Determinants of activity at human toll-like receptors 7 and 8: Quantitative Structure-Activity Relationship (QSAR) of diverse heterocyclic scaffolds

Article abstract of DOI:10.1021/jm500744f

Toll-like receptor (TLR) 7 and 8 agonists are potential vaccine adjuvants, since they directly activate APCs and enhance Th1-driven immune responses. Previous SAR investigations in several scaffolds of small molecule TLR7/8 activators pointed to the strict dependence of the selectivity for TLR7 vis-à-vis TLR8 on the electronic configurations of the heterocyclic systems, which we sought to examine quantitatively with the goal of developing heuristics to define structural requisites governing activity at TLR7 and/or TLR8. We undertook a scaffold-hopping approach, entailing the syntheses and biological evaluations of 13 different chemotypes. Crystal structures of TLR8 in complex with the two most active compounds confirmed important binding interactions playing a key role in ligand occupancy and biological activity. Density functional theory based quantum chemical calculations on these compounds followed by linear discriminant analyses permitted the classification of inactive, TLR8-active, and TLR7/8 dual-active compounds, confirming the critical role of partial charges in determining biological activity.

Full text of DOI:10.1021/jm500744f

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Article
Determinants of Activity at Human Toll-like Receptors 7 and 8: Quantitative
Structure-Activity Relationship (QSAR) of Diverse Heterocyclic Scaffolds
Euna Yoo, Deepak Salunke, Diptesh Sil, Xiaoqiang Guo, Alex Salyer, Alec Hermanson,
Manoj Kumar, Subbalakshmi S. Malladi, Rajalakshmi Balakrishna, Ward Hugh
Thompson, Hiromi Tanji, Umeharu Ohto, Toshiyuki Shimizu, and Sunil A. David
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500744f • Publication Date (Web): 05 Sep 2014
Downloaded from http://pubs.acs.org on September 16, 2014
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Determinants of Activity at Human Toll-like Receptors
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and 8: Quantitative Structure-Activity Relationship
(QSAR) of Diverse Heterocyclic Scaffolds
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Euna Yoo, Deepak B. Salunke, Diptesh Sil, Xiaoqiang Guo,
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Alex C.D. Salyer, Alec R. Hermanson, Manoj Kumar,
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Subbalakshmi S. Malladi, Rajalakshmi Balakrishna, Ward H. Thompson,
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Hiromi Tanji, Umeharu Ohto, Toshiyuki Shimizu,
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and Sunil A. David
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Department of Medicinal Chemistry, University of Kansas
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Department of Chemistry, and Center for Environmentally Beneficial
Catalysis, University of Kansas
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Graduate School of Pharmaceutical Sciences, University of Tokyo
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Running Title: Determinants of TLR7 and TLR8 agonism: QSAR on Heterocyclic Scaffolds
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Keywords: Tollꢀlike receptors; TLR7; TLR8; QSAR; Thiazoloquinolines; Oxazoloquinolines;
Pyrazoloquinoline; Imidazoquinoxaline.
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Abstract
Tollꢀlike receptor (TLR)ꢀ7 and ꢀ8 agonists are potential vaccine adjuvants, since directly activate
APCs and enhance Th1ꢀdriven immune responses. Previous SAR investigations in several
scaffolds of small molecule TLR7/8 activators pointed to the strict dependence of the selectivity
for TLR7 visꢀàꢀvis TLR8 on the electronic configurations of the heterocyclic systems, which we
sought to examine quantitatively with the goal of developing ‘heuristics’ to define structural
requisites governing activity at TLR7 and/or TLR8. We undertook a scaffoldꢀhopping approach,
entailing the syntheses and biological evaluations of thirteen different chemotypes. Crystal
structures of TLR8 in complex with two most active compounds confirmed important binding
interactions playing a key role in ligand occupancy and biological activity. Density functional
theoryꢀbased quantum chemical calculations on these compounds, followed by linear
discriminant analyses permitted the classification of inactive, TLR8ꢀactive and TLR7/8 dualꢀ
active compounds, confirming the critical role of partial charges in determining biological
activity.
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Introduction
The human immune system can be conceived of as comprising of two mutually nonꢀexclusive
subsystems: the innate and adaptive immune limbs work cooperatively to afford protection from
numerous pathogenic microorganisms and toxins. Tollꢀlike receptors (TLRs) are important
pattern recognition receptors (PRRs) of the innate immune system which are expressed on
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leukocytes, as well as epithelial cells of mucosal surfaces. They are activated upon specific
recognition of pathogenꢀassociated molecular patterns (PAMPs) that are distinct to the
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pathogen, providing immediate defense mediated by a variety of effector mechanisms including
the production of proinflammatory cytokines, upꢀregulation of major histocompatibility complex
(
MHC) molecules and costimulatory signals in antigenꢀpresenting cells (APCs), as well as the
activation of natural killer (NK) cells. These events lead to priming and amplification of adaptive
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immune responses mediated via both Tꢀ and Bꢀlymphocyte effector functions.
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Ten functional TLRs have been identified in humans so far; TLRs present in the intracellular
compartments (TLRꢀ3, ꢀ7, ꢀ8, and ꢀ9) sense viral and bacterial nucleic acids and those expressed
on the cell surface (TLRꢀ1, ꢀ2, ꢀ4, ꢀ5, ꢀ6 and ꢀ11) sense outer membrane components of bacteria,
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fungi, and protozoan organisms. Aside from binding to exogenous PAMPs, TLRs are also
known to interact with endogenous molecules released from damaged tissues or dead cells. This
endogenous ligandꢀmediated TLR activation appears to play a central role in pathological
conditions such as tissue injury, repair and regeneration, autoimmune diseases and
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tumorigenesis.
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Several synthetic compounds have been identified as modulators of TLR signaling. Detailed
mechanistic processes underlying TLR signaling that have emerged during the past fifteen years
have not only provided insights in the interfaces between innate and adaptive immune
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responses, but are also paving the way for clinical applications. Given that TLRs are
predominantly expressed on, or in immune cells, appropriate stimulation of the immune cells via
effective targeting of TLRs is increasingly recognized as a key factor in vaccinology. From this
perspective, we have explored in detail structureꢀactivity relationships (SAR) of several
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immunostimulatory chemotypes.
In particular, TLR7/8 agonists have demonstrated potential as vaccine adjuvants, since they
directly activate APCs and can enhance both humoral and cellular immune responses —
especially Th1ꢀbiased responses. TLR7 is expressed in plasmacytoid dendritic cells (pDC) and B
cells whereas TLR8 is mainly expressed in conventional/myeloid dendritic cells (cDC),
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monocytes, macrophages, and neutrophils. The natural ligands for TLR7 and TLR8 are singleꢀ
stranded RNA (ssRNA); these endosomal TLRs can also be activated by synthetic small
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molecule TLR7/8 agonists. Small molecule TLR7/8 activators constitute a small set of
compounds occupying a limited chemical space. Extensive SAR on several TLR7/8ꢀagonistic
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scaffolds such as imidazo[4,5ꢀc]quinolines,
imidazo[4,5ꢀc]pyridines,
thiazolo[4,5ꢀ
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c]quinolines, furo[2,3ꢀc]pyridines as well as furo[2,3ꢀc]quinolines have been reported from
our laboratory (Fig. 1). Recently, a C2ꢀbutyl furo[2,3ꢀc]quinoline (5) having pure TLR8
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agonistic activity was coꢀcrystallized with the human TLR8 ectodomain. This served as the
point of departure toward a focused structureꢀbased ligand design study, leading to the
identification of 3ꢀpentylꢀquinolineꢀ2ꢀamine (6, Fig. 1) as a novel, structurally simple, and highly
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potent human TLR8ꢀspecific agonist. Our SAR investigations in several of these scaffolds,
while continuing to incrementally improve our understanding of the structural features required
for the TLR7/8 activity, pointed strongly also to the strict dependence of the selectivity for TLR7
visꢀàꢀvis TLR8 on the electronic configurations of the heterocyclic systems, the nuances of
which we desired to examine quantitatively with the goal of developing ‘heuristics’ to clearly
define structural requisites governing activity at TLR7 and/or TLR8. In order to systematically
examine the effect of electronic properties on the activity profiles, we undertook a scaffoldꢀ
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hopping approach,
entailing the syntheses and biological evaluations of thirteen different
chemotypes including oxazolo[4,5ꢀc]quinoline, thiazolo/oxazolo[4,5ꢀc]quinolinꢀ2ꢀamines,
thiazolo/oxazolo[5,4ꢀc]quinolines, imidazo[1,2ꢀc]quinazoline, [1,2,4]triazolo[1,5ꢀc]quinazoline,
imidazo[1,2ꢀa]quinoxaline, [1,2,4]triazolo[1,5ꢀa]quinoxaline, [1,2,4]ꢀtriazolo[4,3ꢀa]quinoxaline,
and pyrazolo[3,4ꢀc]quinoline.
