Synthesis and kinase inhibitory potencies of new pyrido[3,4-g ]quinazolines substituted at the 8-position

As part of the structure-activity relationship study undertaken around the pyrido[3,4-g ]quinazoline moiety, new derivatives substituted at the 8-position were synthesized and evaluated regarding their ability to inhibit various protein kinases (CDK5, CLK1, DYRK1A, CK1, GSK3). Most active compound exhibited a nanomolar potency toward CLK1, demonstrating that substitution at 8-position is compatible with CLK1 inhibition.


Introduction
A few years ago, as part of our program dedicated to the identification of new heteroaromatic compounds with kinase inhibitory potencies, we designed and synthesized a new pyrido [3,4-g]quinazoline series for the inhibition of CDC-like kinase 1 (CLK1)/Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A). 1he structure-activity relationship (SAR) studies undertaken around this new series (Figure 1) showed that the kinase inhibition profile was highly dependent on the scaffold substitution.2][3][4] To complete this SAR study, we decided to focus our interest on the 8-position of the pyridoquinazoline scaffold (Figure 1), evaluating the impact of this structural modification on the biological activities.As previously reported, [1][2][3][4] the synthetic pathway was based on the preparation of a tetrasubstituted benzene derivative A, with substituents at the 1-and 2-positions used to construct the isoquinoline moiety (after Sonogashira cross-coupling with TMS-acetylene and subsequent cyclization in the presence of ammonia), while those at the 4-and 5-positions allowed the formation of the aminopyrimidine moiety (after oxidation, nitration and condensation with diversely substituted guanidine/amidine derivatives) (Scheme 1).Scheme 1. Synthetic strategy used to prepare the pyrido [3,4-g]quinazoline tricyclic scaffold.
To generate a new compound library, the functionalization of the 8-position was achieved via a similar approach using, in the Sonogashira coupling step, various alkynes bearing functional groups that could allow post-modifications after the formation of the tricyclic system.

Results and Discussion
Our first idea to prepare pyrido [3,4-g]quinazolines bearing aminoalkyl side chains at the 8-position was the use, in the Sonogashira coupling step, of a but-1-yne derivative bearing at the 4-position a phthalimide protected amine precursor.Therefore compound A was reacted with 2-(but-3-yn-1-yl)isoindoline-1,3-dione 5 in the presence of PdCl 2 (PPh 3 ) 2 , CuI and Et 3 N in DMF to afford 1 in 73% yield. 6Compound 1 was then cyclized to the corresponding isoquinoline 2 in the presence of ammonia in methanol 7 before primary alcohol oxidation with MnO 2 8 , leading to chloroaldehyde 3 (Scheme 2).The next step was the introduction of a nitro group at the 5-position of the isoquinoline moiety in order to allow subsequent formation of the aminopyrimidine moiety.Unfortunately, the nitro derivative was never obtained due to a preferred nitration of the phthalimide moiety.
Thus, we decided to use an azido group as amine precursor.The synthesis of the corresponding isoquinolines 5/6 was performed via the same synthetic sequence using 4-azidobut-1-yne 9 as alkyne partner in the first step (Scheme 2).Regrettably, compound 6 was unstable under the nitration conditions used.Formation of the aminopyrimidine ring directly from 3 or 6 in the presence of guanidine carbonate, without activation of the isoquinoline by an electron-withdrawing group, was unsuccessful.
Because of these failures, the preparation of another intermediate, an isoquinoline bearing at the 3position an ethyl side chain bearing a mesylate terminal group, likely to undergo a nucleophilic displacement with amines, was considered.Therefore, compound A primary alcohol was protected as a THP group before Sonogashira coupling in the presence of but-3-yn-1-ol leading to 8 that was cyclized to the corresponding isoquinoline 9 (Scheme 3).Finally, chloroaldehyde 12 was prepared by mesylation of the primary alcohol, cleavage of the THP group under mild acidic conditions, and oxidation.In this case, the nitration reaction led to the attempted product 13 in 77% yield.In the last step, the formation of the aminopyrimidine moiety performed in the presence of guanidine carbonate, led to tricyclic compound 14.Basic conditions used also led to an elimination reaction (Scheme 3).Due to low solubility of 14, all further double bond transformations assayed (hydroboration, hydroamination, oxidative cleavage) were unsuccessful.Scheme 3. Mesylate strategy: synthesis of compound 14.
Another strategy was to introduce a side chain bearing a nitrile group, that could be reduced to the corresponding amino analogue or hydrolyzed to give the corresponding amide.Therefore, compound A was reacted with pent-4-ynenitrile under Sonogashira coupling conditions to give 15 that was cyclized to the corresponding isoquinoline 16 in the presence of ammonia in methanol under microwave irradiation before primary alcohol oxidation leading to chloroaldehyde 17 (Scheme 4).Again, next step was the introduction of a nitro group at the 5-position of the isoquinoline moiety.Despite numerous efforts using various nitration conditions (HNO 3 , Ac 2 O, AcOH 10 or KNO 3 , H 2 SO 4 /TFA 11 or AgNO 3 , NBS 12  Tricyclic compounds 14, 19 and 20 were then evaluated toward a small panel of protein kinases (CDK5, CLK1, DYRK1A, CK1 and GSK3) using similar procedures as previously described. 4,18As shown in IC 50 values were determined when the residual kinase activity was ≤ 35% at a compound concentration of 1 M.Kinase activities were assayed in triplicate.Typically, the standard deviation of single data points was below 10%.Assays for 19 and 20 were performed using a 32 P radioassay in the presence of 15 M ATP while 14 testing was carried out using the ADP-Glo assay in the presence of 10 M ATP.

Conclusions
In conclusion, various synthetic strategies were carried out to introduce a functionalized side chain at the 8position of pyrido [3,4-g]quinazoline moiety.The use of the mesylate approach led to vinyl derivative 14 while the nitrile approach allowed the preparation of propionamides 19 and 20.The evaluation of these new diversely substituted compounds toward five protein kinases showed that compounds 14 and 20 were potent inhibitors of CLK1 with sub-micromolar/nanomolar potencies.Altogether, the results obtained demonstrated that the substitution at the 8-position of this tricyclic heteroaromatic scaffold is compatible with potent kinase inhibition.

Figure 1 .
Figure 1.Structural modifications performed in the pyrido[3,4-g]quinazoline series as part of our structureactivity relationship study.

Table 1 , most active compounds were 14 and 20 with interesting IC 50 values of 111 nM and 92 nM, respectively, toward CLK1. Table 1. Kinase inhibition assays (% residual kinase activity)
M 10 M 1 M 10 M 1 M 10 M 1 M 10 M 1 M