30 mg, 84%). H NMR (500
MHz, CDCl ) δ 6.36 (s, 1H), 5.11 (s, 2H), 2.49 (t, J = 7.8 Hz, 2H), 2.29 (s, 3H), 1.69 – 1.59 (m,
3
1
3
2
H), 1.37 – 1.28 (m, 4H), 0.88 (t, J = 7.0 Hz, 3H). C NMR (126 MHz, CDCl ) δ 172.04, 167.86,
3
+
1
62.97, 110.12, 37.84, 31.73, 28.68, 23.95, 22.62, 14.11. MS (ESIꢀTOF) for C H N [M + H]
1
0
17
3
calculated 180.1495, found 180.1486.
5-Iodo-4-methyl-6-pentylpyrimidin-2-amine (4k). To a solution of compound 4j (17.9 mg, 0.1
mmol) in anhydrous DMF (1 mL) was added NIS (22.5 mg, 0.1 mmol), and the reaction mixture
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9
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was stirred for 12 h. The reaction mixture was diluted with water and extracted with EtOAc (3 x 10
mL). The combined organic layer was dried over Na SO , concentrated under reduced pressure, and
2
4
the crude material was purified by flash chromatography (40% EtOAc/hexanes) to obtain the
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compound 4k as a white solid (25 mg, 82 %). H NMR (500 MHz, CDCl ) δ 5.02 (s, 2H), 2.77 (t, J
3
13
=
8.0 Hz, 2H), 2.54 (s, 3H), 1.68 – 1.61 (m, 2H), 1.42 – 1.32 (m, 4H), 0.91 (t, J = 7.0 Hz, 3H). C
NMR (126 MHz, CDCl ) δ 172.78, 169.92, 162.02, 84.73, 41.50, 31.83, 29.70, 28.06, 22.61, 14.16.
3
+
MS (ESIꢀTOF) for C H IN [M + H] calculated 306.0462, found 306.0459.
1
0
16
3
Compounds 5a and 5b were synthesized similarly as compound 3.
4
,5-Dichloro-6-methylpyrimidin-2-amine (5a). Compound 2 (143.6 mg, 1 mmol) and Nꢀ
1
chlorosuccinimide (133.5 mg, 1 mmol) were used as reagents. White solid (126 mg, 71%). H NMR
1
3
(
500 MHz, DMSOꢀd ) δ 7.17 (s, 2H), 2.36 (s, 3H). C NMR (126 MHz, DMSOꢀd ) δ 166.74,
6
6
+
1
60.51, 156.68, 113.06, 22.68. MS (ESIꢀTOF) for C H Cl N [M + H] calculated 177.9933, found
5 5 2 3
177.9962
5
-Bromo-4-chloro-6-methylpyrimidin-2-amine (5b). Compound 2 (143.6 mg, 1 mmol) and Nꢀ
1
bromosuccinimide (178 mg, 1 mmol) were used as reagents. White solid (155 mg, 70%). H NMR
13
(
500 MHz, DMSOꢀd ) δ 7.18 (s, 2H), 2.40 (s, 3H). C NMR (126 MHz, DMSOꢀd ) δ 168.47,
6
6
+
1
61.12, 158.71, 103.25, 25.21. MS (ESIꢀTOF) for C H BrClN [M + H] calculated 221.9428,
5 5 3
found 221.9470.
Compounds 6a and 6b were synthesized similarly as compound 4b.
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5
4
N -Butyl-5-chloro-6-methylpyrimidine-2,4-diamine (6a). Compound 5a (35.6 mg, 0.2 mmol),
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Et N (56 µL, 0.4 mmol) and butylamine (39.5 µL, 0.4 mmol) were used as reagents. White solid
3
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(34 mg, 79%). H NMR (500 MHz, CDCl ) δ 5.17 (s, 1H), 4.78 (s, 2H), 3.43 – 3.35 (m, 2H), 2.28
3
1
3
(
s, 3H), 1.62 – 1.52 (m, 2H), 1.44 – 1.33 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H). C NMR (126 MHz,
CDCl ) δ 160.68, 160.30, 158.43, 103.19, 40.83, 31.76, 21.69, 20.20, 13.95. MS (ESIꢀTOF) for
3
+
C H ClN [M + H] calculated 215.1058, found 215.1050.
9
15
4
4
5
-Bromo-N -butyl-6-methylpyrimidine-2,4-diamine (6b). Compound 5b (44.5 mg, 0.2 mmol) ),
Et N (56 µL, 0.4 mmol) and butylamine (39.5 µL, 0.4 mmol) were used as reagents. White solid
3
1
(
45 mg, 87%). H NMR (500 MHz, CDCl ) δ 5.24 (s, 1H), 4.80 (s, 2H), 3.41 – 3.36 (m, 2H), 2.32
3
1
3
(
s, 3H), 1.62 – 1.52 (m, 2H), 1.44 – 1.33 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H). C NMR (126 MHz,
CDCl ) δ 162.57, 160.97, 159.11, 93.92, 41.10, 31.70, 24.23, 20.22, 13.96. MS (ESIꢀTOF) for
3
+
C H BrN [M + H] calculated 259.0553, found 259.0543.