Crystal structures of TLR8 in complex with two most active compounds confirmed important
binding interactions playing a key role in ligand occupancy and biological activity. Reasoning
that stereoꢀelectronic effects of heterocyclic ring systems could have a profound effect on the
biological activity of TLR7/8 modulators, we undertook studies of threeꢀdimensional molecular
electrostatic potential (MESP) in an effort to obtain complementary and/or mechanistic
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information in characterizing active molecules. Density functional theory (DFT) based
quantum chemical calculations and linear discriminant analyses were therefore performed. These
studies allowed, for the first time, a clear delineation of inactive, TLR8ꢀactive and TLR7/8 dualꢀ
active compounds, confirming the critical role of partial charges in determining biological
activity.
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Results and Discussion
As mentioned earlier, a number of leads including pure TLR7 agonists (1 and 2), dual TLR7/8
agonist (e.g., 3), and pure TLR8 agonists (4, 5, and 6) are undergoing preclinical evaluation as
vaccine adjuvants in our laboratory. Our earlier structureꢀactivity relationship studies on the
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imidazo[4,5ꢀc]quinolines,
thiazolo[4,5ꢀc]quinolines, furo[2,3ꢀc]pyridines, as well as
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furo[2,3ꢀc]quinolines had all converged on the optimal chain length for the C2 alkyl substituent
being butyl. Our goal was therefore to examine the electronic effects of heterocyclic
modifications, while holding the substituent at the C2 position invariant at 4 atoms. We
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envisioned that a reagentꢀbased diversification approach could allow us to access to several
different heterocyclic scaffolds (including the thiazolo[4,5ꢀc]quinolines) with substantial
variations in the electronic configurations (Scheme 1). Employing this diversification strategy,
the previously described 2ꢀbutylthiazolo[4,5ꢀc]quinoline was synthesized from aminoquinolinꢀ4ꢀ
ol and valeroyl chloride via a oneꢀpot, sequential reaction involving acylation and subsequent
microwaveꢀaccelerated (120 °C, 600W) cyclization using P S (7, Scheme 1), while replacement
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of P S with P O in this reaction resulted in its congener 8 (2ꢀbutyloxazolo[4,5ꢀc]quinoline) in
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moderate yield. Microwaveꢀassisted cyclization also yielded Nꢀpropylthiazolo[4,5ꢀc]quinolinꢀ2ꢀ
amine 10 using propyl isothiocyanate, whereas conventional heating was unsuccessful. The
synthesis of Nꢀpropyloxazolo[4,5ꢀc]quinolinꢀ2ꢀamine 12 using P O led to the formation of a
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mixture of compounds with very poor yields; substituting Nꢀ(3ꢀdimethylaminopropyl)ꢀN′ꢀ
ethylcarbodiimide (EDC) for P O in this reaction not only worked as a sulfur scavenger, but
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greatly enhanced yields of the desired oxazolo analog 12 (Scheme 1). The C4 amine
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functionality was then installed using conventional methods
to furnish the 2ꢀ
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butyloxazolo[4,5ꢀc]quinolinꢀ4ꢀamine 9, the Nꢀpropylthiazolo[4,5ꢀc]quinolineꢀ2,4ꢀdiamine 11,
and the Nꢀpropyloxazolo[4,5ꢀc]quinolineꢀ2,4ꢀdiamine 13.
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The 2ꢀbutyloxazolo[4,5ꢀc]quinolinꢀ4ꢀamine 9 displayed more potent dual TLR7/8ꢀagonistic
activity compared to the thiazolo[4,5ꢀc]quinolinꢀ4ꢀamine 3, with EC values of 0.55 ꢁM and
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.18 ꢁM in TLR7 and TLR8 assays, respectively (Fig. 2). The Nꢀpropylthiazolo[4,5ꢀc]quinolineꢀ
,4ꢀdiamine 11, however, exhibited comparable TLR7ꢀagonistic activity (EC : 0.73 µM), but a
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tenꢀfold reduction in TLR8 potency (EC : 3.94 µM). Astonishingly, the Nꢀpropyloxazolo[4,5ꢀ
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c]quinolineꢀ2,4ꢀdiamines 13 was entirely devoid of any detectable TLR7ꢀ or TLR8ꢀagonistic
activity. The C2 Nꢀacyl derivative, Nꢀ(4ꢀaminothiazolo[4,5ꢀc]quinolinꢀ2ꢀyl)propionamide 16
and its desꢀacyl analog 17 were synthesized from commerciallyꢀavailable thiazolo[4,5ꢀ
c]quinolinꢀ2ꢀamine (Scheme 2). These compounds were also found to be inactive in cell based
assays (Fig. 2).
The dramatic (and rather unexpected) differences in activity profiles of the closelyꢀrelated
congeners warranted a detailed investigation of analogues with variable electronic properties. We
first synthesized and evaluated the regioisomeric 2ꢀbutylthiazolo[5,4ꢀc]quinolinꢀ4ꢀamine 22a and
2ꢀbutyloxazolo[5,4ꢀc]quinolinꢀ4ꢀamine 22b (Scheme 3). The thiazolo[5,4ꢀc]quinoline derivative
(22a) was completely inactive in both TLR7 and TLR8 agonism assays and the oxazolo[5,4ꢀ
c]quinoline derivative (22b) was found to possess negligibly low TLR8ꢀagonistic activity. This
result, too, was unexpected, given that we had observed prominent and selective TLR8 agonism
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in the 2ꢀbutylfuro[2,3ꢀc]quinolinꢀ4ꢀamine 5, but further strengthened the case for a systematic
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exploration of the role of electron densities in the heterocyclic core in determining TLR7/8
activity.
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Further scaffold modifications were therefore carried out based on the pure TLR7ꢀagonistic lead
molecule 1ꢀbenzylꢀ2ꢀbutylꢀ1Hꢀimidazo[4,5ꢀc]quinolinꢀ4ꢀamine 1 (Fig.1). ‘Repositioning’ of the
nitrogen atoms in the imidazole ring as well as triazole analogues were designed and synthesized
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(Schemes 4ꢀ8).
The novel analogues 2,3ꢀdihydroimidazo[1,2ꢀc]quinazoline 27 and
imidazo[1,2ꢀc]quinazoline 29, with an altered imidazole fused ring (Scheme 4) were entirely
inactive (Fig. 2). We sought to examine if activity could be restored by incorporating an
additional nitrogen atom in ring system, but the triazole analogue 33 (2ꢀbutylꢀ[1,2,4]triazolo[1,5ꢀ
c]quinazolinꢀ5ꢀamine, Scheme 5) was also inactive. On the other hand, the 1,2ꢀ
dihydroimidazo[1,2ꢀa]quinoxaline 37 and the imidazo[1,2ꢀa]quinoxaline 39 shown in Scheme 6
were found to be selective TLR8 agonists with EC50 values of 3.05 ꢁM and 7.99 ꢁM,
respectively (Fig. 2). Transitioning from the imidazo[1,2ꢀa]quinoxaline scaffold to two other
triazolo analogues (46 in Scheme 7 and 49 in Scheme 8) also resulted in complete loss of
activity.
Our scaffoldꢀhopping approach also led us to synthesize 2ꢀbutylꢀ2Hꢀpyrazolo[3,4ꢀc]quinolinꢀ4ꢀ
amine 53 (Scheme 9). Compound 53 was found to be extraordinarily potent as a TLR7 agonist
(
EC : 0.19 ꢀM), significantly greater than that of the thiazoloquinoline 3, (EC : 0.86 ꢀM), the
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oxazoloquinoline 9 (EC : 0.55 ꢀM), and the aminothiazoloquinoline 11 (EC : 0.73 ꢀM), and
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approaching that of our bestꢀinꢀclass, pure TLR7 agonistic imidazoquinoline 1 (EC : 0.059 ꢀM).
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agonism assays (EC50 : 0.056 ꢀM) among all TLR8ꢀactive compounds that we had hitherto
characterized (Fig. 2).
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We confirmed TLR7/8 selectivity and potency of the active compounds in secondary screens
including cytokineꢀinducing properties in human peripheral blood mononuclear cells (hPBMCs),
as well as cellular activation in ex vivo whole human blood. In IFNꢀα induction assays, as
expected and in accordance with our previous SAR, compounds with TLR7 agonistic activity (9,
11, and 53) showed IFNꢀα inducing ability and the TLR8 selective compound 39 did not (Fig.
3). We also found strong Type II interferon (IFNꢀγ), cytokine (ILꢀ1α, ILꢀ1β, ILꢀ6, ILꢀ10, TNFꢀ
α) and chemokine (IPꢀ10/CXCLꢀ10, MCPꢀ1, MIPꢀ1β) induction by the active compounds
consistent with their TLR7/8 selectivity profiles (Fig. 4). The extraordinary potency of 53 was
also manifested in CD69 expression in whole blood assays, showing dramatically enhanced
expression in cytokineꢀinduced killerꢀ, natural killerꢀ, Tꢀ and Bꢀ lymphocytic subsets (Fig. 5).
We were fortunate in also being able to obtain the crystal structures of TLR8 in complex with the
two most active compounds: the oxazoloquinoline 9 and the pyrazoloquinoline 53. An
examination of TLR8 liganded with 9 and 53 confirmed nearꢀidentical binding geometries of the
two compounds (Fig. 6). Major interactions include hydrogen bonding of the amidine group with
Asp543 and the N atom of the oxazole/pyrazole ring with Thr574, πꢀ π interactions of the
quinoline ring with Phe405, and hydrophobic interactions of the alkyl chain in a pocket formed
by Tyr348, Val378, and Phe405.
