Synthesis and biological evaluation of H-8 analogues as PKN3 inhibitors

The protein kinase N3 (PKN3) represents one of the understudied kinases in the human kinome and therefore one of the kinases SGC-UNC wants to target. As part of the UNC-SOLAR program, a summer program for students, Guillermo Correa Otero was working with me in the lab for 9 weeks. Under the supervision of David Drewry, Guillermo’s project aimed at synthesizing small molecules that should explore the SAR around the PKN3 inhibitor H-8.

PKN3 is an N family kinase with potential as an oncology target. PKN3 has been shown to interact with a mediator of tumor invasion and metastasis, RhoC, and promote malignant cell growth in breast and prostate cancer.1 PKN3 acts downstream of phosphoinositide 3-kinase (PI3K) making it an attractive target for therapeutic intervention since its inhibition is less likely to cause negative side effects. During normal cell function PTEN (a tumor suppressor) regulates PI3K activation. When PTEN is aberrantly inactivated PI3K activity is not turned off which can lead to PKN3 upregulation, increased metastatic behavior, and malignant cell growth in prostate cancer.2

As PKN3 can act downstream of PI3K signaling, compounds that selectively inhibit PKN3 will provide valuable insights into its role in cancer biology and represent a promising treatment for cancer.3 A siRNA-based therapy that advanced to the clinic demonstrates the potential of PKN3 silencing. However, potential drawbacks connected to this siRNA approach include immune side effects, challenges in tissue-specific access, and requirements for multiple intravenous injections.4-7

One approach to mitigate these drawbacks would be the development of a small molecule PKN3 inhibitor. Although some potent PKN3 inhibitors (Figure 1) having been disclosed in the literature,structure activity relationships of these compounds and PKN3, to our knowledge, have not been explored.

Figure 1. PKN3 inhibitors described in literature.3

Strategy and compound design

The N family of kinases (PKN1-3) is one of 16 families that comprise the AGC branch on the kinome tree. PKN1-3 are serine/threonine kinases and show high amino acid similarity in the ATP binding pocket.The selection of a lead compound with selectivity for PKN3 will make it easier to delineate the biological roles PKN3 plays.

Isoquinoline sulfonamide H-8 (Figure 2), previously reported as a PKA/PKG8 inhibitor, shows a Kvalue of 10 nM for full-length human PKN3 in an off-chip mobility shift assay.Additionally, H-8 inhibits only 4/69 kinases > 80 % at 10 mM and is 180-fold selective for PKN3 over PKN1 and 2.3, 9 Therefore, H-8 represents a promising lead structure for the development of novel PKN3 selective inhibitors and is easy to obtain in a one-step synthesis for an undergraduate student. To explore structure activity relationships, we synthesized a set of compounds with isoquinoline scaffold. Specifically, we varied the sulfonamide group, the attachment position, and the sulfonamide substitution.

Figure 2. Lead compound H-8 and initial modifications.


Utilizing three different synthesis routes isoquinoline amides and sulfonamides were synthesized (Figure 3, A-C).

Figure 3. Synthesis of new isoquinolines as potential PKN3 inhibitors.

Route A: Acid 8 was reacted with amine 9, T3P and triethylamine (TEA)10 to form the amide bond present in 10. Boc-deprotection was performed under acidic conditions with trifluoroacetic acid (TFA)11 furnishing primary amine 11.

Route B: Boc-protection12 of amine 12 provided intermediate 13 which was further coupled with isoquinoline-5-amine (14) to afford test compound 1510. Boc-deprotection11 of 15 provided secondary amine 16.

Route C: Sulfonamides 19a(Table 1) were obtained by reacting isoquinoline-5-sulfonyl chloride (17) with different aliphatic amines (18af).13

Table 1. H-8 analogues with varied sulfonamide substituents.

Cell-based Potency Evaluation

In a PKN3 NanoBRET cellular target engagement assay established in our lab the inhibitory activity of literature compounds 16, and synthesized molecules 10, 11, 15, 16, and 19af towards PKN3 was measured. Synthesized compounds 10, 15, 16, 19d, 19e and 6 did not show activity up to a screening concentration of 10 mM in intact cells. 11, 19ac, and 19f were weakly active at 10 mM indicating that further chemical optimization needs to be performed to obtain potent PKN3 inhibitors. Some literature reported PKN3 inhibitors demonstrated potency in the PKN3 NanoBRET assay; PP1 showed the lowest IC50 value of 78 nM.

A possible strategy to continue this project could include merging the selective H-8 scaffold with structural parts of more potent literature reported PKN3 inhibitors. Furthermore, the measurement of additional literature compounds in the PKN3 NanoBRET assay will help to guide the decision regarding how to continue this project.

Finally, we want to thank Julie Pickett and Carrow Wells for testing the compounds in the PKN3 NanoBRET cellular target engagement assay.

Structure Image Smiles Mol. Weight
O=C(O)c1ccc2cnccc2c1 173.2

Download SDF file


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