9
15
4
4
N -Butyl-5-fluoro-6-methylpyrimidine-2,4-diamine (6c). To
a
solution of guanidine
hydrochloride (95.5 mg, 1 mmol) in MeOH (5 mL), were added compound 7 (125 µL, 1 mmol) and
Et N (280 µL, 2 mmol). The reaction mixture was refluxed for 12 h. The reaction mixture was
3
cooled to room temperature, and the resulting precipitate was filtered and washed with methanol, to
yield compound 8 as a white solid (100 mg, 69%). A suspension of compound 8 (71.5 mg, 0.5
mmol) in phosphorus(V) oxychloride was placed in a pressure vessel. The reaction mixture was
stirred for 3 h at 85 ºC. The reaction mixture was cooled to room temperature and the solvent was
removed under reduced pressure. The reaction mixture was diluted with water and extracted with
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1
2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
EtOAc (3 x 20 mL). The combined organic layer was dried over Na SO and concentrated under
2
4
reduced pressure, and the crude material was purified by flash chromatography (40%
EtOAc/hexanes) to afford the compound 9 as a white solid (50 mg, 62%). MS (ESIꢀTOF) for
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C H ClFN [M + H] calculated 162.0229, found 162.0278. To a solution of 9 (32.3 mg, 0.2 mmol)
5
5
3
in MeOH (3 mL), was added Et N (56 µL, 0.4 mmol) and butylamine (39.5 µL, 0.4 mmol). The
3
reaction mixture was stirred for 12 h at 70 ºC. The reaction mixture was cooled to room temperature
and the solvent was removed under reduced pressure. The reaction mixture was diluted with water
and extracted with EtOAc (3 x 20 mL). The combined organic layer was dried over Na SO and
2
4
concentrated under reduced pressure, and the crude material was purified by flash chromatography
1
(
80% EtOAc/hexanes) to afford the compound 6c as a white solid (30 mg, 76%). H NMR (500
MHz, CDCl ) δ 4.76 (s, 1H), 4.58 (s, 2H), 3.44 – 3.36 (m, 2H), 2.18 (d, J = 2.9 Hz, 3H), 1.62 – 1.52
3
1
3
(
m, 2H), 1.45 – 1.34 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 158.14 (d, J
3
=
5.1 Hz), 152.99 (d, J = 12.7 Hz), 147.58 (d, J = 13.7 Hz), 139.53 (d, J = 238.8 Hz). 40.31, 31.88,
+
2
0.18, 16.61, 13.95. MS (ESIꢀTOF) for C H FN [M + H] calculated 199.1354, found 199.1347.
9
15
4
Compound 11 was synthesized similarly as compound 3.
4-Chloro-5-iodopyrimidin-2-amine (11). 4ꢀChloropyrimidinꢀ2ꢀamine (10) (129.5 mg, 1 mmol)
and NIS (225 mg, 1 mmol) were used as reagents. White solid (170 mg, 67.5%). MS (ESIꢀTOF) for
+
C H ClIN [M + H] calculated 255.9133, found 255.9205.
4
3
3
Compounds 15-20 were synthesized similarly as compound 4b.
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Journal of Medicinal Chemistry
1
2
4
N -Butyl-6-methylpyrimidine-2,4-diamine (15). 4ꢀChloroꢀ6ꢀmethylpyrimidinꢀ2ꢀamine (2) (28.7
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
mg, 0.2 mmol), Et N (56 µL, 0.4 mmol) and butylamine (39.5 µL, 0.4 mmol) were used as reagents.
3
1
White solid (28 mg, 78%). H NMR (500 MHz, CDCl ) δ 5.61 (s, 1H), 4.81 (s, 2H), 4.72 (s, 1H),
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3.24 – 3.16 (m, 2H), 2.18 (s, 3H), 1.58 – 1.49 (m, 2H), 1.43 – 1.31 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H).
1
3
C NMR (126 MHz, CDCl ) δ 166.28, 164.06, 162.81, 93.09, 41.16, 31.67, 23.97, 20.18, 13.89.
3
+
MS (ESIꢀTOF) for C H N [M + H] calculated 181.1448, found 181.1442.
9
16
4
4
N -Butylpyrimidine-2,4-diamine (16). 4ꢀChloropyrimidinꢀ2ꢀamine (10) (25.9 mg, 0.2 mmol),
Et N (56 µL, 0.4 mmol) and butylamine (39.5 µL, 0.4 mmol) were used as reagents. White solid
3
1
(
25 mg, 75%). H NMR (500 MHz, CDCl ) δ 7.81 (d, J = 5.9 Hz, 1H), 5.74 (d, J = 5.9 Hz, 1H),
3
4
.84 (s, 3H), 3.26 – 3.18 (m, 2H), 1.60 – 1.50 (m, 2H), 1.44 – 1.32 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H).