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Whereas the steric properties of most of these analogues are very similar, their activity profiles
are considerably different and the data, taken together, strongly pointed to electronic densities of
ring system(s) being dominant determinants of occupancy and activation of TLR7 and TLR8 by
these analogues. We therefore undertook quantum chemical calculations of electron densities and
of Mulliken atomic charges with the objective of obtaining insights into the properties of these
molecules, which we hoped would lead to quantitative predictors of selectivity and potency at
TLR7 and TLR8.
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As described earlier, the crystal structures of TLR8 complexed with active analogues showed
key Hꢀbonds between the amidine group of the quinoline moiety with the side chain of Asp543,
and the N atoms of the oxazole or pyrazole moieties of compounds 9 and 53, respectively, with
Thr574, providing major contributions to overall binding interactions (Fig. 6). Consistent with
our expectation that the strength and geometry of the Hꢀbonds are modulated by electron
densities and Mulliken charges on appropriate heteroatoms, we observed clear differences in
atoms known to be involved in Hꢀbonding interactions. The active compounds 3, 9, 11, 39, and
53 (Fig. 7 and Fig. S1) display pronounced negative charges (ꢀ0.24, ꢀ0.24, ꢀ0.29, ꢀ0.27, and ꢀ0.23
electron units, respectively) at position M1 of the fiveꢀmembered ring. Compounds that do not
have the electronegative atom at position M1 (22a and 29; +0.15, 0.0) were found to be inactive.
We also noticed a higher partial positive charge at the M2 position in the oxazolo[4,5ꢀ
c]quinolineꢀ2,4ꢀdiamine 13 (0.33 electron unit, Fig. 7c) compared to other compounds,
attributable to adjacent electronegative heteroatoms (N and O), possibly explaining the lack of
activity. The quinazoline analogues 29 and 33 were unique in that the presence of an additional
electronegative nitrogen atom at position M5 (ꢀ0.25 and ꢀ0.22, respectively) resulted in strong
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positive charge at position M6 (+0.26 and +0.29 electron units, respectively, Fig. S1 and Fig.
7e), again correlating with absence of agonistic activities.
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Given the importance of hydrogen bonding of the amidine group with Asp543, we examined
electrostatic potentials at M7. Active compounds could be differentiated from inactive
compounds (Fig. 8A) with three exceptions: compounds 13, 16 and 22a. The absence of
TLR7/8ꢀagonistic activity in 16 and 22a could be explained readily; the presence of a polar
amide sidechain in 16 is expected to disfavor interactions in the hydrophobic pocket, while the
regioꢀisomeric thiazoloquinoline 22a possesses a bulky sulfur atom at M1. The misclassification
of 13 as an active compound may, as mentioned earlier, be related to the higher partial positive
charge at the M2 position.
The availability of electron density and charge parameters for all atoms in all of the analogues
prompted us to examine if a formal classification of active visꢀàꢀvis inactive compounds could be
arrived at, with our goal of being able to utilize such methodology in prospectively designing
‘bespoke’ compounds with predefined selectivity. Stepwise linear discriminant analyses were
performed with the 13 compounds shown in Fig. 2 as a training set. A linear combination of the
variables corresponding to M1ꢀM6 explained 100% of the variance in two dimensions
(Discriminant Functions 1 and 2, see Fig. 8B), allowing a clearꢀcut classification of inactive
(coded ‘0’), TLR8ꢀactive (coded ‘1’), and TLR7/8 dualꢀactive (coded ‘2’) compounds (Fig. 8B).
2
6
The discriminant functions were utilized to examine a test set of compounds which included 5
3
5
(
Fig. 1), CL097 , as well as nine ‘hypothetical’ compounds (Fig. 8B). Compound 5 and CL097
were correctly classified as being TLR8 (Group 1) and TLR7/8 dualꢀactive (Group 2). All of the
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proposed analogues with the exception of H3 were predicted to be active. It is noteworthy that
the isomeric compounds H8 and H9 (2ꢀbutylꢀcyclopentaquinolinꢀ4ꢀamines) could be considered
2
8
as conformationallyꢀconstrained analogues of 3ꢀalkylꢀquinolineꢀ2ꢀamines (Fig. 1)
which we have recently designed and characterized as pure TLR8 agonists. The
thienoquinolines H1 and H2 as well as the pyrroloquinolines H3 and H4 are of particular
interest, and we are currently evaluating such analogues.
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The question as to why the activity profiles of the oxazoloquinoline 9 and its 2ꢀamino analogue
13 are completely divergent remains unclear, however, and crystallographic observations of the
complex of 9 with TLR8 even in conjunction with electronic structure calculations only allow us
to speculate at the present time as to the role of the water molecule by virtue of its permanent
dipole moment and polarizability on stabilizing (or destabilizing interactions) depending on
electron densities around the fiveꢀmembered ring. We are gratified, nonetheless, that quantum
chemical calculations in conjunction with rigorous multivariate analyses may afford an
empirical, but accessible means to evaluating analogues de novo.
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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. The solvents were removed under reduced
pressure using standard rotary evaporators. Flash column chromatography was carried out using
0
1
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0
RediSep Rf ‘Gold’ high performance silica columns on CombiFlash R instrument unless
f
otherwise mentioned, while thinꢀlayer chromatography was carried out on silica gel CCM preꢀ
coated aluminum sheets. Purity for all final compounds was confirmed to be greater than 97% by
LCꢀMS using a Zorbax Eclipse Plus 4.6 mm x 150 mm, 5 ꢀm analytical reverse phase C18
column with H OꢀCH CN gradients and an Agilent 6520 ESIꢀQTOF Accurate Mass
2
3
spectrometer (mass accuracy of 5 ppm) operating in the positive ion acquisition mode.
2
-Butylthiazolo[4,5-c]quinoline (7): To a solution of 3ꢀaminoquinolinꢀ4ꢀol (12 mg, 0.075
mmol) in pyridine (0.5 mL) was added valeroyl chloride (11 ꢀL, 0.09 mmol) and the resulting
°
mixture was heated in a sealed vial at 50 C for 1 h. P S (33 mg) was added and the mixture was
2
5
°
heated at 120 C for 1 h under microwave irradiation. The solvents were removed and the crude
residue was purified by flash chromatography (SiO , MeOH in CH Cl : 0 to 5%) to give
2
2
2
1
compound 7 (13.4 mg, 79%) as reddish brown solid. [ H NMR (500 MHz, CDCl ) δ 9.44 (s,
3
1
H), 8.24 (d, J = 8.4 Hz, 1H), 7.96 (dd, J = 8.1, 0.9 Hz, 1H), 7.73 (ddd, J = 8.4, 5.4, 1.4 Hz, 1H),
7.63 (ddd, J = 8.1, 7.1, 1.1 Hz, 1H), 3.25–3.19 (m, 2H), 1.97–1.89 (m, 2H), 1.55–1.46 (m, 2H),
13
1
.00 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 173.0, 147.9, 145.8, 144.2, 140.6, 130.6,
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28.8, 128.8, 127.6, 125.0, 123.6, 34.2, 32.0, 22.4, 13.9. MS (ESIꢀTOF, m/z): calculated for
+
C H N S [M + H] 243.0950; found 243.0976 (Data from Org. Biomol. Chem. 2013, 11, 1179].
14 14
2
2-Butyloxazolo[4,5-c]quinoline (8): To a solution of 3ꢀaminoquinolinꢀ4ꢀol (12 mg, 0.075
mmol) in pyridine (0.5 mL) was added valeroyl chloride (11 ꢀL, 0.09 mmol) and the resulting
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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28
29
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48
49
50
51
52
53
54
55
56
57
58
59
60
°
mixture was heated in a sealed vial at 50 C for 1 h. P O (22 mg) was added and the resulting
2
5
°
mixture was heated at 120 C for 1 h under microwave irradiation. The solvents were removed
and the crude residue was purified by flash chromatography (SiO , MeOH in CH Cl : 0 to 5%)
2
2
2
1
to give compound 8 (11.5 mg, 63%) as pale yellow solid. H NMR (500 MHz, CDCl ) δ 9.27 (s,
3
1
H), 8.25 (d, J = 8.4 Hz, 1H), 8.20 (ddd, J = 8.1, 1.5, 0.6 Hz, 1H), 7.75 (ddd, J = 8.5, 7.0, 1.5 Hz,
H), 7.68 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 3.08 (dd, J = 9.0, 6.3 Hz, 2H), 1.96 (ddd, J = 15.2, 7.6,
1
1
3
6.0 Hz, 2H), 1.51 (dt, J = 14.8, 7.4 Hz, 2H), 1.01 (t, J = 7.4 Hz, 3H). C NMR (126 MHz,
CDCl ) δ 167.5, 152.1, 145.9, 143.9, 134.9, 130.1, 128.7, 127.3, 120.1, 116.3, 28.9, 28.4, 22.3,
