Selective Modulation of Gq/Gs pathways by Naphtho Pyrano Pyrimidines as antagonists of the Neuropeptide S Receptor
Antagonists of the Neuropeptide S Receptor have been postulated as promising therapeutics in the treatment of respiratory, sleep, anxiety, and addictive disorders. Here we present the SAR of a new series of orthosteric antagonists. Neuropeptide S Receptor signaling is coupled to both Gq and Gs proteins, and we observe that different analogues in this structural series can selectively antagonize these two pathways. Many G-protein coupled receptors transduce signals through multiple pathways. Selective antagonism of these pathways may lead the way to the development of more targeted pharmacological profiles and therapies.
<!>Chemistry<!>SAR Investigation<!>cAMP assay<!>Intracellular Calcium Assay<!>Radioligand Binding Assay<!>General procedure for the Knoevenagel condensation. Synthesis of 2-arylidenemalononitrile 4<!>Cyclization reaction to obtain 3-amino -1-aryl-1H-benzo[f]chromene-2-carbonitrile 5<!>Synthesis of (E)-Ethyl N-2-cyano-1-aryl-1H-benzo[f]chromen-3-ylformimidate<!>Ring closure of 10-aryl-12-aryl-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine 3<!>10-(2-Morpholinoethyl)-12-(3-methoxyphenyl)-12H-naphtho[ 1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3b)<!>10-(2-Morpholinoethyl)-12-(4-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3c)<!>10-(2-Morpholinoethyl)-12-phenyl-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3d)<!>10-Phenyl-12-phenyl-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3e)<!>10-Phenyl-12-(2-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3f)<!>10-Phenyl-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3g)<!>10-Phenyl-12-(benzo[d][1,3]dioxole)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3h)<!>10-Phenyl-12-(2,3-dihydrobenzofuran-5-yl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3i)<!>10-Phenyl-12-(3,4-diethoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3j)<!>10-Phenyl-12-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3k)<!>10-Phenyl-12-(2,3-dimethoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3l)<!>10-Benzyl-12-phenyl-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3m)<!>10-Benzyl-12-(3,4-dimethoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3n)<!>10-Phenethyl-12-phenyl-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3o)<!>10-Phenethyl-12-(3,4-dimethoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3p)<!>10-Phenylpropyl-12-phenyl-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3q)<!>10-Phenylpropyl-12-(3,4-dimethoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3r)<!>10-Phenylbutyl-12-phenyl-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3s)<!>10-(3-Fluorophenyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3t)<!>10-(4-Fluorophenyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3u)<!>10-(2-Chlorophenyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3v)<!>10-(3-Chlorophenyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3w)<!>10-(2-Bromophenyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3x)<!>10-(3-Bromophenyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3y)<!>10-(4-Bromophenyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3z)<!>10-(2-Cyanophenyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3aa)<!>10-(3-Cyanophenyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3ab)<!>10-(4-Cyanophenyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3ac)<!>10-(2-Pyridyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3ad)<!>10-(3-Pyridyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3ae)<!>10-(4-Pyridyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3af)<!>10-(2-Tolyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3ag)<!>10-(3-Tolyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3ah)<!>10-(4-Tolyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3ai)<!>10-(4-Tert-butylphenyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3aj)<!>10-(2-Methoxyphenyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3ak)<!>10-(3-Methoxyphenyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3al)<!>10-(4-Methoxyphenyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3am)<!>10-(Benzo[d][1,3]dioxole)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3an)<!>10-(2-(Methylthio)phenyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3ao)<!>10-(4-N-Acetylphenyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3ap)<!>10-(Thiophen-2-ylmethyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3aq)<!>10-(Biphenyl-2-yl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3ar)<!>10-(Biphenyl-4-yl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3as)<!>10-(2-Morpholinopropyl)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3at)<!>10-(4-Ethylbenzoate)-12-(3-methoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (3au)<!>Synthesis of 12-(3,4-Dimethoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-amine (6a)<!>Synthesis of 12-Phenyl-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-amine (6d)<!>Synthesis of 8-Aryl-12-(3,4-dimethoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine 7a\xe2\x80\x937c<!>8-Benzyl-12-(3,4-dimethoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (7a)<!>8-Phenethyl-12-(3,4-dimethoxyphenyl)-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-imine (7b)<!>8-(3-Phenylpropyl)-12-(3,4-dimethoxyphenyl)-12H-naphtho[1\',2\':5,6]-pyrano[2,3-d]pyrimidin-11-imine (7c)<!>General method for the synthesis of amide 8<!>Synthesis of 12-Phenyl-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-yl tert butyl carbamate (8a)<!>Synthesis of 12-Phenyl-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-yl phenyl amide (8b)<!>Synthesis of 12-Phenyl-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-yl benzyl amide (8c)<!>Synthesis of 12-Phenyl-12h-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-yl phenethyl amide (8d)<!>Synthesis of 12-Phenyl-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-yl (3-benzyl) urea (8e)<!>Synthesis of 12-Phenyl-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-yl (ethyl propanoate)amide (8f)<!>Synthesis of 12-Phenyl-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-yl (ethyl butanoate)amide (8g)<!>Synthesis of 12-Phenyl-12H-naphtho[1\',2\':5,6]pyrano[2,3-d]pyrimidin-11-yl (ethyl pentanoate)amide (8h)<!>Synthesis of 11-oxo-9 Aryl-12-phenyl-12H-naphtho[1\',2\':5,6]-pyrano[2,3-d]pyrimidine 9a\xe2\x80\x939d<!>11-oxo-9 Benzyl-12-phenyl-12H-naphtho[1\',2\':5,6]-pyrano[2,3-d]pyrimidine (9a)<!>11-oxo-9 Phenethyl-12-phenyl-12H-naphtho[1\',2\':5,6-]pyrano[2,3-d]pyrimidine (9b)<!>11-oxo-9 -((3-Phenyl)-propanyl)-12-phenyl-12H-naphtho[1\',2\':5,6-]pyrano[2,3-d]pyrimidin (9c)<!>11-oxo-9 Methyl-12-phenyl-12H-naphtho[1\',2\':5,6-]pyrano[2,3-d]pyrimidin (9d)<!>Synthesis of 11-(Amino)-9 Aryl-12-phenyl-12H-naphtho[1\',2\':5,6]-pyrano[2,3-d]pyrimidine 10a\xe2\x80\x9310c<!>Synthesis of 11-(Amino)-9 benzyl-12-phenyl-12H-naphtho[1\',2\':5,6]-pyrano[2,3-d]pyrimidine (10a)<!>Synthesis of 11-(Amino)-9 phenethyl-12-phenyl-12H-naphtho[ 1\',2\':5,6-]pyrano[2,3-d]pyrimidine (10b)<!>Synthesis of 11-(Amino)-9-((3-phenyl)-propanyl)-12-phenyl-12H-naphtho[1\',2\':5,6]-pyrano[2,3-d]pyrimidine (10c)<!>Synthesis of 2-Amino-4-(3,4-dimethoxyphenyl)-4H-benzo[h]chromene-3-carbonitrile (11)<!>10-(2-Morpholinoethyl)-12-(3,4-dimethoxyphenyl)-12H-naphtho[1\',2\':3,4]pyrano[2,3-d]pyrimidin-11-imine (12)<!>Synthesis of 2-Amino-4-(3,4-dimethoxyphenyl)-7-Alkyl-4Hchromene-3-carbonitrile 13a\xe2\x80\x9313b<!>2-Amino-4-(3,4-dimethoxyphenyl)-7-methoxy-4H-chromene-3-carbonitrile (13a)<!>2-Amino-7-(benzyloxy)-4-(3,4-dimethoxyphenyl)-4H-chromene-3-carbonitrile (13b)<!>Synthesis of 5-(3,4-Dimethoxyphenyl)-8-alkyl-3-(2-morpholinoethyl)-3H-chromeno[2,3-d]pyrimidin-4(5H)-imine 14a\xe2\x80\x9314b<!>5-(3,4-Dimethoxyphenyl)-8-methoxy-3-(2-morpholinoethyl)-3H-chromeno[ 2,3-d]pyrimidin-4(5H)-imine (14a)<!>8-(Benzyloxy)-5-(3,4-dimethoxyphenyl)-3-(2-morpholinoethyl)-3H-chromeno[2,3-d]pyrimidin-4(5H)-imine (14b)<!>Synthesis of 2,7-Diamino-4-(3,4-dimethoxyphenyl)-4H-chromene-3-carbonitrile (15)<!>Synthesis of 2,7-Dibromo-4-(3,4-dimethoxyphenyl)-4H-chromene-3-carbonitrile<!>Synthesis of 2-Amino-7-bromo-4-(3,4-dimethoxyphenyl)-4H-chromene-3-carbonitrile (16)<!>Synthesis of 5-(3,4-Dimethoxyphenyl)-3-(2-morpholinoethyl)-3H-chromeno[2,3-d]pyrimidin-4(5H)-imine (17)<!>Synthesis of 5-(3,4-Dimethoxyphenyl)-3-(2-morpholinoethyl)-4,5-dihydro-3H-chromeno[ 2,3-d]pyrimidin-4-amine (18)<!>