1
3
C NMR (126 MHz, CDCl ) δ 163.55, 162.96, 156.60, 94.73, 41.13, 31.62, 20.18, 13.89. MS (ESIꢀ
3
+
TOF) for C H N [M + H] calculated 167.1291, found 167.1282.
8
14
4
4
N -Butyl-5-iodopyrimidine-2,4-diamine (17). 4ꢀChloroꢀ5ꢀiodopyrimidinꢀ2ꢀamine (11) (51.1 mg,
0
.2 mmol), Et N (56 µL, 0.4 mmol) and butylamine (39.5 µL, 0.4 mmol) were used as reagents.
3
1
White solid (42 mg, 72%). H NMR (500 MHz, CDCl ) δ 7.99 (s, 1H), 5.07 (s, 1H), 4.78 (s, 2H),
3
1
3
3.44 – 3.36 (m, 2H), 1.63 – 1.53 (m, 2H), 1.46 – 1.34 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H). C NMR
(
126 MHz, CDCl ) δ 162.41, 161.60, 160.26, 65.42, 41.21, 31.56, 20.24, 13.98. MS (ESIꢀTOF) for
3
+
C H IN [M + H] calculated 293.0258, found 293.0251.
8
13
4
4
N -Butylpyrimidine-2,4,6-triamine (18). 6ꢀChloropyrimidineꢀ2,4ꢀdiamine (12) (28.9 mg, 0.2
mmol), K CO (56 mg, 0.4 mmol) and butylamine (39.5 µL, 0.4 mmol) were used as reagents. Pale
2
3
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Page 20 of 52
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
yellow solid (25 mg, 69%). H NMR (500 MHz, CDCl ) δ 4.95 (s, 1H), 4.57 (s, 3H), 4.44 (s, 2H),
3
1
3
3
.15 – 3.08 (m, 2H), 1.59 – 1.49 (m, 2H), 1.44 – 1.32 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H). C NMR
(
126 MHz, CDCl ) δ 164.50, 164.45, 162.68, 74.37, 41.53, 31.56, 20.24, 13.91. MS (ESIꢀTOF) for
3
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C H N [M + H] calculated 182.1400, found 182.1389.
8
15
5
4
N -Butyl-6-methoxypyrimidine-2,4-diamine (19). 4ꢀChloroꢀ6ꢀmethoxypyrimidinꢀ2ꢀamine (13)
(31.9 mg, 0.2 mmol), Et N (56 µL, 0.4 mmol) and butylamine (39.5 µL, 0.4 mmol) were used as
3
1
reagents. White solid (32 mg, 82%). H NMR (500 MHz, CDCl ) δ 5.12 (s, 1H), 4.74 (s, 2H), 4.69
3
(
s, 1H), 3.81 (s, 3H), 3.18 – 3.10 (m, 2H), 1.59 – 1.49 (m, 2H), 1.43 – 1.31 (m, 2H), 0.92 (t, J = 7.3
1
3
Hz, 3H). C NMR (126 MHz, CDCl ) δ 171.75, 165.40, 162.52, 75.70, 53.39, 41.55, 31.51, 20.20,
3
+
1
3.88. MS (ESIꢀTOF) for C H N O [M + H] calculated 197.1397, found 197.1392.
9
16
4
4
N -Butyl-6-chloropyrimidine-2,4-diamine (20). 4,6ꢀDichloropyrimidinꢀ2ꢀamine (14) (32.8 mg,
0
.2 mmol), Et N (56 µL, 0.4 mmol) and butylamine (39.5 µL, 0.4 mmol) were used as reagents.
3
1
White solid (34 mg, 84%). H NMR (500 MHz, CDCl ) δ 5.74 (s, 1H), 5.35 (s, 3H), 3.26 – 3.06 (m,
3
1
3
2
H), 1.57 – 1.47 (m, 2H), 1.40 – 1.29 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H). C NMR (126 MHz,
CDCl ) δ 164.40, 162.57, 159.89, 91.77, 41.30, 31.43, 20.09, 13.81. MS (ESIꢀTOF) for C H ClN
4
3
8
13
+
[M + H] calculated 201.0902, found 201.0895.
Compounds 21-23 were synthesized similarly as compound 4k.
4
N -Butyl-5-iodopyrimidine-2,4,6-triamine (21). Compound 18 (18.1 mg, 0.1 mmol) and NIS
1
(22.5 mg, 0.1 mmol) were used as reagents. Pale yellow solid (15 mg, 49%). H NMR (500 MHz,
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Page 21 of 52
Journal of Medicinal Chemistry
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2
CDCl ) δ 4.92 (s, 1H), 4.81 (s, 2H), 4.60 (s, 2H), 3.44 – 3.35 (m, 2H), 1.62 – 1.52 (m, 2H), 1.45 –
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
1
3
1
.34 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 162.12, 161.66, 161.49,
3
+
4
6.15, 41.67, 32.00, 20.25, 14.02. MS (ESIꢀTOF) for C H IN [M + H] calculated 308.0367,
8
14
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
found 308.0336.
