6-(Arylaminomethyl) Isoquinolines as Enzyme Inhibitors and Their Preparation: A Patent Highlight of Factor XIIa Inhibitors

Graphical Abstract

2. DESCRIPTION OF THE INVENTION

The molecules of this invention are inhibitors of factor Xlla (FXIIa), and, therefore, possess several potential therapeutic uses in which FXlla is implicated. FXIIa is a serine protease that is formed from its zymogen precursor factor XII (FXII). Single-chain FXII has limited proteolytic activity which is enhanced upon interaction with negative surfaces [1]. Proteolytic cleavage of FXII to FXIIa’s light and heavy chains considerably increases its catalytic activity. FXIIa exists in two forms: αFXIIa with a full heavy chain and βFXIla with a small fragment of the heavy chain.

Structurally, FXIIa is different from many other serine proteases. For example, Tyr99 residue partially blocks the S2 pocket in the active site of FXIIa. Other serine proteases containing this Tyr99 residue, for example, tissue plasminogen activator (tPA), factor Xa (FXa), and factor IXa (FIXa), have an open S2 pocket. Furthermore, FXIIa possesses an incomplete “aromatic box” leading to a more open S4 pocket. Nevertheless, many trypsin-like serine proteases have their S4 pocket lined by an “aromatic box”, which contributes to their inhibitors’ P4-driven selectivity and activity [2–5].

FXII, in addition to high molecular weight kininogen and plasma pre-kallikrein (PK), constitutes the contact system. Several mechanisms activate this system. These include interactions with negatively charged surfaces and molecules, artificial surfaces, unfolded proteins, and foreign tissues (biological transplants such as bacteria, organ/tissue transplants, bio-prosthetic heart valves, and biological surfaces including extracellular matrix and endothelium). Additionally, the system can be activated by plasmin. Activation of the contact system results in the activation of the complement system, the kallikrein-kinin system (KKS), and the intrinsic coagulation pathway. FXIIa converts PK to plasma kallikrein (PKa), which positively feedbacks the activation of FXII to FXIla. In addition, FXIIa has several other direct and indirect substrates, including plasminogen, proteinase-activated receptors (PARs), and neuropeptide Y (NPY), which can contribute to the biological activity of FXIla. Thus, FXIIa inhibition provides therapeutic benefits by treating illnesses about these systems.

PAR2 activation by PKa precipitates neuroinflammation and may lead to neuro-inflammatory disorders such as multiple sclerosis [6]. PKa-mediated activation of PAR1 and PAR2 on vascular smooth muscle cells has also been implicated in atherosclerosis and vascular hypertrophy [7]. FXIIa-mediated activation of plasminogen contributes to fibrinolysis [8]. PKa proteolytically cleaves NPY, thus altering its binding to its receptors [9]. Inhibition of FXIIa could provide clinical benefits in diseases caused by PAR signaling, plasminogen activation, and NPY metabolism. Activation of KKS by FXIIa leads to the production of bradykinin (BK), which can mediate pain, inflammation, angioedema, vasodilation, and vascular hyperpermeability [10, 11].

CSL-312, a FXIIa antibody inhibitory, is currently in clinical trials for the treatment and prevention of normal Cl inhibitor as well as Cl inhibitor deficient hereditary angioedema (HAE), which results in intermittent swelling of face, throat, hands, gastrointestinal tract, and genitals [12]. Mutations in FXII that promote its activation to FXIIa were identified as a cause of HAE [13, 14]. Because FXIIa facilitates the generation of PKa, FXIIa inhibitors could provide protective effects against all forms of BK-mediated angioedema, including HAE and nonhereditary bradykinin-mediated angioedema (BK-nHAE) [15–18]. Specific types of BK-nHAE include nonhereditary angioedema with normal Cl Inhibitor (AE-nCl lnh), which can be drug-induced, hormonal, or environmental.

Environmental factors that cause AE-nCl Inh include air pollution [19] and silver nanoparticles, such as the nanoparticles used as antibacterial components in healthcare, biomedical, and consumer products [20]. Studies suggested a link between the contact system, bradykinin pathways, and BK-nHAE [21–24]. For example, BK-medicated angioedema can be attributed to thrombolytic therapy. Furthermore, angioedema that is induced by t-PA is a life-threatening complication following thrombolytic therapy in acute stroke victims [25–29]. It was also reported that certain drugs could cause angioedema [30, 31]. Hermanrud et al. reported recurrent angioedema associated with dipeptidyl peptidase IV inhibitors and also discussed acquired angioedema induced by angiotensin-converting enzyme inhibitors (ACEI) [32]. Kim et al. [33] reported angiotensin Il receptor blocker (ARB)-related angioedema. Reichman et al. also reported angioedema risk for patients taking ARBs, ACEIs, and β-blockers [34]. Diestro et al. also reported a possible association between certain ARBs and angioedemas [35]. Giard et al. reported that bradykinin-mediated angioedema can be precipitated by estrogen contraception, called “oestrogen associated angioedema” [36].