<p>Neuropeptide S (NPS, SFRNGVGTGMKKTSFQRAK, human sequence) is a small peptide produced in the brain predominantly in a group of previously not well-described neurons located between the locus coeruleus (LC), the Barrington nucleus and the parabrachial nuclei. NPS binds specifically with a G-protein coupled receptor expressed in several brain areas, Neuropeptide S receptor (NPSR). Activation of NPSR induces transient increases in intracellular calcium and cAMP, suggesting coupling of this receptor to both Gs and Gq G-proteins. Animal functional studies have linked NPSR with susceptibility for asthma1, the modulation of arousal, anxiety, and in the extinction of conditioned fear.2–6 Moreover, recently Padeña el al.7 have shown that in a dose dependent manner, NPS reinstates cocaine-seeking behavior in a mouse model for addiction. Thus, NPSR may represent a novel drug target for the treatment of sleep, anxiety, and addiction disorders.</p><p>In addition to NPS peptidic analogues8–10, two small molecule NPSR antagonist series have been reported (Figure 1). The first series, exemplified by N-(4-fluorobenzyl)-1-oxo-3,3-diphenyltetrahydro-1H-oxazolo[3,4- a]pyrazine-7(3H)-carboxamide (1), was able to partially reduce the NPS induced hyperlocomotion in mice.11–12 The second series, represented by N-(3-methyl-1-morpholinopentan-3-yl)-N-((1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)methyl)cyclohexanecarboxamide (2), also showed initial pharmacological results that point to its capacity to reduce the NPS induced hyperlocomotion.13</p><p>In parallel with this recently reported work, we developed a new HTS assay for identifying NPSR antagonists.14 The assay was based on the use of HTRF (homogeneous time resolved fluorescence) for the detection of cAMP (Figure 2). Antagonists of NPSR are able to decrease the signal induced by NPS in a dose dependent manner. Active compounds are also evaluated for their capacity to modulate Ca2+ signaling and for their capacity to compete with the NPS peptide in a radiolabel displacement assay.</p><!><p>In a high-throughput screen of 220,877 compounds (see supporting info), we identified 10-(2-morpholinoethyl)-12-(3,4 dimethoxyphenyl)-12H-Naphtho[1',2':5,6]pyrano[2,3-d]pyrimidin-11-imine (Figure 1, 3a) as a strong antagonist of NPSR signaling.</p><p>Compound 3a inhibited cAMP and Ca2+ signaling with an IC50 value of 4.87 and 1.38 µM, respectively. In radiolabeled peptide displacement studies, 3a was able to displace NPS with a Ki of 0.21 µM. Based on this activity, we decided to further explore this series' SAR. Schemes 1 through 5 show the main methodology involved in the synthesis of analogues. Scheme 1 describes analogues with substituents on the phenyl ring at position 12 and the nitrogen at position 10. Thus, amine 5 was synthesized by refluxing 2-naphthol and a suitable 2-arylidenemalononitrile 4 in ethanol with piperidine. Starting materials 2-arylidenemalononitrile 4 were either purchased or obtained by Knoevenagel condensation between the corresponding aldehyde and malononitrile using piperidine as a base. Compound 5 was then heated with triethylorthoformate to give the ethoxyimino intermediate, which was subsequently reacted with the proper amine to obtain compound 3.</p><p>Scheme 2 shows the synthesis of analogues at positions 8 and 11. Thus, amine 5a was dissolved into formamide and heated to give aminopyrimidine 6a. This compound was then reacted with three different alkyl bromides to give compounds 7a–7c. All three compounds were mixtures of the desired products and alkylation products at the other pyrimidine nitrogen, (Ratio: 7a:3n = 8:1; 7b:3p = 8:1, 7c:3r = 3:1). Attempts to separate these mixtures by preparative HPLC were not successful. Alternatively, aminopyrimidine 6d was acylated using an acyl chloride in the presence of diisopropylethylamine to yield compounds with the general structure of 8.</p><p>Additional compounds with pyrimidine ring substitution at position 9 were made by combining 5a with several acyl chlorides and heating them in a microwave oven to obtain pyrimidinones 9a–9d. Next, these pyrimidinones were exposed to phosphorous (V) oxychloride and heated to give chloropyrimidine intermediates, which were then reacted with ammonium hydroxide to give the final aminopyrimidines 10a–10c (Scheme 3).</p><p>In order to investigate the effect of naphthyl regiosubstitution, compound 12 was synthesized in a similar fashion to that of previously described compounds. Scheme 4 shows how 1-naphthol was substituted for 2-naphthol under the same conditions to get 12 in three steps.</p><p>Additional naphthyl ring modifications were explored with compounds 14a and 14b. Scheme 5 shows how they were synthesized using 3-methoxyphenol and 3-benzyloxyphenol respectively as starting material.</p><p>Lastly, Scheme 6 discloses the synthesis of two compounds having an unsubstituted phenyl ring at the original naphthyl position. The methodology was developed starting with similar previously described procedures to obtain diamine 15. The diamine was then converted to a dibromide intermediate with t-butylnitrite and copper (II) bromide. The dibromide intermediate was then selectively converted to the mono-amine with ammonia in isopropanol to give 16. Next, the aminobromide 16 was cyclized by transforming it into the ethoxy-imino intermediate which, parallel to previous methods, was then reacted with morpholinoethylamine. Finally, reduction of the bromide with palladium (II) chloride and triethylsilane gave 17 as well as the over-reduced side-product 18.</p><!><p>Since its discovery by Sato and coworkers15 and its deorphanization by Xu2 in 2004, the neuropeptide S receptor (NPSR) has been implicated in important neurocircuits such as the control of sleep, stress, anxiety, and arousal. At the same time, important technologies have been developed to separately explore signaling of GPCRs through different pathways, by the development of HTRF technologies to measure cAMP responses, and the development of kinetic fluorescence-based plate readers to measure transient calcium signaling.16 We used both to characterize the SAR of this series (PubChem AIDs: 434936, 434931 and 1464). The initial hit from the screen, compound 3a, had antagonistic activity in the low µM range for both the cAMP and calcium pathways. Additional studies disclosed the capacity of the molecule to compete with the natural substrate, having a Ki of 210 nM. As we began characterizing the activity of analogues, we observed interesting variations in the relative modulation of cAMP and calcium signaling. Thus, the ability of a particular substituent to increase antagonistic activity with respect to cAMP did not always correspond with an equivalent effect on calcium signaling. There were compounds that had a greater impact on cAMP signaling, while others preferentially antagonized calcium signaling. It is not clear which one of these two pathways is more important in establishing a neurocircuit response, so we have decided to measure and report on both pathways. Previous authors have focused their reports on calcium signaling.8–12 It is also not clear whether the assays used to measure antagonism of cAMP and calcium signaling are equally sensitive. Regardless, we observe clear, reproducible differences in the relative antagonism of cAMP and calcium signaling for close analogues in the present series, and believe that the relative antagonism of the two signaling pathways will critically affect in vivo activity, and may explain why previous series have only shown a partial reversion of NPS-induced phenotype.</p><p>Initial exploration of the peripheral methoxy functional groups indicate that the elimination of these substituents diminishes the potency of the molecule ten-fold (Table 1, calcium IC50). In addition, the meta-substituted methoxy substituent has the most impact on activity. Interestingly though, compound 3c lacks the calcium selectivity of the other compounds.</p><p>During our SAR studies we found that the introduction of an aromatic ring at the 10 position increased the potency of the molecule. Using this information, different methoxy and biomimetic functional groups were explored (Table 2). 2,3-benzodioxine 3k displayed the lowest calcium IC50 value followed by dihydrobenzofuran 3i and the meta-methoxy analogue 3g, showing the possibility of maintaining activity with more metabolically stable substituents. 