4
N -Butyl-5-iodo-6-methoxypyrimidine-2,4-diamine (22). Compound 19 (19.6 mg, 0.1 mmol) and
1
NIS (22.5 mg, 0.1 mmol) were used as reagents. White solid (22 mg, 68%). H NMR (500 MHz,
CDCl ) δ 5.06 (s, 1H), 4.71 (s, 2H), 3.87 (s, 3H), 3.43 – 3.35 (m, 2H), 1.62 – 1.52 (m, 2H), 1.45 –
3
13
1
5
3
.33 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 167.90, 162.50, 162.34,
3
+
4.44, 46.69, 41.67, 31.94, 20.23, 14.00. MS (ESIꢀTOF) for C H IN O [M + H] calculated
9
15
4
23.0363, found 323.0362.
4
N -Butyl-6-chloro-5-iodopyrimidine-2,4-diamine (23). Compound 20 (20 mg, 0.1 mmol) and NIS
1
(
22.5 mg, 0.1 mmol) were used as reagents. White solid (23 mg, 70%). H NMR (500 MHz,
DMSOꢀd ) δ 6.54 (s, 2H), 6.45 (t, J = 5.8 Hz, 1H), 3.35 – 3.27 (m, 2H), 1.54 – 1.44 (m, 2H), 1.33 –
6
1
3
1
.22 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, DMSOꢀd ) δ 162.48, 161.89, 161.12,
6
+
6
1.91, 40.94, 30.92, 19.56, 13.78. MS (ESIꢀTOF) for C H ClIN [M + H] calculated 326.9868,
8
12
4
found 326.9863.
4
5
-Benzyl-N -butyl-6-methylpyrimidine-2,4-diamine (24). To a solution of compound 4b (30.6
mg, 0.1 mmol), benzylboronic acid pinacol ester (44.4 µL, 0.2 mmol) and K CO (55 mg, 0.4
2
3
mmol) in 4:1 mixture of 1,4ꢀdioxane (2 mL) and water (0.5 mL) was added Pd(dppf)Cl (7.3 mg,
2
0.01 mmol). The reaction mixture was degassed with dry nitrogen for 5 min, then stirred for 12 h at
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Page 22 of 52
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2
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8
5 ºC under nitrogen atmosphere. The reaction mixture was diluted with water and extracted with
EtOAc (3 x 20 mL). The combined organic layer was dried over Na SO concentrated under
2
4,
reduced pressure, and the crude material was purified by flash chromatography (10%
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MeOH/CH Cl ) to obtain the compound 24 as a pale yellow solid (20 mg, 74%). H NMR (500
2
2
MHz, MeOD) δ 7.29 – 7.24 (m, 2H), 7.20 – 7.16 (m, 1H), 7.15 – 7.10 (m, 2H), 3.81 (s, 2H), 3.34 –
1
3
3
.29 (m, 2H), 2.16 (s, 3H), 1.49 – 1.39 (m, 2H), 1.24 – 1.12 (m, 2H), 0.85 (t, J = 7.4 Hz, 3H). C
NMR (126 MHz, MeOD) δ 163.45, 162.37, 162.24, 140.45, 129.59, 128.74, 127.29, 105.17, 41.44,
+
3
2.55, 31.31, 20.93, 20.88, 14.15. MS (ESIꢀTOF) for C H N [M + H] calculated 271.1917,
1
6
22
4
found 271.1917.
4
5
-(3-Aminopropyl)-N -butyl-6-methylpyrimidine-2,4-diamine dihydrochloride (27a). To a
solution of compound 4b (92 mg, 0.3 mmol) in 2:1 mixture of DMF (6 mL) and DIPEA (3 mL)
were added Pd(PPh ) (34.6 mg, 0.03 mmol) and CuI (11.4 mg, 0.06 mmol). The reaction mixture
3
4
was degassed with dry nitrogen for 5 min, then Nꢀbocꢀpropargylamine (93 mg, 0.6 mmol) was
added. The resulting reaction mixture was stirred for 12 h under nitrogen atmosphere. After
completion of the reaction, the mixture was diluted with water and extracted with ethylacetate (3 x
3
0 mL). The combined organic layer was dried over Na SO and concentrated under reduced
2 4
pressure, the crude material was purified by flash chromatography (90% EtOAc/hexanes) to obtain
+
the compound 25a as yellow oil (72 mg, 72%). MS (ESIꢀTOF) for C H N O [M + H] calculated
1
7
27
5
2
3
34.2238, found 334.2254. To a solution of compound 25a (33.3 mg, 0.1 mmol) in anhydrous
EtOAc (10 mL) was added a catalytic amount of Pd/C, and the reaction mixture was subjected to
hydrogenation at 50 psi for 5 h. The reaction mixture was filtered, and the filtrate concentrated
under reduced pressure. The crude material was purified by flash chromatography (20%
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Page 23 of 52
Journal of Medicinal Chemistry
1
2
MeOH/CH Cl ) to obtain Nꢀboc protected alkylamine 26a as a pale oil (24 mg, 71%). MS (ESIꢀ
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
2
+
TOF) for C H N O [M + H] calculated 338.2551, found 338.2698. To a stirred solution of Nꢀ
1
7
31
5
2
boc protected alkylamine 26a (17 mg, 0.05 mmol) in 1,4ꢀdioxane (1 mL) was added hydrogen
chloride (1 mL, 4 M in dioxane), and the reaction mixture was stirred for 3 h at room temperature.