3
+
1
3.7. MS (ESIꢀTOF, m/z): calculated for C H N O [M + H] 227.1179; found 227.1216.
14 14
2
2-Butyloxazolo[4,5-c]quinoline 5-oxide. To a solution of compound 8 (68 mg, 0.30 mmol) in
CHCl (5 mL), was added mꢀCPBA (≤ 77%, 100 mg, 0.45 mmol) and the reaction mixture was
3
stirred at room temperature for 4 h. The solvent was removed under reduced pressure and the
crude residue was purified by flash chromatography (SiO , MeOH in CH Cl : 0 to 5%) to give 2ꢀ
2
2
2
1
butyloxazolo[4,5ꢀc]quinoline 5ꢀoxide. H NMR (500 MHz, CDCl ) δ 8.97 (s, 1H), 8.92 – 8.87
3
(m, 1H), 8.18 (ddd, J = 6.4, 2.2, 0.6 Hz, 1H), 7.83 – 7.77 (m, 2H), 3.06 (dd, J = 9.0, 6.3 Hz, 2H),
13
1
.97 – 1.90 (m, 2H), 1.55 – 1.47 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl )
3
δ 169.5, 144.3, 139.8, 134.6, 130.0, 129.7, 129.6, 121.5, 120.8, 116.6, 28.8, 28.4, 22.3, 13.7. MS
+
(ESIꢀTOF, m/z): calculated for C H N O [M + H] 243.1128; found 243.1146.
1
4
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2
-Butyloxazolo[4,5-c]quinolin-4-amine (9). 2ꢀButyloxazolo[4,5ꢀc]quinoline 5ꢀoxide (60 mg,
0
.25 mmol) was dissolved in anhydrous CH Cl (2 mL). Benzoyl isocyanate (74 mg, 0.50 mmol)
2 2
was added and the resulting mixture was refluxed for 1.5 h. The solvent was removed and the
residue was dissolved in anhydrous methanol (2 mL). Sodium methoxide (27 mg, 0.50 mmol)
was added and the resulting mixture was refluxed for 30 min. The solvent was removed under
0
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2
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4
5
6
7
8
9
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1
2
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4
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7
8
9
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0
reduced pressure and the crude residue was purified by flash chromatography (SiO , MeOH in
2
1
CH Cl : 0 to 10%) to give compound 9 (37 mg, 61%) as white solid. H NMR (500 MHz,
2
2
MeOD) δ 7.95 (ddd, J = 8.0, 1.5, 0.5 Hz, 1H), 7.70 – 7.65 (m, 1H), 7.56 (ddd, J = 8.5, 7.0, 1.5
Hz, 1H), 7.35 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 3.09 – 3.03 (m, 2H), 1.92 (ddd, J = 13.7, 8.2, 6.9
1
3
Hz, 2H), 1.55 – 1.46 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, MeOD) δ 168.0,
154.0, 152.9, 146.4, 130.2, 126.3, 125.6, 124.0, 121.1, 114.3, 30.0, 28.9, 23.3, 14.0. MS (ESIꢀ
+
TOF, m/z): calculated for C H N O [M + H] 242.1288; found 242.1313.
14
15
3
N-Propylthiazolo[4,5-c]quinolin-2-amine (10): To a solution of 3ꢀaminoquinolinꢀ4ꢀol (32 mg,
0
.20 mmol) in pyridine (1 mL) was added propyl isothiocyanate (31 ꢀL, 0.30 mmol) and the
°
resulting mixture was heated in a sealed vial at 50 C for 30 min. P S (89 mg) was added and the
2
5
°
resulting mixture was heated at 120 C for 1 h under microwave irradiation. The solvent was
removed and the crude residue was purified by flash chromatography (SiO , MeOH in CH Cl : 0
2
2
2
1
to 5%) to give compound 10 (34 mg, 70%) as pale brown solid. H NMR (400 MHz, CDCl ) δ
3
9.12 (s, 1H), 8.15 (dd, J = 8.3, 0.8 Hz, 1H), 7.78 – 7.71 (m, 1H), 7.60 (ddd, J = 8.4, 7.0, 1.7 Hz,
1
H), 7.55 (ddd, J = 8.2, 7.0, 1.4 Hz, 1H), 5.85 (s, 1H), 3.47 (dd, J = 11.7, 7.0 Hz, 2H), 1.78 (dd, J
13
=
14.4, 7.3 Hz, 2H), 1.06 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 168.3, 147.0, 143.1,
3
143.1, 134.0, 130.3, 127.1, 127.0, 123.7, 123.6, 47.8, 22.7, 11.4. MS (ESIꢀTOF, m/z): calculated
+
for C H N S [M + H] 244.0903; found 244.0946.
1
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N -Propylthiazolo[4,5-c]quinoline-2,4-diamine (11). H NMR (500 MHz, MeOD) δ 7.57 (d, J
=
8.4 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.42 – 7.36 (m, 1H), 7.26 – 7.19 (m, 1H), 3.42 (t, J = 7.0
1
3
Hz, 2H), 1.72 (h, J = 7.3 Hz, 2H), 1.02 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, MeOD) δ 169.3,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
52.0, 143.2, 137.6, 133.7, 128.3, 125.8, 124.4, 123.9, 121.0, 47.8, 23.5, 11.8. MS (ESIꢀTOF,
+
m/z): calculated for C H N S [M + H] 259.1012; found 259.1054.
1
3
14
4
N-Propyloxazolo[4,5-c]quinolin-2-amine (12): To a solution of 3ꢀaminoquinolinꢀ4ꢀol (32 mg,
0.20 mmol) in pyridine (1 mL) was added propyl isothiocyanate (31 ꢀL, 0.30 mmol) and the
°
resulting mixture was heated in a sealed vial at 50 C for 30 min. EDC (77 mg, 0.4 mmol) was
°
added and the resulting mixture was heated at 120 C for 30 min under microwave irradiation.
The solvent was removed and the crude residue was purified by flash chromatography (SiO2,
1
MeOH in CH Cl : 0 to 5%) to give compound 12 (25 mg, 59%) as brown solid. H NMR (500
2
2
MHz, MeOD) δ 8.85 (s, 1H), 8.08 – 8.03 (m, 1H), 8.04 – 8.00 (m, 1H), 3.51 (q, J = 7.3 Hz, 2H),
1
3
1
1
2
.34 (t, J = 7.3 Hz, 3H). C NMR (126 MHz, MeOD) δ 164.6, 150.2, 145.1, 141.2, 137.8, 129.7,
+
28.7, 128.6, 120.2, 116.7, 38.9, 14.9. MS (ESIꢀTOF, m/z): calculated for C H N O [M + H]
12
11
3
1
14.0957; found 214.1045. H NMR (500 MHz, MeOD) δ 8.83 (s, 1H), 8.06 – 8.02 (m, 1H),
1
3
8.01 – 7.97 (m, 1H), 7.67 – 7.58 (m, 2H), 3.42 (t, J = 7.1 Hz, 2H). C NMR (126 MHz, MeOD)
δ 164.7, 150.1, 145.1, 141.2, 137.8, 129.7, 128.7, 128.6, 120.2, 116.7, 45.9, 23.6, 11.6. MS (ESIꢀ
+
TOF, m/z): calculated for C H N O [M + H] 228.1131; found 228.1164.
13
13
3
1
2-(Propylamino)oxazolo[4,5-c]quinoline 5-oxide. H NMR (500 MHz, DMSOꢀd ) δ 8.83 (s,
6
1
H), 8.58 (t, J = 5.8 Hz, 1H), 8.56 (d, J = 8.8 Hz, 1H), 7.97 (dd, J = 8.3, 0.6 Hz, 1H), 7.75 (ddd,
J = 8.2, 6.9, 1.1 Hz, 1H), 7.69 – 7.64 (m, 1H), 3.36 – 3.30 (m, 10H), 1.69 – 1.59 (m, 2H), 0.94 (t,
1
3
J = 7.4 Hz, 3H). C NMR (126 MHz, DMSOꢀd ) δ 163.8, 139.3, 137.2, 136.8, 129.4, 127.7,
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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5
5
5
5
5
5
5
5
5
6
1
27.3, 120.5, 119.6, 115.3, 44.4, 22.0, 11.3. MS (ESIꢀTOF, m/z): calculated for C H N O [M
13
13
3
2
+
+
H] 244.1081; found 244.0974.
2
1
N -Propyloxazolo[4,5-c]quinoline-2,4-diamine (13). H NMR (500 MHz, DMSOꢀd ) δ 11.96
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
(s, 1H), 8.16 (dd, J = 8.0, 1.2 Hz, 1H), 7.50 – 7.45 (m, 1H), 7.44 (dd, J = 8.2, 1.0 Hz, 1H), 7.16
(ddd, J = 8.1, 6.6, 1.5 Hz, 1H), 7.06 (t, J = 5.9 Hz, 1H), 3.24 (dd, J = 14.1, 6.3 Hz, 2H), 1.63 –
13
1.48 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, DMSOꢀd ) δ 161.7, 155.7, 150.2,
6
1
37.4, 129.0, 124.9, 124.0, 120.2, 117.0, 112.8, 43.9, 22.6, 11.3. MS (ESIꢀTOF, m/z): calculated
+
for C H N O [M + H] 243.1240; found 243.1141.