3e had the lowest cAMP IC50 value and the phenyl substituent at position 12 was kept constant for the carbonyl analogues at position 11 (Table 3)</p><p>During the development of these series, a number of papers were published disclosing the activity of N-(4-fluorobenzyl)-1-oxo-3,3-diphenyltetrahydro-1H-oxazolo[3,4-a]pyrazine-7(3H)-carboxamide (1) and analogues as NPSR antagonists.10–11 Structural studies of that series have shown the importance of the diphenyl subtituent at the 3 position for mantaining activity. In addition, NPS displacement assays demonstrate that both series bind in the same region of the receptor, at the NPS binding site.</p><p>With this in mind, we decided to compare the conformational ensembles of both series for similarity. Figure 3 shows one superposition of these structures maximizing the overlap of the aromatic regions. From this perspective it can be seen that the main structural difference between these two molecules is on the N-(4-fluorobenzyl)-urea region. This region is crucial for the potency of the series, and its elimination yields almost inactive molecules. Thus, we rationalized that the introduction of functional groups within our series mimicking the effects of that benzyl urea region might increase the potency of our series.</p><p>We began by first synthesizing analogues with amine substitutions at position 11. Elimination of the ethylmorpholine substituent at position 10 (compound 6d, Table 3) improved the calcium activity more than 30-fold. In general, amide derivatives of compound 6d reduced the activity of the molecule. However, dicarboxylic aliphatic chains (compounds 8f, 8g and 8h) are reasonably tolerated with only slightly higher calcium IC50 values, which increased along with chain length. Amide substituents having an appending phenyl ring also increase the IC50 value, abolishing activity completely when an aromatic ring is next to the carbonyl functionality (compounds 8b, 8c and 8d). The same depletion of activity can be observed with the t-buty carbamate (compound 8a) as well as the benzyl urea (compound 8e).</p><p>While comparing the activity of compounds 3b (Table 1) and 3g (Table 2), one can see that the introduction of an aromatic ring at the 10 position increases the potency of the molecule. Thus, we decided to explore the impact of phenyl substituents with differing amounts of elongation from the pyrimidine ring. Table 4 shows that only the two carbon chain analogue (compound 3o, calcium IC50) was able to retain activity. Interestingly, introduction of meta- and para-methoxy substituents on the phenyl ring at position 12 shifts the tolerability of the elongated chain to the analogue with a single carbon linker, which was the only one able to maintain activity (compound 3n versus 3p and 3r, calcium IC50). Moreover, the impact of the aromatic ring chain length on calcium signal does not correlate with that of cAMP. In table 4, the cAMP IC50 column shows that the majority of analogues are well tolerated and do not diminish activity (3e, 3m, 3o and 3q) to a large degree. Only the analogue with a butyl elongating chain, 3s, deeply impacts the cAMP activity. Although compounds having meta- and para-methoxy substitutions on the phenyl ring at position 12 have a 5 to 10 times lower potency than the corresponding analogues without methoxy groups, they also show that elongation of the chain does not significantly impact cAMP activity. More importantly, these results showed a GPCR antagonist with selectivity toward either cAMP (3m and 3q) or calcium (3n), thus making them important tools for further characterizing the relationship between different signaling pathways and neurocircuits modulated by NPS.</p><p>In further exploration of the impact of aromatic ring substitution at position 10, we synthesized additional analogues having functionalities on that part of the molecule. Table 5 shows that only the meta-fluoro halogenated analogue 3t improved calcium signaling potency. Although none of the substituents increased antagonism of cAMP activity, it's worth mentioning that a number of analogues had a strong negative impact on it. The ortho-nitrile substituent 3aa (Table 5) had no cAMP activity, giving us a compound completely selective toward the calcium pathway.</p><p>Tables 6 and 7 display the activity of compounds with substitutions at the 8 and 9 position, respectively. It can be seen that substituents on those regions of the molecule negatively impact both the calcium and the cAMP signal.</p><p>Lastly, table 8 shows that the regioselectivity of the naphthyl ring (compounds 3a and 12) is important for maintaining activity in both assays. Replacement of the naphthyl aromatic ring by an unsubstituted phenyl ring (compound 17) maintains the potency of the molecule, while substituents in the meta position are not well tolerated (14a, 14b).</p><p>In conclusion, we disclose a new series of small molecule NPSR antagonists which explore the SAR affecting both calcium and cAMP signaling by the receptor. In general, aromatic substituents at the 10 and 12 positions can positively impact the potency of the molecule, while substitutions at positions 8 and 9 consistently reduce it. We found that elongating the distance between the aromatic ring and the pyrimidine core at position 10 yielded cAMP selective compounds. In comparison, introducing an ortho-nitrile onto a directly attached aromatic ring at the same position produced a selective antagonist of calcium signaling. The development and use of these selective analogues can be relevant to the study of the involvement of different G coupling pathways in the neurobiology of the NPS receptor.</p><!><p>Intracellular cAMP level was measured using LANCE cAMP detection kit (Perkin Elmer, Waltham, MA). CHO-NPSR cells were seeded at 4 µl/well with 2000 cells in white, tissue culture treated 1536-well plates. After overnight incubation at 37 °C 5% CO2, 23 nl of compounds were added to each well by a pintool station, followed by addition of 1 ul stimulation buffer (1X HBSS buffer, 0.1% BSA, 5 mM HEPES, 500 µM RO-201724, 1.5% Alexa-647 conjugated anti-cAMP antibody stock, 100 nM NPS) by a BioRAPTR flying reagent dispenser (Beckman Coulter, Fullerton, CA). Cells were incubated at 37 °C for 1 hour and 1 ul of detection reagent (1X detection buffer provided by the manufacturer, 1% TritionX-100, biotin labeled cAMP 1:250 dilution, Eu-W8044 1:750 dilution) were added. After 2 hour incubation at room temperature, plates were measured using the ViewLux ultraHTS microplate imager (Perkin Elmer) under LANCE setting. The results were expressed as a ratio of the acceptor fluorescence intensity (671/8 nm) divided by the donor fluorescence intensity (618/8 nm)</p><!><p>Intracellular calcium was measure using BD PBX NW calcium assay kit (BD Bioscience, Rockville, MD). Cells were seeded at 3 µl/well with 1500 cells in black, tissue culture treated, clear bottom 1536-well plates. After overnight incubation at 37 °C 5% CO2, cells were loaded with 3 µl of the calcium dye prepared as manufacturer's instruction and incubated for another 1 h. 23 nl of compounds prepared in DMSO were then added using a pintool station (Kalypsys, San Diego, CA). Fluorescence was monitored over time as cells were challenged with EC90 of agonists in a FDSS-7000 detector (Hamamatsu, Bridgewater, NJ).</p><!><p>Radioligand binding assay was conducted as described before (Xu et al., 2004).Y10-NPS labeled with 125I was obtained from NEN Perkin Elmer (Boston, MA). In the displacement binding assay, increasing concentrations of unlabeled human NPS or compounds were used to compete with 0.15 nM [125I]Y10-NPS. Non-specific binding was determined in the presence of 1 µM unlabeled NPS. CHO-NPSR cells were first seeded into 24-well plates and cultured until reaching about 95% confluence. Cells were then washed once with 1 ml PBS and incubated with radioligand with or without compounds in DMEM medium containing 0.1% bovine serine albumin at room temperature for 90 minutes. Cells were washed twice with ice-cold PBS and lysed with 0.5 ml 1N NaOH. Bound radioactivity was counted in gamma counter. Data from duplicate were analyzed using GraphPad Prism (GraphPad, San Diego, CA).</p><!><p>The synthesis of several non-commercially available intermediates 2-arylidenemalononitriles 4 was carried using the same reaction conditions described for the following synthesis of 2-benzylidenemalononitrile and with final yields between 85 to 95%.</p><p>Piperidine (6.25 g, 73.4 mmol) was added to a stirred solution of benzaldehyde (7.4 g, 73.4 mmol), and malononitrile (4.85 g, 73.4 mmol) in Ethanol (50 ml). After stirring overnight, the reaction mixture was concentrated in vacuum prior to the addition of dilute HCl (200 ml, 0.1 M) and the reaction products extracted into dichloromethane. The combined extracts were dried (Na2SO4) and concentrated in vacuum, subsequent recrystallization from dichloromethane/hexane afforded 2-benzylidenemalononitrile (93% yield) which was quickly taken on to the cyclization reaction.