Excess solvent was removed under reduced pressure and the resulted residue was thoroughly
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
washed with diethyl ether to obtain the desired compound 27a as a white solid (12 mg, 77%). H
NMR (500 MHz, , MeOD) δ 3.53 (t, J = 7.3 Hz, 2H), 3.05 (t, J = 7.6 Hz, 2H), 2.56 (t, J = 8.0 Hz,
2
3
2
2
H), 2.32 (s, 3H), 1.84 – 1.73 (m, 2H), 1.68 – 1.58 (m, 2H), 1.43 – 1.32 (m, 2H), 0.96 (t, J = 7.4 Hz,
1
3
H). C NMR (126 MHz, MeOD) δ 163.44, 155.71, 149.22, 107.20, 42.26, 40.08, 32.19, 27.02,
+
2.19, 21.14, 16.50, 14.17. MS (ESIꢀTOF) for C H N [M + H] calculated 238.2026, found
12
23
5
38.2021.
4
5
-(4-Aminobutyl)-N -butyl-6-methylpyrimidine-2,4-diamine dihydrochloride (27b). To a
solution of compound 4b (92 mg, 0.3 mmol) in 2:1 mixture of DMF (6 mL) and DIPEA (3 mL)
were added Pd(PPh ) (34.6 mg, 0.03 mmol) and CuI (11.4 mg, 0.06 mmol). The reaction mixture
3
4
was degassed with dry nitrogen for 5 min, then tertꢀbutyl butꢀ3ꢀynꢀ1ꢀylcarbamate (101.4 mg, 0.6
mmol) was added. The resulting reaction mixture was stirred for 12 h under nitrogen atmosphere.
After completion of the reaction, the mixture was diluted with water and extracted with ethylacetate
(
3 x 30 mL). The combined organic layer was dried over Na SO and concentrated under reduced
2 4
pressure, the crude material was purified by flash chromatography (90% EtOAc/hexanes) to obtain
+
the compound 25b as yellow oil (75 mg, 72%). MS (ESIꢀTOF) for C H N O [M + H] calculated
1
8
29
5
2
3
48.2394, found 348.2407. To a solution of compound 25b (34.7 mg, 0.1 mmol) in anhydrous
EtOAc (10 mL) was added a catalytic amount of Pd/C, and the reaction mixture was subjected to
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Journal of Medicinal Chemistry
Page 24 of 52
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
hydrogenation at 50 psi for 5 h. The reaction mixture was filtered, and the filtrate concentrated
under reduced pressure. The crude material was purified by flash chromatography (20%
MeOH/CH Cl ) to obtain Nꢀboc protected alkylamine 26b as a pale oil (25 mg, 71%). MS (ESIꢀ
2
2
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TOF) for C H N O [M + H] calculated 352.2707, found 352.2755. To a stirred solution of Nꢀ
18 33 5 2
boc protected alkylamine 26b (17.6 mg, 0.05 mmol) in 1,4ꢀdioxane (1 mL) was added hydrogen
chloride (1 mL, 4 M in dioxane), and the reaction mixture was stirred for 3 h at room temperature.
Excess solvent was removed under reduced pressure and the resulted residue was thoroughly
1
washed with diethyl ether to obtain the desired compound 27b as a white solid (13 mg, 80%). H
NMR (500 MHz, MeOD) δ 3.52 (t, J = 7.3 Hz, 2H), 2.96 (t, J = 7.7 Hz, 2H), 2.51 (t, J = 8.0 Hz,
2
2
4
H), 2.30 (s, 3H), 1.81 – 1.71 (m, 2H), 1.67 – 1.59 (m, 2H), 1.55 – 1.46 (m, 2H), 1.43 – 1.32 (m,
13
H), 0.96 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, MeOD) δ 163.60, 155.67, 148.81, 108.22,
2.26, 40.60, 32.21, 28.20, 26.17, 24.58, 21.16, 16.56, 14.19. MS (ESIꢀTOF) for C H N [M +
13 25
5
+
H] calculated 252.2183, found 252.2182.