1
3
14
4
2
-Propionamidothiazolo[4,5-c]quinoline 5-oxide (15). To a solution of thiazolo[4,5ꢀc]quinolinꢀ
2ꢀamine (100 mg, 0.497 mmol) in pyridine (2 mL) was added propionyl chloride (54 ꢀL, 0.62
mmol) and the resulting mixture was stirred at room temperature for 1 h. The solvent was
removed and the crude residue was purified by flash chromatography (SiO , MeOH in CH Cl : 0
2
2
2
to 5%) to give compound Nꢀ(thiazolo[4,5ꢀc]quinolinꢀ2ꢀyl)propionamide 14 as impure solid,
which was dissolved in CHCl (4 mL). m-CPBA (≤ 77%, 224 mg, 1.0 mmol) was added and the
3
reaction mixture was stirred at room temperature for 4 h and then concentrated. The crude
residue was purified by flash chromatography (SiO , MeOH in CH Cl : 0 to 5%) to give
2
2
2
1
compound 15 (98 mg, 72%) as white solid. H NMR (500 MHz, DMSOꢀd ) δ 12.75 (s, 1H), 9.11
6
(s, 1H), 8.68 – 8.62 (m, 1H), 8.24 – 8.17 (m, 1H), 7.86 – 7.76 (m, 2H), 2.56 (q, J = 7.5 Hz, 2H),
1
3
1.14 (t, J = 7.5 Hz, 3H). C NMR (126 MHz, DMSOꢀd ) δ 173.3, 160.0, 142.9, 137.8, 129.8,
6
1
29.7, 128.7, 125.1, 124.6, 123.6, 120.3, 28.4, 8.8. MS (ESIꢀTOF, m/z): calculated for
+
C H N O S [M + H] 274.0645; found 274.0667.
13 11
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N-(4-Aminothiazolo[4,5-c]quinolin-2-yl)propionamide (16). Compound 15 (55 mg, 0.20
mmol) was dissolved in anhydrous CH Cl (2 mL). Benzoyl isocyanate (59 mg, 0.40 mmol) was
2
2
added and the resulting mixture was refluxed for 1.5 h. The solvent was removed under reduced
pressure and the residue was dissolved in anhydrous MeOH (2 mL). Sodium methoxide (22 mg,
0.40 mmol) was added and the reaction mixture was refluxed for 30 min. The solvent was
removed and the crude residue was purified by flash chromatography (SiO , MeOH in CH Cl : 0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
2
2
1
to 10%) to give compound 16 (41 mg, 79%) as white solid. H NMR (500 MHz, DMSOꢀd ) δ
6
12.53 (s, 1H), 7.80 (dd, J = 8.0, 1.0 Hz, 1H), 7.61 (dd, J = 8.3, 0.6 Hz, 1H), 7.48 (ddd, J = 8.4,
7.0, 1.4 Hz, 1H), 7.26 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 2.55 (q, J = 7.5 Hz, 2H), 1.14 (t, J = 7.5
1
3
Hz, 3H). C NMR (126 MHz, DMSOꢀd ) δ 173.1, 157.0, 151.5, 144.2, 133.8, 133.1, 127.8,
6
+
125.9, 123.7, 122.2, 119.1, 28.3, 9.0. MS (ESIꢀTOF, m/z): calculated for C H N OS [M + H]
13
12
4
273.0805; found 273.0832. The sodium methoxide solvolysis also resulted in small amount of
thiazolo[4,5-c]quinoline-2,4-diamine (17) (4 mg, 9%) which was isolated from the crude
1
mixture as white solid. H NMR (500 MHz, MeOD) δ 7.58 (ddd, J = 8.4, 1.0, 0.5 Hz, 1H), 7.52
(ddd, J = 8.0, 1.4, 0.5 Hz, 1H), 7.41 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.24 (ddd, J = 8.1, 7.1, 1.1
13
Hz, 1H). C NMR (126 MHz, MeOD) δ 169.6, 152.0, 143.6, 137.3, 134.7, 128.4, 126.0, 124.4,
+
1
23.9, 121.1. MS (ESIꢀTOF, m/z): calculated for C H N S [M + H] 217.0542; found 217.0569.
10
8
4
N-(Quinolin-4-yl)valeramide (18). To a solution of 4ꢀaminoquinoline (302 mg, 2.1 mmol) in
pyridine (6 mL) was added valeroyl chloride (0.28 mL, 2.4 mmol). The mixture was heated at 65
°
C for 90 min and then concentrated. The crude residue was purified by flash chromatography
(
SiO , MeOH in CH Cl : 0 to 10%) to give compound 18 (382 mg, 80%) as a dark brown solid.
2
2
2
1
H NMR (500 MHz, DMSOꢀd ) δ 11.11 (s, 1H), 9.07 (d, J = 6.5 Hz, 1H), 8.95 (dd, J = 8.7, 1.1
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1
2
2
2
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2
2
2
2
2
2
3
3
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Hz, 1H), 8.70 (d, J = 6.4 Hz, 1H), 8.32 (dd, J = 8.5, 1.1 Hz, 1H), 8.12 (ddd, J = 8.4, 6.9, 1.1 Hz,
1H), 7.93 (ddd, J = 8.4, 6.9, 1.1 Hz, 1H), 2.80 (t, J = 7.4 Hz, 2H), 1.66 (tt, J = 7.5 Hz, 2H), 1.39
1
3
(
tq, J = 7.4 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, DMSOꢀd ) δ 174.2, 149.6,
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
145.0, 139.0, 133.8, 128.1, 123.8, 121.2, 119.0, 108.9, 36.4, 26.6, 21.6, 13.7. MS (ESIꢀTOF,
+
m/z): calculated for C H N O [M + H] 229.1341; found 229.1306.
1
4
16
2
N-(3-Bromoquinolin-4-yl)valeramide (19). To a suspension of compound 19 (151 mg, 0.66
mmol) in benzene (anhydrous, 10 mL) was added Nꢀbromosuccinimide (NBS, 143 mg, 0.81
mmol) and azobisisobutyronitrile (AIBN, 99 mg, 0.60 mmol). The mixture was refluxed for 5 h
and then concentrated. The crude residue was purified by flash chromatography (SiO , EtOAc in
2
1
hexane: 0 to 30%) to give compound 19 (86 mg, 42%) as a light brown solid. H NMR (500
MHz, DMSOꢀd ) δ 10.31 (s, 1H), 9.05 (s, 1H), 8.08 (dd, J = 8.5, 1.2 Hz, 1H), 7.95 (dd, J = 8.4,
6
1.4 Hz, 1H), 7.84 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.70 (ddd, J = 8.2, 6.8, 1.3 Hz, 1H), 2.54ꢀ2.46
1
3
(
m, 2H), 1.68 (quin, J = 7.5 Hz, 2H), 1.43 (sex, J = 7.4 Hz, 2H), 0.95 (t, J = 7.4 Hz, 3H). C
NMR (126 MHz, DMSOꢀd ) δ 171.4, 152.1, 147.1, 141.5, 130.2, 129.1, 127.8, 126.6, 123.8,
6
+
116.8, 35.2, 27.3, 21.9, 13.8. MS (ESIꢀTOF, m/z): calculated for C H BrN O [M + H]
14 15 2
307.0446; found 307.0254.
2-Butylthiazolo[5,4-c]quinoline (20a). To a solution of compound 19 (60.8 mg, 0.20 mmol) in
pyridine (4 mL) was added Lawesson’s reagent (234 mg, 0.58 mmol). The resulting mixture was
heated in a sealed vial under microwave irradiation (500 W, 140 °C) for 35 min and then
concentrated. The crude residue was purified by flash chromatography (SiO , EtOAc in hexanes:
2
1
0
to 10%) to give compound 20a (15 mg, 31%) as brown oil. H NMR (500 MHz, CDCl ) δ 9.33
3
(s, 1H), 8.71 (dd, J = 8.1, 1.5 Hz, 1H), 8.22 (brd, J = 8.3 Hz, 1H), 7.76 (ddd, J = 8.3, 7.0, 1.5 Hz,
1H), 7.70 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 3.27 (t, J = 7.7 Hz, 2H), 1.95 (tt, J = 7.6, 7.7 Hz, 2H),
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.52 (qt, J = 7.3, 7.4 Hz, 2H), 1.01 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 177.3,
3
1
54.9, 145.9, 143.8, 129.4, 128.7, 127.8, 127.2, 123.6, 123.4, 34.3, 32.0, 22.3, 13.8. MS (ESIꢀ
+
TOF, m/z): calculated for C H N S [M + H] 243.0956; found 243.1019.