</p><!><p>The synthesis of all intermediates 3-amino -1-aryl-1H-benzo[f]chromene-2-carbonitrile 5 was carried using the same reaction conditions described for the synthesis of 3-amino-1-phenyl-1H-benzo[f]chromene-2-carbonitrile and with final yields between 45 to 94%.</p><p>A solution of 2-benzylidenemalononitrile (5.35 g, 34.7 mmol), in ethanol (140 mL) was treated with naphthalen-2-ol (5 g, 34.7 mmol) and piperidine (17.13 mL, 173 mmol). The reaction mixture was refluxed for 45 minutes until completed precipitation. The solid product was collected by filtration and washed with cold ethanol to yield 3-amino-1-phenyl-1H-benzo[f]chromene-2-carbonitrile (82% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.89 – 7.96 (m, 2 H), 7.81 – 7.87 (m, 1 H), 7.38 – 7.47 (m, 2 H), 7.34 (d, J=9.00 Hz, 1 H), 7.26 (t, J=7.43 Hz, 2 H), 7.11 – 7.21 (m, 3 H), 6.96 (s, 2 H), 5.29 (s, 1 H).</p><!><p>The synthesis of all intermediates (E)-ethyl N-2-cyano-1-Aryl-1H-benzo[f]chromen-3-ylformimidate was carried using the same reaction conditions described for the synthesis of (E)-ethyl N-2-cyano-1-phenyl-1H-benzo[f]chromen-3-ylformimidate and with final yields between 73 to 89%:</p><p>A mixture of 3-amino-1-phenyl-1H-benzo[f]chromene-2-carbonitrile 5d (3.5 g, 11.73 mmol), triethyl orthoformate (1.739 g, 11.73 mmol) and acetic anhydride (47 mL) was refluxed for 4 hours. The solvent was removed under reduced pressure. The crude from the reaction was titrated with toluene and the separated solid was filtered and recrystallized from toluene to yield (E)-ethyl N-2-cyano-1-phenyl-1H-benzo[f]chromen-3-ylformimidate which was taken on crude to the ring closure reaction.</p><!><p>The synthesis of all final compounds of10-Aryl-12-aryl-12H-Naphtho[1',2':5,6]pyrano[2,3-d]pyrimidin-11-imine 3 was carried using the same reaction conditions described for the synthesis of 10-(2-morpholinoethyl)-12-(3,4-dimethoxyphenyl)-12H-naphtho[1',2':5,6]pyrano[2,3-d]pyrimidin-11-imine 3a and with final yields between 30 to 77%.</p><p>LDA (7.24 mL, 14.48 mmol, 2M ) was added over a THF (48 mL) solution of (E)-ethyl N-2-cyano-1-(3,4-dimethoxyphenyl)-1H-benzo[f]chromen-3-ylformimidate (5 g, 12.06 mmol) and 2-morpholinoethanamine (1.57 g, 12.06 mmol) cooled down to −78 °C under nitrogen atmosphere. The reaction was stirred at room temperature for 16 hours. The precipitate was filtered, washed with cold EtOH and dried under vacuum to yield 0.531 g of clean desired 10-(2-morpholinoethyl)-12-(3,4-dimethoxyphenyl)-12H-Naphtho[1',2':5,6]pyrano[2,3-d]pyrimidin-11-imine 3a (8.83%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.30 (d, J=8.61 Hz, 1 H), 7.94 – 8.03 (m, 2 H), 7.83 – 7.93 (m, 3 H), 7.51 (t, J=7.24 Hz, 1 H), 7.36 – 7.46 (m, 1 H), 7.23 (d, J=1.96 Hz, 1 H), 6.85 (dd, J=8.41, 2.15 Hz, 1 H), 6.72 (d, J=8.61 Hz, 1 H), 5.81 (s, 1 H), 3.97 – 4.05 (m, 1 H), 3.84 – 3.93 (m, 1 H), 3.64 (s, 1 H), 3.57 (s, 1 H), 3.49 – 3.55 (m, 6 H), 3.45 (t, J=4.50 Hz, 2 H), 3.11 – 3.20 (m, 4 H), 2.99 – 3.00 (m, 1 H), 2.45 – 2.49 (m, 1 H); HRMS (ESI): m/z calcd for C29H31N4O4 [M+H]+ 499.2345, found 499.2351.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 8.28 (d, J=8.22 Hz, 1 H), 7.93 – 8.02 (m, 2 H), 7.83 – 7.94 (m, 2 H), 7.51 (t, J=7.04 Hz, 1 H), 7.36 – 7.47 (m, 2 H), 7.03 – 7.17 (m, 2 H), 6.95 (d, J=7.83 Hz, 1 H), 6.63 (dd, J=7.83, 2.35 Hz, 1 H), 5.72 (s, 1 H), 3.94 – 4.06 (m, 1 H), 3.88 (dd, J=13.30, 5.87 Hz, 1 H), 3.61 (s, 3 H), 3.49 – 3.57 (m, 2 H), 3.40 – 3.48 (m, 3 H), 3.17 (q, J=6.52 Hz, 2 H), 2.51 – 2.60 (m, 1 H), 2.41 – 2.50 (m, 2 H); HRMS (ESI): m/z calcd for C28H29N4O3 [M+H]+ 469.2239, found 469.2240.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 8.25 (d, J=8.61 Hz, 1 H), 7.95 – 8.04 (m, 2 H), 7.82 – 7.92 (m, 2 H), 7.32 – 7.54 (m, 5 H), 6.72 (d, J=9.00 Hz, 2 H), 5.83 (s, 1 H), 3.95 – 4.08 (m, 1 H), 3.89 (dd, J=13.30, 5.87 Hz, 1 H), 3.58 (s, 3 H), 3.49 – 3.56 (m, 2 H), 3.41 – 3.48 (m, 3 H), 3.09 – 3.22 (m, 2 H), 2.50 – 2.61 (m, 1 H), 2.41 – 2.50 (m, 2 H); HRMS (ESI): m/z calcd for C28H29N4O3 [M+H]+ 469.2239, found 469.2233.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 8.26 (d, J=8.61 Hz, 1 H), 7.95 – 8.02 (m, 2 H), 7.82 – 7.93 (m, 2 H), 7.34 – 7.55 (m, 5 H), 7.16 (t, J=7.63 Hz, 2 H), 7.04 (t, J=7.43 Hz, 1 H), 5.89 (s, 1 H), 3.99 (dt, J=13.60, 5.53 Hz, 1 H), 3.80 – 3.93 (m, 1 H), 3.48 – 3.56 (m, 3 H), 3.39 – 3.49 (m, 2 H), 3.10 – 3.22 (m, 2 H), 2.49 – 2.60 (m, 1 H), 2.40 – 2.49 (m, 2 H); HRMS (ESI): m/z calcd for C27H27N4O2 [M+H]+ 439.2134, found 439.2133.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 10.66 (br. s., 1 H), 8.29 (d, J=8.61 Hz, 1 H), 8.16 (s, 1 H), 7.79 – 7.92 (m, 2 H), 7.59 – 7.76 (m, 5 H), 7.40 – 7.59 (m, 4 H), 7.34 (d, J=7.83 Hz, 1 H), 7.23 – 7.30 (m, 2 H), 7.14 – 7.22 (m, 1 H), 6.46 (s, 1 H); HRMS (ESI): m/z calcd for C27H20N3O [M+H]+ 402.1606, found 402.1611.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 10.39 (br. s., 1 H), 8.07 (s, 1 H), 7.82 (d, J=9.00 Hz, 1 H), 7.75 – 7.80 (m, 1 H), 7.66 – 7.73 (m, 1 H), 7.59 (br. s., 3 H), 7.35 – 7.46 (m, 3 H), 7.26 (br. s., 1 H), 7.19 (s, 1 H), 7.09 – 7.17 (m, 1 H), 6.89 – 6.97 (m, 2 H), 6.76 (t, J=7.63 Hz, 1 H), 6.10 (s, 1 H), 4.06 (s, 3 H); HRMS (ESI): m/z calcd for C28H22N3O2 [M+H]+ 432.1712, found 432.1712.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.26 (d, J=8.22 Hz, 1 H), 8.04 (s, 1 H), 7.68 – 7.79 (m, 2 H), 7.56 (br. s., 3 H), 7.45 (t, J=7.24 Hz, 1 H), 7.31 – 7.40 (m, 3 H), 7.27 (s, 2 H), 7.01 – 7.10 (m, 2 H), 6.58 – 6.65 (m, 1 H), 6.44 (s, 1 H), 3.64 (s, 3 H); HRMS (ESI): m/z calcd for C28H22N3O2 [M+H]+ 432.1712, found 432.1714.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 11.16 (br.s, 1 H), 8.20 (d, J=8.61 Hz, 1 H), 8.05 (s, 1 H), 7.74 (dd, J=11.35, 8.61 Hz, 2 H), 7.59 (br. s., 3 H), 7.45 (t, J=7.63 Hz, 1 H), 7.24 – 7.41 (m, 4 H), 7.13 (d, J=7.83 Hz, 1 H), 6.98 (s, 1 H), 6.56 (d, J=8.22 Hz, 1 H), 6.43 (s, 1 H), 5.73 (s, 1 H), 5.65 (s, 1 H); HRMS (ESI): m/z calcd for C28H20N3O3 [M+H]+ 446.1504, found 446.1504.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.20 (d, J=8.61 Hz, 1 H), 8.05 (s, 1 H), 7.64 – 7.79 (m, 3 H), 7.57 (br. s., 3 H), 7.22 – 7.46 (m, 7 H), 6.53 (d, J=8.22 Hz, 1 H), 6.37 (s, 1 H), 4.25 – 4.43 (m, 2 H), 2.90 – 3.06 (m, 2 H); HRMS (ESI): m/z calcd for C29H22N3O2 [M+H]+ 444.1712, found 444.1711.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 8.73 (s, 1 H), 8.21 (d, J=8.61 Hz, 1 H), 7.95 – 8.09 (m, 2 H), 7.59 – 7.74 (m, 6 H), 7.45 – 7.57 (m, 2 H), 7.28 (d, J=1.96 Hz, 1 H), 6.74 – 6.87 (m, 3 H), 6.29 (s, 1 H), 3.79 – 4.00 (m, 4 H), 1.15 – 1.30 (m, 6 H); HRMS (ESI): m/z calcd for C31H28N3O3 [M+H]+ 490.2125, found 490.2130.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 8.73 (s, 1 H), 8.20 (d, J=8.61 Hz, 2 H), 7.96 – 8.07 (m, 2 H), 7.61 – 7.74 (m, 6 H), 7.53 (td, J=7.53, 0.98 Hz, 1 H), 6.98 – 7.05 (m, 1 H), 6.91 (dd, J=8.41, 2.15 Hz, 1 H), 6.69 – 6.77 (m, 1 H), 6.29 (s, 1 H), 4.04 – 4.17 (m, 4 H); HRMS (ESI): m/z calcd for C29H22N3O3 [M+H]+ 460.1656, found 460.1664.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.33 – 8.97 (m, 1 H), 8.08 (s, 1 H), 7.94 (d, J=8.22 Hz, 1 H), 7.77 (dd, J=17.61, 8.61 Hz, 2 H), 7.55 (br. s., 3 H), 7.31 – 7.46 (m, 4 H), 7.22 (d, J=3.91 Hz, 1 H), 6.99 (d, J=7.43 Hz, 1 H), 6.83 (t, J=8.02 Hz, 1 H), 6.68 (d, J=7.04 Hz, 1 H), 6.13 (s, 1 H), 3.84 (s, 3 H), 3.69 (s, 3 H); HRMS (ESI): m/z calcd for C29H24N3O3 [M+H]+ 462.1817, found 462.1818.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.04 (d, J=8.22 Hz, 1 H), 7.75 – 7.83 (m, 3 H), 7.42 – 7.50 (m, 4 H), 7.28 – 7.41 (m, 5 H), 7.21 – 7.27 (m, 4 H), 7.13 (t, J=7.24 Hz, 1 H), 5.53 (s, 1 H), 5.27 – 5.36 (m, 1 H), 4.87 (d, J=14.87 Hz, 1 H); HRMS (ESI): m/z calcd for C28H22N3O [M+H]+ 416.1763, found 416.1754.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.07 (br. s., 1 H), 8.88 (s, 1 H), 8.17 (d, J=8.41 Hz, 1 H), 7.99 (dd, J=14.87, 8.61 Hz, 2 H), 7.58 – 7.66 (m, 2 H), 7.45 – 7.55 (m, 1 H), 7.26 – 7.36 (m, 3 H), 7.16 – 7.25 (m, 3 H), 6.82 – 6.90 (m, 1 H), 6.76 – 6.82 (m, 1 H), 6.19 (s, 1 H), 5.45 (q, 2 H), 3.61 (d, 6 H); HRMS (ESI): m/z calcd for C30H26N3O3 [M+H]+ 476.1974, found 476.1978.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.08 (d, J=8.61 Hz, 1 H), 7.77 (t, J=8.02 Hz, 2 H), 7.34 – 7.54 (m, 6 H), 7.16 – 7.30 (m, 6 H), 7.13 (t, J=7.43 Hz, 1 H), 6.94 – 7.01 (m, 2 H), 5.54 (s, 1 H), 4.16 (dt, J=13.21, 6.50 Hz, 1 H), 3.98 (dt, J=13.40, 6.80 Hz, 1 H), 2.93 – 3.14 (m, 2 H); LCMS(4 min method): (electrospray +ve), m/z 430.1 (MH)+; HPLC: tR = 3.20 min, UV254 = 100%.