Compounds 27c was synthesized similarly as compound 27b.
4
5
-(5-Aminopentyl)-N -butyl-6-methylpyrimidine-2,4-diamine
dihydrochloride
(27c).
Intermediate compound 25c. Compound 4b (92 mg, 0.3 mmol), Pd(PPh ) (34.6 mg, 0.03 mmol),
3
4
CuI (11.4 mg, 0.06 mmol) and tertꢀbutyl pentꢀ4ꢀynꢀ1ꢀylcarbamate (109.8 mg, 0.6 mmol) were used
+
as reagents. Yellow oil (75 mg, 69%). MS (ESIꢀTOF) for C H N O [M + H] calculated
19 31
5
2
3
62.2551, found 362.2578.
+
Intermediate compound 26c. Pale oil (28 mg, 77%). MS (ESIꢀTOF) for C H N O [M + H]
19
35
5
2
calculated 366.2864, found 366.2897.
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Journal of Medicinal Chemistry
1
2
1
Compound 27c. White solid (13 mg, 77%). H NMR (500 MHz, MeOD) δ 3.52 (t, J = 7.3 Hz, 2H),
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
2
.94 (t, J = 7.6 Hz, 2H), 2.49 (t, J = 7.0 Hz, 2H), 2.30 (s, 3H), 1.74 – 1.69 (m, 2H), 1.64 – 1.57 (m,
13
H), 1.54 – 1.46 (m, 4H), 1.43 – 1.31 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H). C NMR (126 MHz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MeOD) δ 163.62, 155.63, 148.53, 108.60, 42.18, 40.59, 32.21, 28.76, 28.51, 27.05, 24.85, 21.14,
+
1
6.57, 14.19. MS (ESIꢀTOF) for C H N [M + H] calculated 266.2339, found 266.2335.
1
4
27
5
4
N -Butyl-6-phenylpyrimidine-2,4-diamine (28). To a solution of compound 20 (400 mg, 2 mmol),
phenylboronic acid (366 mg, 3 mmol) and K CO (828 mg, 6 mmol) in 4:1 mixture of 1,4ꢀdioxane
2
3
(8 mL) and water (2 mL) was added Pd(dppf)Cl (73.1 mg, 0. 1 mmol). The reaction mixture was
2
degassed with dry nitrogen for 5 min, then stirred for 12 h at 85 ºC under nitrogen atmosphere. The
reaction mixture was diluted with water and extracted with EtOAc (3 x 50 mL). The combined
organic layer was dried over Na SO and concentrated under reduced pressure, and the crude
2
4
material was purified by flash chromatography (80% EtOAc/hexanes) to obtain the compound 28 as
1
a white solid (420 mg, 87%). H NMR (500 MHz, CDCl ) δ 7.93 – 7.87 (m, 2H), 7.47 – 7.38 (m,
3
3H), 6.16 (s, 1H), 4.78 (s, 2H), 4.73 (s, 1H), 3.36 – 3.28 (m, 2H), 1.65 – 1.56 (m, 2H), 1.49 – 1.37
1
3
(m, 2H), 0.96 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 164.70, 164.47, 163.28, 138.70,
3
+
1
29.76, 128.62, 126.96, 90.86, 41.30, 31.70, 20.22, 13.93. MS (ESIꢀTOF) for C H N [M + H]
14 18 4
calculated 243.1604, found 243.1596.
Compound 29 was synthesized similarly as compound 3.
4
N -Butyl-5-iodo-6-phenylpyrimidine-2,4-diamine (29). Compound 28 (242.3 mg, 1 mmol) and
1
NIS (225 mg, 1 mmol) were used as reagents. White solid (300 mg, 82%). H NMR (500 MHz,
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Journal of Medicinal Chemistry
Page 26 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CDCl ) δ 7.46 – 7.45 (m, 2H), 7.43 – 7.37 (m, 3H), 5.48 (t, J = 5.5 Hz, 1H), 4.92 (s, 2H), 3.49 –
3
13
3
.41 (m, 2H), 1.67 – 1.57 (m, 2H), 1.49 – 1.38 (m, 2H), 0.98 (t, J = 7.3 Hz, 3H). C NMR (126
MHz, CDCl ) δ 167.91, 162.20, 161.53, 142.01, 128.86, 128.55, 128.15, 67.28, 41.74, 31.58, 20.29,
3
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
14.01. MS (ESIꢀTOF) for C H IN [M + H] calculated 369.0571, found 369.0572.
1
4
17
4
Compounds 32a-c were synthesized similarly as compound 27a.
4
5-(3-Aminopropyl)-N -butyl-6-phenylpyrimidine-2,4-diamine
dihydrochloride
(32a).