14
14
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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48
49
50
51
52
53
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55
56
57
58
59
60
2
-Butylthiazolo[5,4-c]quinoline 5-oxide (21a). To a solution of compound 20a (14.5 mg, 0.060
mmol) in CH Cl (1 mL) was added mꢀCPBA (≤ 77%, 35.2 mg). The reaction mixture was
2
2
stirred at room temperature for 2.5 h and then concentrated. The crude residue was purified by
flash chromatography (SiO , MeOH in CH Cl : 0 to 5%) to give compound 21a (13.5 mg, 87%)
2
2
2
1
as brown oil. H NMR (500 MHz, CDCl ) δ 9.04 (s, 1H), 8.90ꢀ8.76 (m, 1H), 8.78ꢀ8.58 (m, 1H),
3
7.95ꢀ7.72 (m, 2H), 3.25 (t, J = 7.7 Hz, 2H), 1.94 (tt, J = 7.6, 7.7 Hz, 2H), 1.63ꢀ1.44 (m, 2H), 1.02
13
(t, 3H, J = 7.3 Hz). C NMR (126 MHz, CDCl ) δ 176.4, 147.8, 140.1, 134.3, 132.6, 130.0,
3
129.5, 128.0, 124.4, 120.3, 34.1, 31.9, 22.3, 13.8. MS (ESIꢀTOF, m/z): calculated for
+
C H N OS [M + H] 259.0827; found 259.0755.
14
14
2
2
-Butylthiazolo[5,4-c]quinolin-4-amine (22a). To a solution of compound 21a (10.1 mg, 0.039
mmol) in CH Cl (0.2 mL) was added benzoyl isocyanate (13.2 mg, 0.090 mmol). The mixture
2
2
was heated to reflux for 3 h and then concentrated. The crude residue was purified by flash
chromatography (SiO , EtOAc in hexanes: 0 to 15%) to give intermediate (8.8 mg, 63%) as light
2
brown oil. Benzamide (8.8 mg, 0.024 mmol) was dissolved in NaOMe solution (0.5 mL, 0.5 M
in MeOH). The mixture was heated to reflux overnight and then concentrated. The crude residue
was purified by flash chromatography (SiO , MeOH in CH Cl : 0 to 5%) to give compound 22a
2
2
2
1
(5.6 mg, 89%) as a yellow solid. H NMR (500 MHz, CDCl ) δ 8.50 (dd, J = 8.1, 1.2 Hz, 1H),
3
7
1
7
.82 (brd, J = 8.4 Hz, 1H), 7.62 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.44 (ddd, J = 8.0, 7.0, 1.1 Hz,
H), 5.15 (brs, 2H), 3.25 (t, J = 7.7 Hz, 2H), 2.00ꢀ1.87 (m, 2H), 1.61ꢀ1.43 (m, 2H), 1.02 (t, J =
13
.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 175.7, 157.0, 150.9, 145.8, 129.2, 125.7, 123.6,
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5
5
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5
5
5
5
5
5
6
1
23.5, 120.2, 116.7, 34.2, 32.0, 22.3, 13.8. MS (ESIꢀTOF, m/z): calculated for C H N S [M +
14
15
3
+
H] 258.1065; found 258.0964.
2-Butyloxazolo[5,4-c]quinoline (20b). To a solution of compound 19 (50.4 mg, 0.16 mmol) in
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
pyridine (2 mL) was added CuI (66 mg, 0.35 mmol) and K CO (47 mg, 0.34 mmol). The
2
3
resulting mixture was heated in a sealed vial under microwave irradiation (500 W, 140 °C) for 35
min and then concentrated. The crude residue was purified by flash chromatography (SiO2,
1
EtOAc in hexanes: 0 to 10%) to give compound 20b (28 mg, 76%) as a brown solid. H NMR
(500 MHz, CDCl ) δ 9.20 (bs, 1H), 8.46 (bs, 1H), 8.25 (bs, 1H), 7.76 (bt, J = 6.0 Hz, 1H), 7.72 –
3
7.64 (m, 1H), 3.09 (t, J = 7.6 Hz, 2H), 2.00 – 1.91 (m, 2H), 1.55 – 1.43 (m, 2H), 1.00 (t, J = 7.4
13
Hz, 3H). C NMR (126 MHz, CDCl ) δ 169.8, 145.5, 143.7, 135.1, 129.9, 128.3, 127.4, 122.2,
3
+
29.0, 28.7, 22.3, 13.7. MS (ESIꢀTOF, m/z): calculated for C H N O [M + H] 227.1184; found
1
4
14
2
227.1051.
2-Butyloxazolo[5,4-c]quinoline 5-oxide (21b). To a solution of compound 20b (28 mg, 0.124
mmol) in CH Cl (2 mL) was added m-CPBA (≤ 77%, 83.5 mg). The reaction mixture was
2
2
stirred at room temperature for 4 h and then concentrated. The crude residue was purified by
flash chromatography (SiO , MeOH in CH Cl : 0 to 5%) to give compound 21b (22 mg, 73%) as
2
2
2
1
a red solid. H NMR (500 MHz, CDCl ) δ 8.93 (s, 1H), 8.87ꢀ8.79 (m, 1H), 8.46ꢀ8.41 (m, 1H),
3
7.84ꢀ7.75 (m, 2H), 3.06 (t, J = 7.6 Hz, 2H), 1.93 (tt, J = 7.7, 7.6 Hz, 2H), 1.50 (qt, J = 7.4, 7.3
1
3
Hz, 2H), 1.01 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 169.9, 143.9, 140.1, 135.7,
3
129.6, 129.3, 123.6, 122.8, 121.8, 120.8, 28.9, 28.5, 22.3, 13.7. MS (ESIꢀTOF, m/z): calculated
+
for C H N O [M + H] 243.1134; found 243.1032.
1
4
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-Butyloxazolo[5,4-c]quinolin-4-amine (22b). To a solution of compound 21b (17.8 mg, 0.073
mmol) in CH Cl (0.5 mL) was added benzoyl isocyanate (38.4 mg, 0.26 mmol). The mixture
2
2
was heated to reflux for 4 h and then concentrated. The crude residue was purified by flash
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
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56
57
58
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60
chromatography (SiO , EtOAc in hexanes: 0 to 20%) to give intermediate (8.7 mg, 34%) as
2
colorless oil. Benzamide (6 mg, 0.017 mmol) was dissolved in NaOMe solution (0.5 mL, 0.5 M
in MeOH). The mixture was heated to reflux for 8 h and then concentrated. The crude residue
was purified by flash chromatography (SiO , MeOH in CH Cl : 0 to 5%) to give compound 22b
2
2
2
1
(3.3 mg, 79%) as a yellow solid. H NMR (500 MHz, CDCl ) δ 8.22 (dd, J = 8.2, 1.5 Hz, 1H),
3
7.80 (dd, J = 8.7, 1.5 Hz, 1H), 7.59 (ddd, J = 8.5, 7.0, 1.6 Hz, 1H), 7.42 (ddd, J = 8.2, 7.0, 1.1
Hz, 1H), 5.15 (bs, 2H), 3.05 (t, J = 7.7 Hz, 2H), 2.01ꢀ1.85 (m, 2H), 1.58ꢀ1.43 (m, 2H), 1.00 (t, J
13
= 7.4 Hz, 3H). C NMR (126 MHz, CDCl ) δ 168.6, 144.9, 144.0, 135.2, 128.5, 127.3, 126.2,
3
1
23.5, 122.0, 119.0, 29.2, 28.6, 22.3, 13.7. MS (ESIꢀTOF, m/z): calculated for C H N O [M +
14 15 3
+
H] 242.1293; found 242.1240.
Quinazoline-2,4(1H,3H)-dione (23). The mixture of anthranilic acid (500 mg, 3.65 mmol) and
urea (2.2 g, mol) was heated at 150 ºC for 6 h. The reaction mixture was cooled to room
temperature and then water (50 mL) was added to quench the reaction. The crude product was
obtained by filtration, and then washed with water (20 mL × 3). The residue was dissolved in hot
aq. NaOH and cooled to 0 ºC and pH was adjusted to 5ꢀ6 using dilute HCl and stirred for 30 min.
The crude mixture was filtered and washed with water and dried under vacuum to yield
1
compound 23 as white solid (500 mg, 85%). H NMR (500 MHz, DMSOꢀd ) δ 11.28 (s, 1H),
6
11.14 (s, 1H), 7.88 (dd, J = 7.8, 1.1 Hz, 1H), 7.65 – 7.60 (m, 1H), 7.17 (ddd, J = 8.2, 6.1, 1.8 Hz,
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2
2
2
2
2
2
2
3
3
3
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3
3
3
3
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
2
H). C NMR (126 MHz, DMSOꢀd ) δ 162.9, 150.3, 140.9, 135.0, 127.0, 122.4, 115.3, 114.4.
6
+
MS (ESIꢀTOF, m/z): calculated for C H N O [M + H] 163.0502; found 163.0491.