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.24 (s, 1 H), 8.32 (s, 1 H), 8.20 (d, J=8.41 Hz, 1 H), 7.99 (t, J=9.19 Hz, 2 H), 7.65 (ddd, J=8.41, 7.04, 1.37 Hz, 1 H), 7.57 (d, J=9.00 Hz, 1 H), 7.48 – 7.54 (m, 1 H), 7.26 (d, J=1.37 Hz, 1 H), 7.10 – 7.21 (m, 3 H), 6.90 – 6.97 (m, 2 H), 6.84 – 6.89 (m, 2 H), 6.24 (s, 1 H), 4.34 – 4.57 (m, 2 H), 3.68 (s, 3 H), 3.61 (s, 3 H), 2.97 (t, J=6.94 Hz, 2 H); HRMS (ESI): m/z calcd for C31H28N3O3 [M+H]+ 490.2130, found 490.2135.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.06 (d, J=8.22 Hz, 1 H), 7.77 (dd, J=8.61, 5.09 Hz, 2 H), 7.63 (s, 1 H), 7.33 – 7.52 (m, 6 H), 7.06 – 7.32 (m, 7 H), 5.50 (s, 1 H), 5.28 (s, 1 H), 3.96 – 4.05 (m, 1 H), 3.64 – 3.79 (m, 1 H), 2.65 (t, J=7.43 Hz, 2 H), 2.06 – 2.18 (m, 2 H); HRMS (ESI): m/z calcd for C30H26N3O [M+H]+ 444.2076, found 444.2076.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.13 (br. s., 1 H), 8.74 (s, 1 H), 8.20 (d, J=8.41 Hz, 1 H), 7.92 – 8.06 (m, 2 H), 7.56 – 7.69 (m, 2 H), 7.47 – 7.55 (m, 1 H), 7.26 (d, J=2.15 Hz, 1 H), 7.15 – 7.23 (m, 2 H), 7.02 – 7.14 (m, 3 H), 6.81 – 6.87 (m, 1 H), 6.75 – 6.81 (m, 1 H), 6.19 (s, 1 H), 4.23 (t, J=7.83 Hz, 2 H), 3.66 (s, 3 H), 3.59 (s, 3 H), 2.54 – 2.65 (m, 2 H), 1.91 – 2.04 (m, 2 H); HRMS (ESI): m/z calcd for C32H30N3O3 [M+H]+ 504.2287, found 504.2293.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.06 (d, J=8.61 Hz, 1 H), 7.78 (dd, J=8.41, 6.46 Hz, 2 H), 7.68 (s, 1 H), 7.35 – 7.52 (m, 6 H), 7.07 – 7.30 (m, 7 H), 5.48 – 5.54 (m, 1 H), 5.07 (s, 1 H), 4.03 (ddd, J=13.60, 7.92, 5.87 Hz, 1 H), 3.65 – 3.77 (m, 1 H), 2.57 – 2.67 (m, 2 H), 1.71 – 1.87 (m, 2 H), 1.64 (quin, J=7.73 Hz, 2 H); HRMS (ESI): m/z calcd for C31H28N3O [M+H]+ 458.2232, found 458.2235.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.23 (s, 1 H), 8.70 (s, 1 H), 8.20 (d, J=8.41 Hz, 1 H), 7.95 – 8.08 (m, 2 H), 7.83 (br. s., 2 H), 7.60 – 7.70 (m, 2 H), 7.48 – 7.58 (m, 3 H), 7.12 – 7.23 (m, 2 H), 6.97 (dd, J=7.34, 1.27 Hz, 1 H), 6.70 – 6.78 (m, 1 H), 6.35 (s, 1 H), 3.60 – 3.67 (m, 3 H); LCMS: (electrospray +ve), m/z 448.1 (MH)+; HPLC: tR = 4.69 min, UV254 = 100%.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.24 (br. s. 1 H), 8.73 (s, 1 H), 8.20 (d, J=8.41 Hz, 1 H), 8.02 (dd, J=18.78, 8.61 Hz, 2 H), 7.69 – 7.81 (m, 2 H), 7.61 – 7.69 (m, 2 H), 7.46 – 7.59 (m, 3 H), 7.10 – 7.22 (m, 2 H), 6.94 – 7.00 (m, 1 H), 6.69 – 6.78 (m, 1 H), 6.34 (s, 1 H), 3.60 – 3.67 (m, 3 H); HRMS (ESI): m/z calcd for C28H21N3O2 [M+H]+ 450.1618, found 450.1617.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.26 – 9.39 (m, 1 H), 8.81 – 8.91 (m, 2 H), 8.77 (br. s., 1 H), 8.52 (br. s., 1 H), 8.11 – 8.23 (m, 1 H), 7.91 – 8.09 (m, 2 H), 7.72 (br. s., 1 H), 7.61 – 7.69 (m, 2 H), 7.49 – 7.58 (m, 1 H), 7.10 – 7.23 (m, 2 H), 6.95 – 7.02 (m, 1 H), 6.75 (dd, J=8.22, 2.15 Hz, 1 H), 6.35 (s, 1 H), 3.62 – 3.68 (m, 3 H); HRMS (ESI): m/z calcd for C28H21N3O2Cl [M+H]+ 466.1322, found 466.1318.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.27 (br. s., 1 H), 8.76 (d, J=6.85 Hz, 1 H), 8.38 (br. s., 1 H), 8.20 (d, J=8.41 Hz, 1 H), 8.02 (dd, J=18.49, 8.51 Hz, 2 H), 7.76 – 7.81 (m, 1 H), 7.59 – 7.75 (m, 4 H), 7.48 – 7.56 (m, 1 H), 7.10 – 7.22 (m, 2 H), 6.93 – 7.02 (m, 1 H), 6.75 (dd, J=8.02, 2.15 Hz, 1 H), 6.35 (d, J=6.06 Hz, 1 H), 3.60 – 3.66 (m, 3 H); HRMS (ESI): m/z calcd for C28H21N3O2Cl [M+H]+ 466.1322, found 466.1318.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.45 (br. s., 1 H), 8.83 (br. s., 1 H), 8.16 (d, J=19.37 Hz, 1 H), 7.99 (br. s., 3 H), 7.83 (br. s., 1 H), 7.65 (br. s., 3 H), 7.52 (br. s., 1 H), 7.16 (br. s., 2 H), 7.00 (br. s., 1 H), 6.88 (br. s., 1 H), 6.76 (br. s., 1 H), 6.39 (br. s., 1 H), 3.60 – 3.67 (m, 3 H); HRMS (ESI): m/z calcd for C28H21N3O2Br [M+H]+ 510.0817, found 510.0810.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.27 (br. s., 1 H), 8.76 (d, J=6.85 Hz, 1 H), 8.20 (d, J=8.61 Hz, 1 H), 7.94 – 8.08 (m, 3 H), 7.87 – 7.93 (m, 1 H), 7.59 – 7.70 (m, 4 H), 7.48 – 7.57 (m, 1 H), 7.10 – 7.22 (m, 2 H), 6.97 (d, J=6.65 Hz, 1 H), 6.75 (dd, J=8.02, 2.15 Hz, 1 H), 6.34 (d, J=5.09 Hz, 1 H), 3.61 – 3.68 (m, 3 H); HRMS (ESI): m/z calcd for C28H21N3O2Br [M+H]+ 510.0817, found 510.0819.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.29 (br. s., 1 H), 8.71 – 8.82 (m, 1 H), 8.20 (d, J=8.41 Hz, 1 H), 8.02 (dd, J=19.27, 8.51 Hz, 2 H), 7.71 (br. s., 2 H), 7.46 – 7.69 (m, 5 H), 7.09 – 7.22 (m, 2 H), 6.94 – 7.02 (m, 1 H), 6.75 (dd, J=7.92, 2.25 Hz, 1 H), 6.35 (br. s., 1 H), 3.60 – 3.67 (m, 3 H); LCMS: (electrospray +ve), m/z 511.9 (MH)+; HPLC: tR= 3.23 min,UV254 = 90%.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 10.15 (s, 1 H), 9.98 – 10.08 (m, 1 H), 8.82 (d, J=8.61 Hz, 1 H), 8.55 (dd, J=8.31, 1.27 Hz, 1 H), 8.18 (ddd, J=8.75, 7.29, 1.37 Hz, 1 H), 8.09 (d, J=9.00 Hz, 1 H), 7.92 – 8.05 (m, 3 H), 7.71 (d, J=9.00 Hz, 1 H), 7.63 (ddd, J=8.36, 6.99, 1.27 Hz, 1 H), 7.52 (ddd, J=8.12, 6.94, 0.98 Hz, 1 H), 7.17 – 7.24 (m, 1 H), 7.10 (t, J=8.02 Hz, 1 H), 6.97 (d, J=8.41 Hz, 1 H), 6.63 – 6.70 (m, 1 H), 6.37 (s, 1 H), 3.62 – 3.67 (m, 3 H); HRMS (ESI): m/z calcd for C29H21N4O2 [M+H]+ 457.1664, found 457.1665.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.32 (br. s., 1 H), 8.71 – 8.80 (m, 1 H), 8.17 – 8.23 (m, 2 H), 7.96 – 8.08 (m, 3 H), 7.93 (br. s., 1 H), 7.61 – 7.70 (m, 2 H), 7.48 – 7.57 (m, 2 H), 7.10 – 7.25 (m, 2 H), 6.91 – 7.03 (m, 1 H), 6.75 (d, J=8.41 Hz, 1 H), 6.34 (br. s., 1 H), 3.61 – 3.69 (m, 3 H); HRMS (ESI): m/z calcd for C29H21N4O2 [M+H]+ 457.1664, found 457.1664.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.31 (br. s., 1 H), 8.74 – 8.80 (m, 1 H), 8.19 (m, 3 H), 7.84 – 8.08 (m, 4 H), 7.61 – 7.70 (m, 2 H), 7.47 – 7.58 (m, 1 H), 7.09 – 7.24 (m, 2 H), 6.92 – 7.01 (m, 1 H), 6.72 – 6.79 (m, 1 H), 6.34 (s, 1 H), 3.59 – 3.67 (m, 3 H); HRMS (ESI): m/z calcd for C29H21N4O2 [M+H]+ 457.1664, found 457.1661.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 8.43 (s, 1 H), 8.35 – 8.41 (m, 2 H), 8.13 (d, J=8.41 Hz, 1 H), 7.94 (t, J=9.00 Hz, 2 H), 7.74 – 7.82 (m, 1 H), 7.51 – 7.61 (m, 2 H), 7.41 – 7.48 (m, 1 H), 7.16 – 7.21 (m, 1 H), 7.04 – 7.13 (m, 2 H), 6.89 – 6.95 (m, 1 H), 6.71 (s, 1 H), 6.59 – 6.66 (m, 1 H), 3.59 (s, 3 H); LCMS: (electrospray +ve), m/z 433.1 (MH)+; HPLC: tR = 5.01 min,UV254 = 90%.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.31 (br. s., 1 H), 8.83 – 8.91 (m, 1 H), 8.77 (br. s., 1 H), 8.52 (br. s., 1 H), 8.12 – 8.23 (m, 2 H), 7.91 – 8.08 (m, 2 H), 7.72 (br. s., 1 H), 7.60 – 7.69 (m, 2 H), 7.48 – 7.58 (m, 1 H), 7.12 – 7.22 (m, 2 H), 6.94 – 7.03 (m, 1 H), 6.75 (dd, J=8.22, 2.15 Hz, 1 H), 6.35 (s, 1 H), 3.61 – 3.67 (m, 3 H); HRMS (ESI): m/z calcd for C27H21N4O2 [M+H]+ 433.1664, found 433.1666.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 10.40 (br. s., 1 H), 8.63 (s, 1 H), 8.58 (d, J=7.04 Hz, 1 H), 8.11 – 8.23 (m, 2 H), 7.93 – 8.04 (m, 2 H), 7.55 – 7.67 (m, 3 H), 7.44 – 7.53 (m, 1 H), 7.02 – 7.11 (m, 2 H), 6.85 – 6.93 (m, 1 H), 6.72 – 6.79 (m, 1 H), 6.57 – 6.65 (m, 2 H), 3.53 – 3.59 (m, 3 H); HRMS (ESI): m/z calcd for C27H21N4O2 [M+H]+ 433.1664, found 433.1665.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.39 (br. s., 1 H), 8.71 (d, J=7.63 Hz, 1 H), 8.20 (d, J=8.61 Hz, 2 H), 7.94 – 8.10 (m, 2 H), 7.43 – 7.70 (m, 5 H), 7.14 – 7.23 (m, 2 H), 6.88 – 7.03 (m, 2 H), 6.70 – 6.78 (m, 1 H), 6.40 (d, J=18.98 Hz, 1 H), 3.61 – 3.68 (m, 3 H), 2.10 (s, 3 H); HRMS (ESI): m/z calcd for C29H24N3O2 [M+H]+ 446.1868, found 446.1863.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.24 (br. s., 1 H), 8.72 (s, 1 H), 8.21 (d, J=8.41 Hz, 1 H), 8.02 (dd, J=17.51, 8.31 Hz, 2 H), 7.60 – 7.68 (m, 2 H), 7.38 – 7.60 (m, 5 H), 7.11 – 7.21 (m, 2 H), 6.98 (d, J=7.43 Hz, 1 H), 6.74 (dd, J=8.02, 2.15 Hz, 1 H), 6.36 (s, 1 H), 3.59 – 3.68 (m, 3 H), 2.33 – 2.42 (m, 3 H); HRMS (ESI): m/z calcd for C29H24N3O2 [M+H]+ 446.1868, found 446.1867.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.23 (br. s., 1 H), 8.69 (s, 1 H), 8.20 (d, J=8.22 Hz, 2 H), 7.95 – 8.07 (m, 2 H), 7.59 – 7.69 (m, 2 H), 7.43 – 7.56 (m, 4 H), 7.11 – 7.21 (m, 2 H), 6.95 – 7.01 (m, 1 H), 6.70 – 6.78 (m, 1 H), 6.36 (s, 1 H), 3.60 – 3.67 (m, 3 H), 2.41 (s, 3 H); HRMS (ESI): m/z calcd for C29H24N3O2 [M+H]+ 446.1868, found 446.1865.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.46 (br. s., 1 H), 8.90 (s, 1 H), 8.14 – 8.22 (m, 1 H), 7.95 – 8.09 (m, 2 H), 7.74 – 7.85 (m, 1 H), 7.59 – 7.70 (m, 3 H), 7.43 – 7.58 (m, 3 H), 7.11 – 7.21 (m, 3 H), 6.68 – 6.78 (m, 1 H), 6.36 – 6.43 (m, 1 H), 3.60 – 3.68 (m, 3 H), 1.13 – 1.21 (m, 3 H), 0.81 – 0.88 (m, 6 H); HRMS (ESI): m/z calcd for C32H30N3O2 [M+H]+ 488.2338, found 488.2337.