Intermediate compound 30a. Compound 29 (110.4 mg, 0.3 mmol), Pd(PPh ) (34.6 mg, 0.03
3
4
mmol), CuI (11.4 mg, 0.06 mmol) and Nꢀbocꢀpropargylamine (93 mg, 0.6 mmol) were used as
+
reagents. Yellow oil (90 mg, 76%). MS (ESIꢀTOF) for C H N O [M + H] calculated 396.2394,
2
2
29
5
2
found 396.2447.
Intermediate compound 31a. Pale oil (32 mg, 80%, MS (ESIꢀTOF) for C H N O [M + H]
+
22 33
5
2
calculated 400.2707, found 400.2723).
1
Compound 32a. White solid (15 mg, 81%). H NMR (500 MHz, MeOD) δ 7.64 – 7.58 (m, 3H),
7.56 – 7.49 (m, 2H), 3.62 (t, J = 7.3 Hz, 2H), 2.81 (t, J = 7.9 Hz, 2H), 2.49 (t, J = 8.1 Hz, 2H), 1.83
1
3
–
1.74 (m, 2H), 1.73 – 1.65 (m, 2H), 1.47 – 1.38 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H). C NMR (126
MHz, MeOD) δ 163.69, 155.80, 150.93, 133.01, 131.88, 130.47, 129.63, 107.90, 42.48, 40.04,
+
32.16, 27.43, 22.94, 21.23, 14.23. MS (ESIꢀTOF) for C H N [M + H] calculated 300.2183,
1
7
25
5
found 300.2177.
4
5-(4-Aminobutyl)-N -butyl-6-phenylpyrimidine-2,4-diamine
dihydrochloride
(32b).
Intermediate compound 30b. Compound 29 (110.4 mg, 0.3 mmol), Pd(PPh ) (34.6 mg, 0.03
3
4
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Page 27 of 52
Journal of Medicinal Chemistry
1
2
mmol), CuI (11.4 mg, 0.06 mmol) and tertꢀbutyl butꢀ3ꢀynꢀ1ꢀylcarbamate (101.4 mg, 0.6 mmol)
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
were used as reagents. Yellow oil (75 mg, 61%). MS (ESIꢀTOF) for C H N O [M + H]
23
31
5
2
calculated 410.2551, found 410.2596.
Intermediate compound 31b. Pale oil (28 mg, 68%). MS (ESIꢀTOF) for C H N O [M + H]
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
23
35
5
2
calculated 414.2864, found 414.2882.
1
Compound 32b. White solid (16 mg, 83%). H NMR (500 MHz, MeOD) δ 7.63 – 7.57 (m, 3H),
7.55 – 7.48 (m, 2H), 3.60 (t, J = 7.3 Hz, 2H), 2.76 (t, J = 7.1 Hz, 2H), 2.44 (t, J = 7.3 Hz, 2H), 1.73
13
–
1.63 (m, 2H), 1.55 – 1.48 (m, 4H), 1.46 – 1.38 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H). C NMR (126
MHz, MeOD) δ 163.86, 155.75, 150.59, 133.28, 131.80, 130.37, 129.69, 108.87, 42.45, 40.31,
+
3
3
2.16, 28.05, 26.47, 25.13, 21.22, 14.21. MS (ESIꢀTOF) for C H N [M + H] calculated
1
8
27
5
14.2339, found 314.2335.
4
5-(5-Aminopentyl)-N -butyl-6-phenylpyrimidine-2,4-diamine
dihydrochloride
(32c).
Intermediate compound 30c. Compound 29 (110.4 mg, 0.3 mmol), Pd(PPh ) (34.6 mg, 0.03
3
4
mmol), CuI (11.4 mg, 0.06 mmol) and tertꢀbutyl pentꢀ4ꢀynꢀ1ꢀylcarbamate (109.8 mg, 0.6 mmol)
+
were used as reagents. Yellow oil (80 mg, 63%). MS (ESIꢀTOF) for C H N O [M + H]
24
33
5
2
calculated 424.2707, found 424.2770.
+
Intermediate compound 31c. Pale oil (30 mg, 70%). MS (ESIꢀTOF) for C H N O [M + H]
24
37
5
2
calculated 428.3020, found 428.3051.
1
Compound 32c. White solid (16 mg, 80%). H NMR (500 MHz, MeOD) δ 7.61 – 7.57 (m, 3H),
7
.53 – 7.49 (m, 2H), 3.60 (t, J = 7.3 Hz, 2H), 2.81 (t, J = 7.7 Hz, 2H), 2.41 (t, J = 8.0 Hz, 2H), 1.73
1
3
– 1.63 (m, 2H), 1.57 – 1.36 (m, 6H), 1.34 – 1.23 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H). C NMR (126
MHz, MeOD) δ 163.89, 155.70, 150.27, 133.34, 131.71, 130.30, 129.67, 109.34, 42.36, 40.45,
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Journal of Medicinal Chemistry
Page 28 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
3
3
2.15, 29.03, 28.13, 26.91, 25.34, 21.20, 14.21. MS (ESIꢀTOF) for C H N [M + H] calculated
19 29 5
28.2496, found 328.2493.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Human TLR8-specific reporter gene assays (NF-κB induction), and TLR-2/-3/-4/-5/-7/-9
counter-screens: The induction of NFꢀκB was quantified using human TLRꢀ2/ꢀ3/ꢀ4/ꢀ5/ꢀ7/ꢀ8/ꢀ9ꢀ
specific, rapidꢀthroughput, liquid handlerꢀassisted reporter gene assays as previously described by
3
1, 29, 37, 38
us.