8
6
2
2
2-((2-Chloroquinazolin-4-yl)amino)hexan-1-ol (25). POCl (5 mL) and DIPEA (430 ꢀL, 2.47
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
mmol) were added to compound 23 (200 mg, 1.23 mmol) and the reaction mixture was heated to
reflux for 4 h. The excess POCl was removed by evaporation. The residue was dissolved in ice
3
water, and then the suspension was filtered and washed with water to afford compound 24 as
white solid (220 mg, 90%). To a solution of compound 24 (200 mg, 1 mmol) in DMF (5 mL),
was added DLꢀ2ꢀaminoꢀ1ꢀhexanol (194 ꢀL, 1.5 mmol), the resulting mixture was heated at 100
ºC for 1 h, and allowed to cool, and concentrated. The residue was purified by flash
chromatography (SiO , EtOAc in Hexane: 0 to 50%) to give compound 25 as a yellow solid (95
2
1
mg, 34 %). H NMR (500 MHz, DMSOꢀd ) δ 8.37 (dd, J = 8.4, 0.8 Hz, 1H), 8.24 (d, J = 8.4 Hz,
6
1
H), 7.78 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.60 (dd, J = 8.3, 0.8 Hz, 1H), 7.52 (ddd, J = 8.2, 7.0,
1.2 Hz, 1H), 4.80 (t, J = 5.7 Hz, 1H), 4.35 (qd, J = 5.7, 10.6 Hz, 1H), 3.56 – 3.46 (m, 2H), 1.74 –
.65 (m, 1H), 1.62 – 1.52 (m, 1H), 1.29 (dddd, J = 14.8, 10.3, 8.2, 3.2 Hz, 4H), 0.85 (t, J = 6.7
1
1
3
Hz, 3H). C NMR (126 MHz, DMSOꢀd ) δ 161.4, 157.1, 150.4, 133.6, 126.6, 125.8, 123.5,
6
113.6, 62.8, 52.9, 30.1, 27.8, 22.1, 14.0. MS (ESIꢀTOF, m/z): calculated for C H ClN O [M +
14
18
3
+
H] 280.1211; found 280.1279.
2
-Butyl-2,3-dihydroimidazo[1,2-c]quinazolin-5-amine (27). To a solution of compound 25 (50
mg, 0.18 mmol) in CH Cl (2 mL) was added triethylamine (38 ꢀL, 0.27 mmol) and
2
2
methanesulfonyl chloride (17 ꢀL, 0.22 mmol) and the resulting mixture was stirred at room
temperature overnight. CH Cl (20 mL) was added and the organic layer was washed with water
2
2
(
10 mL x 2), dried over anhydrous sodium sulfate and evaporated to furnish the crude residue of
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compound 26 (45 mg, 96% crude yield). Ammonia in methanol (2M, 1 mL) was added to
compound 26 (10 mg, 38 ꢀmol) and the reaction mixture was heated at 80 ºC for 2 h and
concentrated. The residue was purified by flash chromatography (SiO , MeOH in CH Cl : 0 to
2
2
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
1
0%) to give compound 27 (6 mg, 66%) as a yellow solid. H NMR (500 MHz, MeOD) δ 7.88
(dd, J = 8.0, 1.2 Hz, 1H), 7.53 (ddd, J = 8.5, 7.2, 1.5 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H), 7.12
ddd, J = 8.1, 7.2, 1.0 Hz, 1H), 4.42 – 4.35 (m, 1H), 4.21 (t, J = 10.4 Hz, 1H), 3.77 (dd, J = 10.3,
.6 Hz, 1H), 1.87 – 1.78 (m, 1H), 1.66 (ddd, J = 11.0, 6.6, 3.7 Hz, 1H), 1.50 – 1.37 (m, 4H), 0.97
(
7
1
3
(
t, J = 7.1 Hz, 3H). C NMR (126 MHz, MeOD) δ 156.9, 151.8, 150.6, 135.4, 126.5, 124.6,
23.6, 113.2, 64.5, 51.9, 37.3, 28.6, 23.7, 14.4. MS (ESIꢀTOF, m/z): calculated for C H N [M
1
1
4
18
4
+
+ H] 243.1604; found 243.1589.
2-Butylimidazo[1,2-c]quinazolin-5-amine (29). To a solution of compound 26 (28 mg, 0.11
mmol) in toluene (2 mL) was added MnO (47 mg, 0.54 mmol) and heated at reflux for 20 h.
2
Additional MnO was added and the reaction mixture was refluxed for another 48 h. The mixture
2
was allowed to cool, filtered and purified by column chromatography (SiO , EtOAc in Hexane: 0
2
to 50%) to obtain compound 28 (12 mg, 41%) as a pale yellow solid. Compound 28 (10 mg) in
ammonia solution (2M in ammonia, 1 mL) was heated at 80 ºC for 2 h, concentrated and purified
by column chromatography (SiO , MeOH in CH Cl : 0 to 10%) to obtain compound 29 (4 mg,
2
2
2
1
3
6%) as a pale yellow solid. H NMR (500 MHz, MeOD) δ 8.28 (dd, J = 8.0, 0.9 Hz, 1H), 7.70
(s, 1H), 7.58 – 7.51 (m, 2H), 7.35 (ddd, J = 8.1, 6.8, 1.5 Hz, 1H), 2.84 – 2.78 (m, 2H), 1.79 (ddd,
13
J = 13.1, 8.5, 6.6 Hz, 2H), 1.48 (dq, J = 14.8, 7.4 Hz, 2H), 1.01 (t, J = 7.4 Hz, 3H). C NMR
(
126 MHz, MeOD) δ 131.5, 125.5, 124.7, 123.5, 108.3, 32.4, 29.1, 23.5, 14.2. MS (ESIꢀTOF,
+
m/z): calculated for C H N [M + H] 241.1448; found 241.1466.
1
4
16
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Methyl (2-cyanophenyl)carbamate (30). 2ꢀAminobenzonitrile (200 mg, 1.69 mmol) and
sodium carbonate (359 mg, 3.39 mmol) was heated to reflux in methyl chloroformate (7 mL) for
4
h. The reaction mixture was concentrated and purified by column chromatography (SiO2,
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
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7
8
9
0
1
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
EtOAc in Hexane: 0 to 20%) to obtain compound 30 (276 mg, 93%) as a white solid. H NMR
(500 MHz, DMSOꢀd ) δ 9.77 (s, 1H), 7.79 (dd, J = 7.8, 1.3 Hz, 1H), 7.67 (ddd, J = 8.2, 7.6, 1.6
6
1
3
Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.33 (td, J = 7.6, 1.1 Hz, 1H), 3.69 (s, 3H). C NMR (126
MHz, DMSOꢀd ) δ 154.5, 140.5, 133.9, 133.3, 125.5, 125.2, 116.8, 107.3, 52.2. MS (ESIꢀTOF,
6
+
m/z): calculated for C H N O [M + H] 177.0659; found 177.0694.
9
8
2
2
2-Butyl-[1,2,4]triazolo[1,5-c]quinazolin-5(6H)-one (31). To a solution of compound 30 (200
mg, 1.14 mmol) in Nꢀmethylꢀ2ꢀpyrrolidone (NMP, 5 mL) was added valeric acid hydrazide (158
o
mg, 1.36 mmol) and the reaction mixture was heated at 180 C for 3 h. Crushed ice was added to
the reaction mixture and the solid obtained was filtered and purified by column chromatography
1
(
SiO , EtOAc in hexanes: 0 to 50%) to obtain compound 31 (270 mg, 98%) as white solid. H
2
NMR (500 MHz, DMSOꢀd ) δ 12.21 (s, 1H), 8.11 (dd, J = 7.9, 1.1 Hz, 1H), 7.69 – 7.64 (m, 1H),
6
7
–
1
.41 (d, J = 8.2 Hz, 1H), 7.39 – 7.33 (m, 1H), 2.82 (t, J = 7.6 Hz, 2H), 1.79 – 1.72 (m, 2H), 1.43
1
3
1.33 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, DMSOꢀd ) δ 166.5, 152.7, 143.8,
6
36.9, 132.6, 124.0, 123.5, 116.0, 110.3, 29.6, 27.7, 21.8, 13.7. MS (ESIꢀTOF, m/z): calculated
+
for C H N O [M + H] 243.1240; found 243.1350.
1
3
14
4
2-Butyl-5-chloro-[1,2,4]triazolo[1,5-c]quinazoline (32). To a suspension of compound 31 (18
mg, 74 ꢀmol) in POCl (1 mL) was added DIPEA (29 ꢀL, 0.15 mmol) and the resulting mixture
3
was heated at 110 ºC for 18 h. The mixture was cooled to room temperature and concentrated.
The residue was purified by flash chromatography (SiO , EtOAc in hexane: 0 to 30%) to give
2
1
compound 32 as a pale yellow solid (15 mg, 79%). H NMR (500 MHz, DMSOꢀd ) δ 8.41 (d, J
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=
8.0 Hz, 1H), 7.99 (d, J = 8.3 Hz, 1H), 7.94 (t, J = 7.7 Hz, 1H), 7.82 (t, J = 7.5 Hz, 1H), 2.93 (t,
13
J = 7.6 Hz, 2H), 1.84 – 1.76 (m, 2H), 1.47 – 1.37 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H). C NMR
(126 MHz, DMSOꢀd ) δ 167.0, 152.0, 142.3, 135.3, 132.7, 129.1, 127.6, 123.6, 116.8, 29.7, 27.8,
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
2
1.8, 13.7. MS (ESIꢀTOF, m/z): calculated for C H ClN [M + H] 261.0902; found 261.1021.