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.36 (br. s., 1 H), 8.68 – 8.74 (m, 1 H), 8.16 – 8.24 (m, 1 H), 7.96 – 8.08 (m, 2 H), 7.58 – 7.72 (m, 3 H), 7.47 – 7.56 (m, 1 H), 7.36 (ddd, J=13.60, 8.51, 0.98 Hz, 1 H), 7.09 – 7.27 (m, 3 H), 6.94 – 7.02 (m, 1 H), 6.84 – 6.91 (m, 1 H), 6.71 – 6.80 (m, 1 H), 6.35 – 6.43 (m, 1 H), 3.59 – 3.68 (m, 6 H); HRMS (ESI): m/z calcd for C29H24N3O3 [M+H]+ 462.1817, found 462.1815.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.26 (br. s., 1 H), 8.74 (d, J=1.96 Hz, 1 H), 8.28 (br. s., 1 H), 8.21 (d, J=8.41 Hz, 1 H), 7.46 – 7.69 (m, 4 H), 7.37 (m, 1 H), 7.09 – 7.31 (m, 5 H), 6.98 (br. s., 1 H), 6.74 (dd, J=8.02, 2.15 Hz, 1 H), 6.32 – 6.40 (m, 1 H), 3.78 (s, 3 H), 3.61 – 3.67 (m, 3 H); HRMS (ESI): m/z calcd for C29H24N3O3 [M+H]+ 462.1817, found 462.1812.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.22 (br. s., 1 H), 8.69 (s, 1 H), 8.20 (d, J=8.22 Hz, 2 H), 7.96 – 8.07 (m, 2 H), 7.60 – 7.68 (m, 3 H), 7.46 – 7.60 (m, 2 H), 7.11 – 7.25 (m, 3 H), 6.93 – 7.00 (m, 1 H), 6.71 – 6.78 (m, 1 H), 6.35 (s, 1 H), 3.81 – 3.86 (m, 3 H), 3.60 – 3.66 (m, 3 H); HRMS (ESI): m/z calcd for C29H24N3O3 [M+H]+ 462.1817, found 462.1814.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.25 (br. s., 1 H), 8.71 (d, J=3.33 Hz, 1 H), 8.24 – 8.36 (m, 1 H), 8.19 (d, J=8.61 Hz, 1 H), 7.96 – 8.07 (m, 2 H), 7.59 – 7.69 (m, 2 H), 7.48 – 7.56 (m, 1 H), 7.30 (d, J=1.96 Hz, 1 H), 7.06 – 7.24 (m, 3 H), 6.97 (d, J=8.41 Hz, 1 H), 6.74 (d, J=8.61 Hz, 1 H), 6.34 (d, J=7.43 Hz, 1 H), 6.12 – 6.23 (m, 2 H), 3.59 – 3.67 (m, 3 H); HRMS (ESI): m/z calcd for C29H22N3O4 [M+H]+ 476.1610, found 476.1614.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.40 (br. s., 1 H), 8.74 (s, 1 H), 8.42 (br. s., 1 H), 8.20 (t, J=9.29 Hz, 1 H), 7.96 – 8.09 (m, 2 H), 7.59 – 7.74 (m, 5 H), 7.41 – 7.58 (m, 3 H), 7.11 – 7.22 (m, 2 H), 6.99 (dd, J=7.83, 0.78 Hz, 1 H), 6.87 (dd, J=7.34, 1.27 Hz, 1 H), 6.75 (dd, J=7.92, 2.25 Hz, 1 H), 6.40 (d, J=2.74 Hz, 1 H), 3.58 – 3.68 (m, 3 H), 2.29 – 2.36 (m, 3 H); HRMS (ESI): m/z calcd for C29H24N3O2S [M+H]+ 478.1589, found 478.1589.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 8.69 (s, 1 H), 8.21 (d, J=8.41 Hz, 1 H), 8.02 (dd, J=18.19, 8.61 Hz, 2 H), 7.61 – 7.73 (m, 4 H), 7.47 – 7.59 (m, 3 H), 7.12 – 7.22 (m, 2 H), 6.93 – 7.01 (m, 1 H), 6.69 – 6.79 (m, 1 H), 6.38 (s, 1 H), 4.06 (d, J=5.28 Hz, 1 H), 3.64 (s, 3 H), 3.13 (d, J=5.09 Hz, 3 H); LCMS: (electrospray +ve), m/z 488.2 (MH)+; HPLC: tR = 5.40 min,UV254 = 100%.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.29 (br. s., 1 H), 8.93 (s, 1 H), 8.16 (d, J=8.61 Hz, 1 H), 7.94 – 8.05 (m, 2 H), 7.55 – 7.68 (m, 3 H), 7.50 (t, J=7.34 Hz, 1 H), 7.23 (d, J=2.74 Hz, 1 H), 7.09 – 7.18 (m, 2 H), 7.00 (dd, J=5.09, 3.52 Hz, 1 H), 6.92 (d, J=8.41 Hz, 1 H), 6.71 (dd, J=8.12, 2.05 Hz, 1 H), 6.26 (s, 1 H), 5.62 (s, 2 H), 3.58 – 3.67 (m, 3 H); HRMS (ESI): m/z calcd for C27H22N3O2S [M+H]+ 452.1432, found 452.1432.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.30 (br. s., 1 H), 8.77 (s, 1 H), 8.49 (br. s., 1 H), 8.08 – 8.15 (m, 1 H), 7.94 – 8.05 (m, 2 H), 7.72 – 7.84 (m, 2 H), 7.55 – 7.68 (m, 3 H), 7.44 – 7.54 (m, 1 H), 7.28 – 7.35 (m, 1 H), 7.19 – 7.27 (m, 1 H), 7.15 (t, J=8.02 Hz, 1 H), 7.00 – 7.10 (m, 1 H), 6.85 – 6.98 (m, 3 H), 6.79 (dd, J=8.22, 2.35 Hz, 1 H), 6.66 – 6.75 (m, 1 H), 6.25 (s, 1 H), 3.64 – 3.69 (m, 3 H); HRMS (ESI): m/z calcd for C34H26N3O2 [M+H]+ 508.2025, found 508.2018.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.30 (br. s., 1 H), 8.75 – 8.81 (m, 1 H), 8.31 – 8.43 (m, 1 H), 8.22 (d, J=8.41 Hz, 1 H), 7.94 – 8.08 (m, 4 H), 7.71 – 7.80 (m, 4 H), 7.60 – 7.69 (m, 2 H), 7.47 – 7.56 (m, 2 H), 7.39 – 7.46 (m, 1 H), 7.13 – 7.22 (m, 2 H), 6.99 (dd, J=7.73, 0.88 Hz, 1 H), 6.70 – 6.79 (m, 1 H), 6.39 (s, 1 H), 3.61 – 3.68 (m, 3 H); HRMS (ESI): m/z calcd for C34H26N3O2 [M+H]+ 508.2025, found 508.2021.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 8.76 (s, 1 H), 8.20 (d, J=8.41 Hz, 1 H), 8.01 (dd, J=16.82, 8.61 Hz, 2 H), 7.59 – 7.69 (m, 2 H), 7.52 (t, J=7.53 Hz, 1 H), 7.12 – 7.20 (m, 2 H), 6.91 – 6.98 (m, 1 H), 6.73 (dd, J=8.02, 2.15 Hz, 1 H), 6.29 (s, 1 H), 4.23 (t, J=7.04 Hz, 2 H), 3.93 (br. s., 2 H), 3.65 (s, 3 H), 3.58 (br. s., 2 H), 2.83 – 3.23 (m, 8 H); HRMS (ESI): m/z calcd for C29H31N4O3 [M+H]+ 483.2391, found 483.2396.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.28 (br. s., 1 H), 8.76 (s, 1 H), 8.20 (d, J=8.61 Hz, 3 H), 7.97 – 8.08 (m, 2 H), 7.81 (t, J=8.12 Hz, 2 H), 7.60 – 7.69 (m, 2 H), 7.48 – 7.57 (m, 1 H), 7.11 – 7.22 (m, 2 H), 6.94 – 7.01 (m, 1 H), 6.68 – 6.80 (m, 1 H), 6.35 (s, 1 H), 4.31 – 4.43 (m, 2 H), 3.60 – 3.66 (m, 3 H), 1.27 – 1.37 (m, 3 H); HRMS (ESI): m/z calcd for C31H26N3O4 [M+H]+ 504.1923, found 504.1924.</p><!><p>Compound 5a (200 mg, 0.558 mmol, 1.0 equiv) was taken up in formamide (8 mL) and heated to 220 °C for 20 min. Upon completion, the mixture was concentrated and taken up in ethyl acetate and water. The layers were separated and the water layer was extracted with ethyl acetate. The combined organic extracts were then washed with water and brine, then dried over Na2SO4, and concentrated to give a brown solid, which was chromatographed with 0–7% MeOH/DCM gradient elution to give 12-(3,4-dimethoxyphenyl)-12H-naphtho[1',2':5,6]pyrano[2,3-d]pyrimidin-11-amine 6a (124 mg, 57.7 % yield) as a tan solid. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.31 (s, 1 H), 8.06 (d, J=8.41 Hz, 1 H), 7.79 (dd, J=8.31, 4.01 Hz, 2 H), 7.43 – 7.53 (m, 2 H), 7.39 (t, J=7.53 Hz, 1 H), 7.09 (dd, J=8.02, 1.96 Hz, 1 H), 6.76 (d, J=8.02 Hz, 1 H), 6.71 (d, J=1.96 Hz, 1 H), 5.46 (s, 1 H), 5.11 (s, 2 H), 3.78 (s, 3 H), 3.68 (s, 3 H).</p><!><p>A mixture of 3-amino-1-phenyl-1H-benzo[f]chromene-2-carbonitrile 5d (3.5 g, 11.73 mmol) and formamide was refluxed for 3 hours. The solvent was removed under vacuum and the solid was recrystallized from toluene to yield 3.2 g of pure product (84%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.21 (d, J=8.22 Hz, 1 H), 8.11 (s, 1 H), 7.90 – 7.98 (m, 2 H), 7.55 – 7.62 (m, 1 H), 7.40 – 7.54 (m, 4 H), 7.15 – 7.25 (m, 4 H), 7.04 – 7.13 (m, 1 H), 6.05 (s, 1 H); HRMS (ESI): m/z calcd for C21H16N3O [M+H]+ 326.1293, found 326.1293.</p><!><p>To compound 6.1 (20 mg, 0.052 mmol) in acetonitrile (1 mL) was added the bromide (0.078 mmol, 1.5 equiv). The mixture was heated to 90 °C in a sealed tube for 4 h. The mixture was dried down and taken up in methanol and purified via HPLC to give products 7a (8:1 ratio 7a:3n), 7b (8:1 ratio 7b:3p), and 7c (3:1 ratio 7c:3r) with yields from 20–50%.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.36 (s, 1 H), 9.03 (s, 1 H), 8.78 (s, 1 H), 8.14 (d, J=8.41 Hz, 1 H), 7.99 (dd, J=18.00, 8.22 Hz, 2 H), 7.56 – 7.67 (m, 3 H), 7.48 – 7.55 (m, 1 H), 7.39 – 7.46 (m, 2 H), 7.31 – 7.38 (m, 1 H), 7.00 (dd, J=8.31, 2.05 Hz, 1 H), 6.87 (d, J=2.15 Hz, 1 H), 6.77 (d, J=8.41 Hz, 1 H), 6.03 (s, 1 H), 5.56 (d, J=1.96 Hz, 2 H), 3.59 (s, 3 H), 3.44 (s, 3 H); HRMS (ESI): m/z calcd for C30H26N3O3 [M+H]+ 476.1974, found 476.1974.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.24 (s, 1 H), 8.68 (br. s., 1 H), 8.59 (s, 1 H), 8.14 (d, J=8.22 Hz, 1 H), 8.02 (dd, J=18.88, 8.31 Hz, 2 H), 7.66 (d, J=8.80 Hz, 2 H), 7.48 – 7.57 (m, 1 H), 7.29 (d, J=7.43 Hz, 5 H), 6.74 – 6.82 (m, 1 H), 6.71 (d, J=9.98 Hz, 1 H), 6.00 (s, 1 H), 4.60 (m, 2H), 3.68 (s, 3 H), 3.62 (s, 3 H), 3.14 – 3.29 (m, 2 H); HRMS (ESI): m/z calcd for C31H28N3O3 [M+H]+ 490.2130, found 490.2129.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.22 (s, 1 H), 8.72 (s, 1 H), 8.65 (s, 1 H), 8.15 (d, J=8.61 Hz, 1 H), 7.94 – 8.08 (m, 2 H), 7.65 (ddd, J=8.41, 7.04, 1.17 Hz, 1 H), 7.48 – 7.57 (m, 2 H), 7.19 – 7.27 (m, 3 H), 7.13 – 7.18 (m, 2 H), 6.93 (dd, J=8.31, 2.25 Hz, 1 H), 6.78 (d, J=8.61 Hz, 1 H), 6.00 (s, 1 H), 4.25 – 4.46 (m, 2 H), 3.56 – 3.64 (m, 6 H), 2.69 – 2.79 (m, 2 H), 2.16 – 2.27 (m, 2 H); HRMS (ESI): m/z calcd for C32H30N3O3 [M+H]+ 504.2287, found 504.2293.</p><!><p>LDA (0.37 mL, 2M, 0.74 mmol) was added to a cooled down (−78 °C) THF solution (3 mL) of 12-Phenyl-12H-naphtho[1',2':5,6]pyrano[2,3-d]pyrimidin-11-amine 6d (200mg, 0.61 mmol) and the corresponding acid chloride (0.74 mmol). The reaction was allowed to reach room temperature and stirred for 24h. Then, the mixture was poured in ice water and the extracted with dichloromethane. The organic layer was then collected and dried over Na2SO4, filtered, and concentrated under vacuum. The crude material was chromatographed over silica gel to yield the corresponding acylated amide with yields between 20 to 40%.