HEK293 cells stably coꢀtransfected with the appropriate hTLR and secreted alkaline
phosphatase (sAP) were maintained in HEKꢀBlue™ Selection medium. Stable expression of
secreted alkaline phosphatase (sAP) under control of NFꢀκB/APꢀ1 promoters is inducible by
appropriate TLR agonists, and extracellular sAP in the supernatant is proportional to NFꢀκB
5
induction. Reporter cells were incubated at a density of ~10 cells/ml in a volume of 80 µl/well, in
3
84ꢀwell, flatꢀbottomed, cell cultureꢀtreated microtiter plates in the presence of graded
concentrations of stimuli. sAP was assayed spectrophotometrically using an alkaline phosphataseꢀ
specific chromogen (present in HEKꢀdetection medium as supplied by InvivoGen) at 620 nm.
Immunoassays for cytokines. Fresh human peripheral blood mononuclear cells (hPBMC) were
isolated from human blood obtained by venipuncture in Cell Preparation Tubes (CPT, Becktonꢀ
Dickinson) with informed consent and as per guidelines approved by the University of Minnesota
5
Human Subjects Experimentation Committee. Aliquots of PBMCs (10 cells in 100
ꢀ
L/well) were
stimulated for 16 h with graded concentrations of test compounds. Supernatants were isolated by
centrifugation, and were assayed in duplicates using analyteꢀspecific multiplexed
cytokine/chemokine bead array assays (HCYTMAGꢀ60KꢀPX29 MILLIPLEX MAP Human
Cytokine/Chemokine Magnetic Bead Panel, EMD Millipore, Billerica, MA) as reported by us
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Page 29 of 52
Journal of Medicinal Chemistry
1
2
2
3
previously. The following analytes were quantified: sCD40L, VEGF, TNFꢀβ, TNFꢀα, TGFꢀα,
RANTES, PDGFꢀAB/BB, PDGFꢀAA, MIPꢀ1β, MIPꢀ1α, MDC (CCL22), MCPꢀ3, MCPꢀ1, IPꢀ10,
ILꢀ17A, ILꢀ15, ILꢀ13, ILꢀ12 (p70), ILꢀ12 (p40), ILꢀ10, ILꢀ9, ILꢀ8, ILꢀ7, ILꢀ6, ILꢀ5, ILꢀ4, ILꢀ3, ILꢀ
2, ILꢀ1ra, ILꢀ1β, ILꢀ1α, IFNꢀγ, IFNꢀα2, GRO, GMꢀCSF, GꢀCSF, fractalkine, Fltꢀ3 ligand, FGFꢀ2,
eotaxin, EGF.
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Flow-cytometric immunostimulation experiments: Cell surface marker upregulation was
2
3
determined by flow cytometry using protocols published by us previously. Briefly, heparinꢀ
anticoagulated whole blood samples were obtained by venipuncture from healthy human volunteers
with informed consent and as per guidelines approved by the University of Minnesota Human
Subjects Experimentation Committee. Serial dilutions of selected compounds were performed using
a BioꢀTek Precision 2000 XS liquid handler in sterile 96ꢀwell polypropylene plates, to which were
o
added 100 µL aliquots of anticoagulated whole human blood. The plates were incubated at 37 C for
1
6 h. Negative (DMSO) controls were included in each experiment. The following fluorochromeꢀ
conjugated antibodies were used: CD14ꢀFITC, CD40ꢀAPC, CD80ꢀPEꢀCy7, CD86ꢀV450 (Bectonꢀ
Dickinson Biosciences, San Jose, CA). Following incubation, 2.5 µg of each antibody was added to
o
wells with a liquid handler, and incubated at 4 C in the dark for 60 min. Following staining,
erythrocytes were lysed and leukocytes fixed by mixing 200 µL of the samples in 800 µL preꢀ
warmed Whole Blood Lyse/Fix Buffer (BectonꢀDickinson Biosciences, San Jose, CA) in 96 deepꢀ
well plates. After washing the cells twice at 300 g for 10 minutes in RPMI, the cells were
transferred to a 96ꢀwell plate. Flow cytometry was performed using a BD FACSVerse instrument
for acquisition on 200,000 gated events. Compensation for spillover was computed for each
experiment on singlyꢀstained UltraComp Beads (eBioscience, Inc., San Diego, CA).
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