1
3
13
4
2
-Butyl-[1,2,4]triazolo[1,5-c]quinazolin-5-amine (33). Compound 32 (10 mg, 38 ꢀmol) was
treated with 2M ammonia in methanol (1 mL), heated at 80 ºC for 20 h and concentrated. The
residue was purified by flash chromatography (SiO , MeOH in CH Cl : 0 to 10%) to give
2
2
2
1
compound 33 as a white solid (5 mg, 55%). H NMR (500 MHz, MeOD) δ 8.21 (ddd, J = 8.0,
.5, 0.5 Hz, 1H), 7.65 (ddd, J = 8.5, 7.1, 1.5 Hz, 1H), 7.57 (dd, J = 8.4, 0.5 Hz, 1H), 7.37 (ddd, J
8.1, 7.1, 1.1 Hz, 1H), 2.97 – 2.89 (m, 2H), 1.86 (dt, J = 15.3, 7.6 Hz, 2H), 1.50 – 1.38 (m, 2H),
1
=
1
3
0.98 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, MeOD) δ 167.9, 153.1, 146.3, 146.1, 133.4, 126.0,
1
24.8, 124.5, 114.4, 31.4, 29.2, 23.4, 14.1. MS (ESIꢀTOF, m/z): calculated for C H N [M +
1
3
15
5
+
H] 242.1400; found 242.1399.
2
,3-Dichloroquinoxaline (34). Quinoxalineꢀ2,3ꢀdiol (500 mg, 3.08 mmol) and POCl (5 mL) in
3
°
DMF (5 mL) were heated at 100 C for 1.5 h, allowed to cool, and concentrated. The residue was
1
treated with ice water and filtered to give compound 34 (418 mg, 68%) as off white solid. H
13
NMR (500 MHz, DMSOꢀd ) δ 8.10 – 8.06 (m, 2H), 7.96 – 7.91 (m, 2H). C NMR (126 MHz,
6
+
DMSOꢀd ) δ 144.7, 140.1, 131.8, 128.0. MS (ESIꢀTOF, m/z): calculated for C H Cl N [M + H]
6
8
4
2
2
198.9824; found 198.9853.
2
-((3-Chloroquinoxalin-2-yl)amino)hexan-1-ol (35). To a solution of compound 34 (200 mg, 1
mmol) in EtOH (5 mL), was added DLꢀ2ꢀaminoꢀ1ꢀhexanol (194 ꢀL, 1.5 mmol) in EtOH (3 mL),
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heated at 90 ºC for 18 h, allowed to cool, and concentrated. The residue was purified by flash
chromatography (SiO , EtOAc in Hexane: 0 to 20%) to give compound 35 as a yellow solid (160
2
1
mg, 57%). H NMR (500 MHz, CDCl ) δ 7.80 (dd, J = 8.2, 1.0 Hz, 1H), 7.66 (dd, J = 8.3, 0.9
3
0
1
2
3
4
5
6
7
8
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0
1
2
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0
Hz, 1H), 7.58 (ddd, J = 8.4, 7.0, 1.4 Hz, 1H), 7.40 (ddd, J = 8.3, 7.0, 1.4 Hz, 1H), 5.65 (d, J = 6.6
Hz, 1H), 4.30 – 4.22 (m, 1H), 3.90 (dd, J = 11.1, 2.8 Hz, 1H), 3.76 (dd, J = 11.1, 6.3 Hz, 1H),
1
3
3
.60 (s, 1H), 1.78 – 1.64 (m, 2H), 1.48 – 1.35 (m, 4H), 0.93 (t, J = 7.1 Hz, 3H). C NMR (126
MHz, CDCl ) δ 148.5, 140.6, 138.1, 136.6, 130.5, 128.1, 125.7, 125.4, 66.8, 54.6, 31.3, 28.5,
3
+
22.7, 14.1. MS (ESIꢀTOF, m/z): calculated for C H ClN O [M + H] 280.1211; found
1
4
18
3
280.1296.
2-Butyl-4-chloro-1,2-dihydroimidazo[1,2-a]quinoxaline (36). To a solution of compound 35
(100 mg, 0.36 mmol) in CHCl (1 mL), was added thionyl chloride (1 mL) at 0 ºC, and heated to
3
reflux for 2 h. The mixture was cooled to room temperature and concentrated. The residue was
purified by flash chromatography (SiO , MeOH in CH Cl : 0 to 10%) to give compound 36 (75
2
2
2
1
mg, 80%) as a yellow solid. H NMR (500 MHz, DMSOꢀd ) δ 8.02 (dd, J = 8.1, 0.7 Hz, 1H),
6
7.94 – 7.89 (m, 1H), 7.67 (dd, J = 14.6, 7.6 Hz, 2H), 4.90 (m, 1H), 4.62 – 4.51 (m, 2H), 1.90 –
1
3
1
.82 (m, 1H), 1.78 – 1.70 (m, 1H), 1.47 – 1.33 (m, 4H), 0.92 (t, J = 7.1 Hz, 3H). C NMR (126
MHz, DMSOꢀd ) δ 147.5, 136.5, 134.3, 133.3, 129.0, 128.8, 126.8, 115.5, 57.2, 53.4, 33.9, 26.3,
6
+
2
1.9, 13.9. MS (ESIꢀTOF, m/z): calculated for C H ClN [M + H] 262.1106; found 262.1173.
1
4
16
3
2-Butyl-1,2-dihydroimidazo[1,2-a]quinoxalin-4-amine (37). Compound 36 (29 mg, 0.11
mmol) was dissolved in 2M ammonia in methanol (1 mL) and heated in a sealed vial at 90 ºC for
2
0 h and concentrated. The residue was purified by flash chromatography (SiO , MeOH in
2
1
CH Cl : 0 to 10%) to give compound 37 (18 mg, 67%) as a yellow solid. H NMR (500 MHz,
2
2
MeOD) δ 7.25 (dd, J = 7.9, 1.2 Hz, 1H), 7.13 (td, J = 7.9, 1.4 Hz, 1H), 7.00 (td, J = 7.8, 1.3 Hz,
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H), 6.85 (dd, J = 8.0, 1.2 Hz, 1H), 4.38 – 4.30 (m, 1H), 4.26 – 4.20 (m, 1H), 3.76 (dd, J = 10.3,
8.6 Hz, 1H), 1.79 – 1.71 (m, 1H), 1.68 – 1.57 (m, 1H), 1.53 – 1.37 (m, 4H), 0.95 (t, J = 7.1 Hz,
1
3
3H). C NMR (126 MHz, MeOD) δ 149.4, 147.7, 134.7, 130.9, 125.9, 125.2, 123.0, 112.8, 65.8,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
5
2.8, 37.8, 28.9, 23.8, 14.4. MS (ESIꢀTOF, m/z): calculated for C H N [M + H] 243.1604;
1
4
18
4
found 243.1677.
2
-Butyl-4-chloroimidazo[1,2-a]quinoxaline (38). To a solution of compound 36 (357 mg, 1.36
mmol) in toluene (15 mL) was added MnO (593 mg, 6.82 mmol) and heated to reflux for 24 h.
2
Additional MnO (593 mg, 6.82 mmol) was added and after another 48 h, the mixture was
2
allowed to cool and filtered. The solvent was evaporated and the crude product was purified by
flash chromatography (SiO , EtOAc in hexane: 0 to 50%) to give compound 38 (99 mg, 28%) as
2
1
a yellow solid. H NMR (500 MHz, DMSOꢀd ) δ 8.73 (s, 1H), 8.33 (dd, J = 8.3, 0.9 Hz, 1H),
6
7
.97 (dd, J = 8.2, 1.2 Hz, 1H), 7.78 – 7.73 (m, 1H), 7.67 – 7.62 (m, 1H), 2.80 (t, J = 7.6 Hz, 2H),
1
3
1
.72 (dd, J = 15.2, 7.6 Hz, 2H), 1.44 – 1.34 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H). C NMR (126
MHz, DMSOꢀd ) δ 158.1, 150.8, 144.0, 143.5, 138.8, 138.4, 136.3, 136.3, 125.5, 122.8, 40.3,
6
+
3
7.6, 31.4, 23.3. MS (ESIꢀTOF, m/z): calculated for C H ClN [M + H] 260.0949; found
14
14
3
2
60.0900.
2-Butylimidazo[1,2-a]quinoxalin-4-amine (39). Compound 38 (70 mg, 0.27 mmol) was
dissolved in 2M ammonia in methanol (2 mL), heated in a sealed vial at 90 ºC for 20 h and
concentrated. The residue was purified by flash chromatography (SiO , EtOAc in hexane: 0 to
2
1
100%) to give compound 39 (21 mg, 33%) as a pale yellow solid. H NMR (500 MHz, MeOD) δ
8
7
1
.18 (s, 1H), 7.94 (dd, J = 8.1, 1.2 Hz, 1H), 7.60 (dd, J = 8.1, 1.2 Hz, 1H), 7.46 – 7.41 (m, 1H),
.36 (ddd, J = 8.5, 7.3, 1.4 Hz, 1H), 2.86 – 2.80 (m, 2H), 1.80 (ddd, J = 13.1, 8.4, 6.7 Hz, 2H),
13
.47 (dd, J = 15.0, 7.5 Hz, 2H), 1.00 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, MeOD) δ 149.5,
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