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.77 (s, 1 H), 7.94 (d, J=8.61 Hz, 1 H), 7.77 – 7.87 (m, 2 H), 7.43 – 7.53 (m, 2 H), 7.39 (t, J=6.85 Hz, 1 H), 7.31 (d, J=7.04 Hz, 2 H), 7.19 (t, J=7.63 Hz, 2 H), 7.06 – 7.13 (m, 1 H), 5.69 (s, 1 H), 1.20 (br. s., 9 H); HRMS (ESI): m/z calcd for C26H24N3O3 [M+H]+ 426.1817, found 426.1797.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.96 (br. s., 1 H), 8.48 (s, 1 H), 7.87 – 8.01 (m, 3 H), 7.80 (t, J=9.59 Hz, 2 H), 7.64 (t, J=7.43 Hz, 1 H), 7.45 – 7.59 (m, 3 H), 7.38 (dt, J=13.99, 6.90 Hz, 2 H), 7.06 – 7.24 (m, 4 H), 6.29 (s, 1 H), 5.28 (s, 1 H); HRMS (ESI): m/z calcd for C28H20N3O2 [M+H]+ 430.1550, found 430.1543.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.54 (s, 1 H), 8.12 (s, 1 H), 7.77 – 7.90 (m, 3 H), 7.37 – 7.54 (m, 8 H), 7.03 – 7.14 (m, 3 H), 6.88 – 6.98 (m, 2 H), 5.87 (s, 1 H), 3.89 – 4.09 (m, 2 H); HRMS (ESI): m/z calcd for C29H22N3O2 [M+H]+ 444.1712, found 444.1713.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 9.13 (br. s., 1 H), 8.52 (s, 1 H), 7.93 (d, J=8.61 Hz, 1 H), 7.75 – 7.86 (m, 2 H), 7.18 – 7.54 (m, 9 H), 6.97 – 7.15 (m, 4 H), 6.06 (s, 1 H), 2.90 – 3.21 (m, 3 H), 2.71 (t, J=7.83 Hz, 1 H); HRMS (ESI): m/z calcd for C30H24N3O2 [M+H]+ 458.1868, found 458.1867.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 9.89 (t, J=5.67 Hz, 1 H), 8.44 (s, 1 H), 8.11 – 8.22 (m, 2 H), 7.78 – 7.86 (m, 2 H), 7.55 (d, J=7.43 Hz, 2 H), 7.36 – 7.51 (m, 3 H), 7.31 (t, J=7.63 Hz, 2 H), 7.14 – 7.25 (m, 5 H), 5.96 (s, 1 H), 4.46 – 4.63 (m, 2 H); HRMS (ESI): m/z calcd for C29H23N4O2 [M+H]+ 459.1821, found 459.1817.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.59 (s, 1 H), 8.08 (d, J=8.22 Hz, 1 H), 7.80 (dd, J=8.41, 4.11 Hz, 2 H), 7.31 – 7.54 (m, 6 H), 7.07 – 7.25 (m, 3 H), 6.14 (s, 1 H), 3.70 – 3.90 (m, 2 H), 3.41 – 3.51 (s, 1 H), 1.20 – 1.36 (m, 3 H); HRMS (ESI): m/z calcd for C26H22N3O4 [M+H]+ 440.1610, found 440.1612.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.92 (s, 1 H), 8.56 (s, 1 H), 8.06 (d, J=8.22 Hz, 1 H), 7.75 – 7.84 (m, 2 H), 7.34 – 7.51 (m, 4 H), 7.18 – 7.30 (m, 2 H), 7.08 – 7.17 (m, 1 H), 6.08 (s, 1 H), 5.28 (s, 1 H), 4.21 (q, J=7.04 Hz, 2 H), 2.89 – 3.11 (m, 2 H), 2.64 – 2.89 (m, 2 H), 1.21 – 1.32 (m, 3 H); HRMS (ESI): m/z calcd for C27H24N3O4 [M+H]+ 454.1767, found 454.1762.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.70 (s, 1 H), 8.58 (s, 1 H), 8.05 (d, J=8.22 Hz, 1 H), 7.76 – 7.83 (m, 2 H), 7.43 – 7.50 (m, 2 H), 7.39 (t, J=6.85 Hz, 1 H), 7.33 (d, J=7.04 Hz, 2 H), 7.22 (t, J=7.63 Hz, 2 H), 7.12 (t, J=7.43 Hz, 1 H), 6.12 (s, 1 H), 4.11 – 4.20 (m, 2 H), 2.80 (t, J=7.24 Hz, 2 H), 2.44 (t, J=7.24 Hz, 2 H), 2.02 – 2.14 (m, 2 H), 1.22 – 1.29 (m, 3 H); HRMS (ESI): m/z calcd for C28H26N3O4 [M+H]+ 468.1923, found 468.1927.</p><!><p>Compound 4a (100 mg, 0.279 mmol, 1.0 equiv) and the acetyl chloride (0.419 mmol, 1.5 equiv) were taken up in THF (2 ml) with pyridine (0.558 mmol, 2.0 equiv). The mixture was stirred at 120 °C for 30 min. Upon consumption of the starting material, the reaction mixture was concentrated in vacuo and the residue was taken up in acetonitrile and submitted for HPLC purification to give products 9a–9d with yields from 10– 40%.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.90 (d, J=7.83 Hz, 1 H), 7.77 – 7.84 (m, 2 H), 7.26 – 7.48 (m, 8 H), 6.98 (d, J=2.15 Hz, 1 H), 6.83 (dd, J=8.31, 2.05 Hz, 1 H), 6.64 (d, J=8.41 Hz, 1 H), 5.81 (s, 1 H), 4.02 (d, J=1.57 Hz, 2 H), 3.69 – 3.75 (m, 6 H); HRMS (ESI): m/z calcd for C30H25N2O4 [M+H]+ 477.1814, found 477.1811.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 11.64 (br. s., 1 H), 7.93 (d, J=8.41 Hz, 1 H), 7.76 – 7.87 (m, 2 H), 7.36 – 7.52 (m, 4 H), 7.27 – 7.36 (m, 4 H), 6.92 (d, J=2.15 Hz, 1 H), 6.84 (dd, J=8.31, 2.05 Hz, 1 H), 6.55 (d, J=8.41 Hz, 1 H), 5.83 (s, 1 H), 3.60 – 3.73 (m, 6 H), 3.12 – 3.23 (m, 2 H), 2.99 (dd, J=10.37, 6.06 Hz, 2 H); HRMS (ESI): m/z calcd for C31H27N2O4 [M+H]+ 491.1971, found 491.1962.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 12.85 (br. s., 1 H), 7.94 (d, J=8.22 Hz, 1 H), 7.82 (d, J=8.61 Hz, 2 H), 7.35 – 7.52 (m, 4 H), 7.25 (m, 3 H) 7.10 – 7.20 (m, 1 H), 6.78 – 6.96 (m, 2 H), 6.60 (d, J=8.22 Hz, 1 H), 5.79 (s, 1 H), 3.59 – 3.73 (m, 6 H), 2.69 – 2.89 (m, 4 H), 2.16 – 2.34 (m, 2 H); HRMS (ESI): m/z calcd for C32H29N2O4 [M+H]+ 505.2127, found 505.2113.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 12.51 (br. s., 1 H), 7.94 (d, J=8.41 Hz, 1 H), 7.80 (d, J=8.80 Hz, 2 H), 7.35 – 7.48 (m, 3 H), 6.95 (d, J=1.76 Hz, 1 H), 6.87 (dd, J=8.31, 2.05 Hz, 1 H), 6.65 (d, J=8.22 Hz, 1 H), 5.81 (s, 1 H), 3.73 (d, J=1.57 Hz, 6 H), 2.51 (s, 3 H); HRMS (ESI): m/z calcd for C24H21N2O4 [M+H]+ 401.1501, found 401.1502.</p><!><p>Compound 9.1, 9.2, or 9.3 (0.059 mmol, 1.0 equiv) was taken up in phosphorous (V) oxychloride (1.0 mL) and heated to 120 °C for 20 min. Upon completion, the mixture was poured over crushed ice. It was then taken up in ethyl acetate, and to it was added saturated sodium bicarbonate. The layers were separated, and the organic layer was washed with sodium bicarbonate. The combined aqueous layers were extracted with ethyl acetate. The combined organic extracts were then washed with water and brine, dried over MgSO4, and concentrated to give a yellow oil which was taken up in THF (1.0 ml). Ammonium hydroxide (6.92 µl, 0.178 mmol, 3.0 equiv) was added to the mixture and it was heated to 130 °C for 30 min in a microwave oven. Upon completion, the mixture was concentrated in vacuo and submitted for purification to give products 10a–10c with yields from 10–50%.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 8.17 (d, J=8.41 Hz, 1 H), 7.89 (d, J=8.80 Hz, 2 H), 7.54 (td, J=7.68, 1.27 Hz, 1 H), 7.38 – 7.48 (m, 2 H), 7.25 (d, J=3.13 Hz, 3 H), 7.22 – 7.23 (m, 1 H), 7.21 (d, J=2.15 Hz, 2 H), 7.13 – 7.19 (m, 2 H), 6.79 (dd, J=8.41, 2.15 Hz, 1 H), 6.68 – 6.75 (m, 1 H), 5.88 (s, 1 H), 3.83 (s, 2 H), 3.62 (s, 3 H), 3.56 (s, 3 H); HRMS (ESI): m/z calcd for C30H26N3O3 [M+H]+ 476.1974, found 476.1979.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 8.18 (d, J=8.61 Hz, 1 H), 7.90 (d, J=9.00 Hz, 2 H), 7.51 – 7.59 (m, 1 H), 7.38 – 7.50 (m, 2 H), 7.09 – 7.26 (m, 8 H), 6.83 (dd, J=8.31, 2.05 Hz, 1 H), 6.74 (d, J=8.41 Hz, 1 H), 5.90 (s, 1 H), 3.55 – 3.67 (m, 6 H), 2.92 – 3.03 (m, 2 H), 2.81 (t, J=7.92 Hz, 2 H); HRMS (ESI): m/z calcd for C31H28N3O3 [M+H]+ 490.2130, found 490.2133.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 8.17 (d, J=8.22 Hz, 1 H), 7.90 (d, J=8.80 Hz, 2 H), 7.55 (ddd, J=8.41, 6.94, 1.08 Hz, 1 H,) 7.37 – 7.49 (m, 2 H), 7.09 – 7.29 (m, 8 H), 6.82 (dd, J=8.41, 2.15 Hz, 1 H), 6.73 (d, J=8.41 Hz, 1 H), 5.90 (s, 1 H), 3.62 (s, 3 H), 3.57 (s, 3 H), 2.49 – 2.64 (m, 4 H), 1.90 – 2.01 (m, 2 H); HRMS (ESI): m/z calcd for C32H30N3O3 [M+H]+ 504.2287, found 504.2287.</p><!><p>To 3,4-dimethoxybenzaldehyde (100 mg, 0.602 mmol, 1.0 equiv) and malononitrile (0.038 ml, 0.602 mmol, 1.0 equiv) in ethanol (4 ml) was added 1-naphthol (87 mg, 0.602 mmol, 1.0 equiv) and piperidine (0.298 ml, 3.01 mmol, 5.0 equiv). The mixture was heated to 90 °C in a sealed vial for 18 h, Upon completion, The mixture was concentrated in vacuo and chromatographed with 0–40% ethyl acetate/hexanes gradient elution to give 2-amino-4-(3,4-dimethoxyphenyl)-4H-benzo[h]chromene-3-carbonitrile 11 (84 mg, 38.9 % yield) as a tan solid. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.16 (dd, J=8.22, 0.78 Hz, 1 H), 7.73 – 7.82 (m, 1 H), 7.46 – 7.62 (m, 3 H), 7.02 (d, J=8.41 Hz, 1 H), 6.74 – 6.83 (m, 2 H), 6.71 (d, J=1.96 Hz, 1 H), 4.82 (s, 1 H), 4.70 (s, 2 H), 3.76 – 3.86 (m, 6 H);</p><!><p>Compound 11 (84 mg, 0.234 mmol, 1.0 equiv) was taken up in triethyl orthoformate (1.5 mL) and heated to 155 °C for 48 h in a sealed tube. Upon completion, the mixture was concentrated in vacuo to give a yellow oil which was was taken up in THF (1.5 mL). To it was added 2-morpholinoethylamine (0.037 mL, 0.281 mmol, 1.2 equiv) followed by DBU (0.042 mL, 0.281 mmol, 1.2 equiv). The mixture was heated to 75 °C in a sealed tube for 24 h. Upon completion, the mixture was concentrated in vacuo and submitted for HPLC purification to give compound 12 as a tan solid (12 mg, 15%). 1H NMR (400 MHz, DMSO-d6) δ ppm 9.73 (br. s., 1 H), 8.34 (s, 1 H), 8.26 (d, J=7.83 Hz, 1 H), 7.89 (d, J=7.83 Hz, 1 H), 7.60 – 7.69 (m, 2 H), 7.53 – 7.60 (m, 1 H), 7.37 (d, J=8.61 Hz, 1 H), 7.08 (d, J=2.15 Hz, 1 H), 6.79 (d, J=8.41 Hz, 1 H), 6.65 (dd, J=8.31, 2.05 Hz, 1 H), 5.27 (s, 1 H), 3.73 – 3.84 (m, 4 H), 3.71 (s, 3 H), 3.59 – 3.65 (m, 3 H), 3.24 (br. s., 4 H), 3.00 (br. s., 2 H) 2.87 (br. s., 2 H); HRMS (ESI): m/z calcd for C29H31N4O4[M+H]+ 499.2345, found 499.2346.</p><!><p>To 3,4-dimethoxybenzaldehyde (200 mg, 1.204 mmol, 1.0 equiv) and malononitrile (0.076 ml, 1.204 mmol, 1.0 equiv) in ethanol (8 ml) was added 3-methoxyphenol or 3-(Benzyloxy)phenol (1.204 mmol, 1.0 equiv), and piperidine (0.596 ml, 6.02 mmol, 5.0 equiv). The mixture was heated in a sealed vial to 90 °C for 18 h. Upon completion, the mixture was concentrated in vacuo and chromatographed with 10% ethyl acetate/dichloromethane gradient elution to give compounds 13a and 13b as yellow solids with 16% and 20% yields respectively.</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 6.86 (d, J=8.61 Hz, 1 H), 6.76 – 6.82 (m, 1 H), 6.69 – 6.74 (m, 1 H), 6.66 (d, J=1.96 Hz, 1 H), 6.60 (dd, J=8.61, 2.54 Hz, 1 H), 6.52 (d, J=2.54 Hz, 1 H), 4.62 (s, 1 H), 4.53 (s, 2 H), 3.82 (d, J=8.41 Hz, 6 H), 3.77 (s, 3 H);</p><!><p>1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.30 – 7.42 (m, 5 H), 6.84 – 6.89 (m, 1 H), 6.77 – 6.81 (m, 1 H), 6.64 – 6.75 (m, 3 H), 6.59 (d, J=2.54 Hz, 1 H), 5.02 (s, 2 H), 4.62 (s, 1 H), 4.52 (s, 2 H), 3.80 – 3.86 (m, 6 H);</p><!><p>Compound 13a or 13b (0.201 mmol, 1.0 equiv) was taken up in triethyl orthoformate (2 mL) and heated to 150 °C in a sealed tube overnight. Upon completion, the mixture was concentrated in vacuo to give a yellow oil which was taken up in THF (2 ml). To it was added 2-morpholinoethylamine (0.032 ml, 0.243 mmol, 1.2 equiv) followed by DBU (0.037 ml, 0.243 mmol, 1.2 equiv). The mixture was heated to 75 °C in a sealed tube for 24 h. Upon completion, the mixture was concentrated in vacuo and submitted for HPLC purification to give compounds 14a and 14b as tan solids with yields of 15% and 20% respectively.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 9.54 (br. s., 1 H), 8.26 (s, 1 H), 7.17 (d, J=8.61 Hz, 1 H), 6.94 (d, J=2.15 Hz, 1 H), 6.74 – 6.81 (m, 2 H), 6.67 (dd, J=8.61, 2.54 Hz, 1 H), 6.57 (dd, J=8.41, 1.96 Hz, 1 H), 5.05 (s, 1 H), 3.70 (d, J=12.52 Hz, 6 H), 3.62 (s, 3 H), 3.20 (br. s., 8 H), 2.96 (br. s., 4 H); HRMS (ESI): m/z calcd for C26H32N4O5[M+H]+ 480.2372, found 480.2375.</p><!><p>1H NMR (400 MHz, DMSO-d6) δ ppm 8.63 (s, 1 H), 7.26 – 7.44 (m, 6 H), 7.23 (d, J=8.61 Hz, 1 H), 6.99 (dd, J=9.39, 2.15 Hz, 2 H), 6.85 (dd, J=8.51, 2.45 Hz, 1 H), 6.79 (d, J=8.22 Hz, 1 H), 6.62 (dd, J=8.31, 2.05 Hz, 1 H), 5.31 (s, 1 H), 5.10 (s, 2 H), 3.69 (s, 3 H), 3.63 (s, 3 H), 3.47 (br. s., 8 H), 2.95 (br. s., 4 H); HRMS (ESI): m/z calcd for C32H35N4O5[M+H]+ 555.2607, found 555.2611.</p><!><p>To 3,4-dimethoxybenzaldehyde (2 g, 12.04 mmol, 1.0 equiv) and malononitrile (0.758 ml, 12.04 mmol, 1.0 equiv) in ethanol (80 ml) was added 3-aminophenol (1.313 g, 12.04 mmol, 1.0 equiv), and piperidine (5.96 ml, 60.2 mmol, 5.0 equiv). The mixture was heated to reflux for 6 h. Upon completion, the mixture was concentrated in vacuo and taken up in methanol, after which a precipitate formed which was filtered and washed with cold methanol to compound 15 (1.967 g, 6.08 mmol, 50.5 % yield) as a tan solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 6.83 (dd, J=8.41, 2.74 Hz, 1 H), 6.65 – 6.74 (m, 3 H), 6.57 – 6.65 (m, 2 H), 6.23 (dt, J=8.41, 2.54 Hz, 1 H), 6.15 (t, J=2.45 Hz, 1 H), 5.17 (s, 2 H), 4.43 (d, J=2.15 Hz, 1 H), 3.67 (dd, J=4.60, 2.84 Hz, 6 H);</p><!><p>To compound 15 (417 mg, 1.290 mmol, 1.0 equiv) in acetonitrile (20 ml) at 0 °C was added tert-butyl nitrite (0.375 ml, 2.84 mmol, 2.2 equiv) followed by copper(II) bromide (634 mg, 2.84 mmol, 2.2 equiv). The mixture stirred at 0 °C for 1 h. Upon completion, the mixture was taken up in sat. ammonium chloride and ethyl acetate. The layers were separated and the aqueous layer was extracted with ethyl acetate. The combined organic extracts were then washed with water and brine, dried over Na2SO4, and concentrated in vacuo to give a black oil which was chromatographed with 0–20% ethylacetate/hexanes gradient elution to give 2,7-dibromo-4-(3,4-dimethoxyphenyl)-4H-chromene-3-carbonitrile (164 mg, 0.364 mmol, 28.2 % yield) as a yellow solid. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.27 (d, J=1.96 Hz, 1 H), 7.23 (d, J=1.96 Hz, 1 H), 7.21 (d, J=1.96 Hz, 1 H), 6.83 (dd, J=8.31, 6.36 Hz, 2 H), 6.68 – 6.73 (m, 1 H), 6.64 (d, J=2.15 Hz, 1 H), 4.72 (s, 1 H), 3.79 – 3.88 (m, 6 H).</p><!><p>2,7-dibromo-4-(3,4-dimethoxyphenyl)-4H-chromene-3-carbonitrile (164 mg, 0.364 mmol, 1.0 equiv) was taken up in ammonia in isopropanol (5 ml). The mixture was heated in the microwave at 150 °C for 1 h. Upon completion, the mixture was concentrated in vacuo and chromatographed with 2–7% methanol/dichloromethane gradient elution to give compound 16 (39 mg, 0.101 mmol, 27.7 % yield) as a yellow oil which was taken on quickly to the next reaction.</p><!><p>Compound 16 (155 mg, 0.400 mmol, 1.0 equiv) was taken up in triethyl orthoformate (4 mL) and heated to 155 °C in the microwave for 2 h. Upon completion, the mixture was concentrated in vacuo to give a yellow oil, which was taken up in THF (5 ml). To it was added 2-morpholinoethylamine (0.062 ml, 0.479 mmol, 1.2 equiv) followed by DBU (0.072 ml, 0.479 mmol, 1.2 equiv). The mixture was heated to 75 °C in the microwave for 1 h. Upon completion, the mixture was concentrated in vacuo and chromatographed with 2–7% methanol/dichloromethane gradient elution to give 8-bromo-5-(3,4-dimethoxyphenyl)-3-(2-morpholinoethyl)-3H-chromeno[2,3-d]pyrimidin-4(5H)-imine (11 mg, 0.021 mmol, 5.22 % yield) as a tan solid, which was taken up in dichloromethane (1 ml). To it was added palladium(II) chloride (0.740 mg, 4.17 µmol, 0.2 equiv) and triethylsilane (4.00 µl, 0.025 mmol, 1.2 equiv). The mixture stirred at r.t. for 24 h. Upon completion, the mixture was concentrated in vacuo and submitted for HPLC purification to give compound 17. 1H NMR (400 MHz, DMSO-d6) δ ppm 9.65 (br. s., 1 H), 8.27 (s, 1 H), 7.14 – 7.32 (m, 3 H), 7.01 – 7.13 (m, 2 H), 6.98 (d, J=2.15 Hz, 1 H), 6.78 (d, J=8.41 Hz, 1 H), 6.58 (dd, J=8.31, 2.05 Hz, 1 H), 5.15 (s, 1 H), 3.81 (br. s., 2 H), 3.66 – 3.72 (m, 3 H), 3.62 (s, 3 H), 3.58 (br. s., 2 H), 3.22 (br. s., 4 H), 2.96 (br. s., 4 H); HRMS (ESI): m/z calcd for C25H30N4O4[M+H]+ 450.2267, found 450.2272.</p><!><p>Synthesis of compound 18 was the same as compound 17, except 2.2 equivalents of triethylsilane was used. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.98 (br. s., 1 H), 8.23 (br. s., 2 H), 7.18 – 7.31 (m, 2 H), 7.11 – 7.18 (m, 2 H), 6.92 (s, 1 H), 6.74 – 6.88 (m, 2 H), 5.24 (s, 1 H), 4.87 (d, J=11.15 Hz, 1 H), 4.53 (dd, J=11.15, 2.74 Hz, 1 H), 3.80 (d, J=11.54 Hz, 2 H), 3.61 – 3.72 (m, 6 H), 3.51 – 3.61 (m, 2 H), 3.13 (d, J=0.78 Hz, 3 H), 2.75 – 3.08 (m, 4 H); HRMS (ESI): m/z calcd for C25H31N4O4[M+H]+ 451.2345, found 451.2345.</p><!><p>Previously described NPSR antagonists and structure of our lead compound.</p><p>Schematic illustration of the assay principle of the HTRF cAMP assay.</p><p>Molecular modeling overlap between 1 and 5.1 (green)</p><p>Analogues at positions 10 and 12. Reagents and conditions: (a) 2-naphthol, piperidine, EtOH, reflux; (b) CH(OEt)3, 155 °C; (c) R2NH2, DBU, THF, 75 °C</p><p>Analogues at positions 8 and 11. Reagents and conditions: (a) Formamide, 220 °C; (b) 7.a: Benzyl Bromide, MeCN, 100 °C; 7. b and 7.c: R3Br, DMF, 150 °C; (c) R4COCl, iPr2NEt.</p><p>Analogues at the 9 position. Reagents and conditions: (a) R5COCl, THF, Pyridine, MW, 120 °C; (b) POCl3, 120 °C; (c) NH4OH, THF, 130 °C.</p><p>Modification of the naphthyl ring. Reagents and conditions: (a) 1-naphthol, malononitrile, piperidine, EtOH, reflux; (b) CH(OEt)3, 155 °C; (c) 2-Morpholinoethylamine, DBU, THF, 75 °C.</p><p>Phenyl analogues at the naphthyl region. Reagents and conditions: (a) 3-methoxyphenol (or 3-benzyloxyphenol), malononitrile, piperidine, EtOH, reflux; (b) CH(OEt)3, 155 °C; (c) 2-Morpholinoethylamine, DBU, THF, 75 °C.</p><p>Additional phenyl analogues of the naphthyl ring. Reagents and conditions: (a) 3-aminophenol, malononitrile, piperidine, EtOH, reflux; (b) t-butyl nitrite, CuBr2, MeCN, 0 °C; (c) NH3, iPrOH, 150 °C; (d) CH(OEt)3, 155 °C; (e) 2-Morpholinoethylamine, DBU, THF, 75 °C; (f) PdCl2, Et3SiH, DCM.</p><p>Methoxy scan at the 12 position.</p><p>Analogues at the 12 position maintaining a phenyl ring at the 10 position.</p><p>Carbonyl analogues at the 11 position.</p><p>Aromatic scan at the 10 position.</p><p>Analogues with aromatic substitution at position 10</p><p>Analogues with modification at the 8 position.</p><p>Phenyl scan at the 9 position.</p><p>Modifications at the naphthyl ring.</p>