Activation of KKS by the contact system has also been linked to diabetic retinopathy and retinal edema [37]. FXIIa concentrations are elevated in the vitreous fluid of patients with diabetic macular edema (DME) or advanced diabetic retinopathy [38, 39]. FXIIa appears to be a mediating factor in both vascular endothelial growth factor (VEGF)-independent [40, 41] and -dependent DME [40]. FXII deficiency is protective against VEGF-induced retinal edema in mice. Thus, it has been proposed that FXIIa inhibition can provide some benefits regarding diabetic retinopathy and retinal edema caused by retinal vascular hyperpermeability, including DME, age-related macular degeneration (AMD), and retinal vein occlusion. The contact system can be activated by bacteria, and thus, FXIIa has been implicated in the treatment of bacterial sepsis [42]. Overall, FXIIa inhibitors potentially carry therapeutic benefits in treating bacterial sepsis, sepsis, and disseminated intravascular coagulation (DIC).

Activation of KKS and BK production, which are mediated by FXIIa, has been implicated in neurodegenerative diseases including epilepsy, multiple sclerosis, Alzheimer’s disease, and migraine [43–46]. Therefore, FXIIa inhibitors can provide therapeutic benefits in decreasing the clinical symptoms and the progression of the above neurodegenerative diseases. FXIIa has also been implicated in anaphylaxis; therefore, its inhibitors can be therapeutically beneficial in mitigating the incidence and clinical severity of anaphylactic reactions [47, 48].

Plasma FXIIa’s role in the coagulation process was identified more than 50 years ago [49]. Activation of factor Xl (FXI) by FXIIa stimulates the intrinsic coagulation pathway. Furthermore, FXIIa can promote coagulation in an FXI-independent fashion [50, 51]. Studies on both experimental animal models and humans have exhibited that FXII deficiency prolongs activated partial prothrombin time (APTT) without increasing bleeding [52, 53]. Pharmacological inhibition of FXIIa also prolongs APTT without adversely affecting hemostasis [54]. These data suggest that FXIIa inhibition can prevent and/or treat thrombosis without causing bleeding. FXIIa inhibitors have been proposed to treat several prothrombotic conditions, including deep vein thrombosis, cancer-associated thrombosis, pulmonary embolism, complications caused by artificial heart valves, extracorporeal membrane oxygenation, catheters, left ventricular assisted devices, cardiopulmonary bypass, dialysis, joint arthroplasty, and sickle cell disease as well as tPA-induced thrombosis, Budd-Chari syndrome, and Paget-Schroetter syndrome. FXIIa inhibitors may also be useful for treating or preventing thromboembolism by decreasing the tendency of devices that come in contact with blood to clot. Examples of such devices are stents, in-dwelling and external catheters, vascular grafts, extracorporeal circulation systems, and orthopedic and cardiac prostheses. Preclinical studies showed that FXIIa contributes to stroke and its complications [55–59]. Furthermore, FXII deficiency decreased the formation of atherosclerotic lesions in ApoE−/− mice [60]. As a result, FXIIa inhibition has been proposed to improve clinical neurological outcomes of the treatment of patients with stroke or atherosclerosis.

Several FXIIa inhibitors have been described before [61–66]. Nevertheless, there remains a need to develop new FXIIa inhibitors that will have utility to treat a wide range of disorders. The patent in this highlight claimed 6- (arylaminomethyl) isoquinolines as a new class of FXIIa inhibitors. Fig. (1) provides specific examples.

Fig. (1).

Key derivatives of 6-(Arylaminomethyl) Isoquinolines are claimed as FXIIa inhibitors in this patent.

3. DEFINITIONS

More details are in the patent.

1. W, X, Y, and Z can be C and N such that the ring containing W, X, Y, and Z can be benzene, pyridine, pyrimidine, pyridazine, triazine, and pyrazine

2. R₁, R₄, and R₅ can be absent or selected from H, alkoxy, alkyl, -OH, CF₃, halo, -CN, -COOR₁₂, and -CONR₁₄R₁₅

3. when X is C, one of R₂ and R₃ is -L-V-R₁₃, and the other of R₂ and R₃ can be selected from H, alkoxy, alkyl, -OH, CF₃, halo, -CN, -COOR₁₂, and -CONR₁₄R₁₅

4. when X is N, R₂ is -L-V-R₁₃, and R₃ is absent

5. R₆-₁₀ can be selected from H, alkoxy, alkyl, -OH, CF₃, halo, -CN, -COOR₁₂, and -CONR₁₄R₁₅

6. L is selected from a bond, -C(O)-, and alkylene

7. V is absent or selected from NR₁₂ and O

8. R₁₂ can be selected from alkyl and H

9. R₁₃ is (CH₂)_0-3(heterocyclyl)

10. alkyl is a linear saturated hydrocarbon having up to 4 carbon atoms (C₁-C₄) or a branched saturated hydrocarbon of 3 or 4 carbon atoms (C₃-C₄); alkyl can be substituted with 1 or 2 substituents selected from (C₁-C₃) alkoxy, -CN, halo, -OH, -NR₁₄R₁₅, -NHCOCH₃, -COOR₁₂, and- CONR₁₄R₁₅

11. alkyl is a linear saturated hydrocarbon having up to 4 carbon atoms (C₁-C₄) or a branched saturated hydrocarbon of 3 or 4 carbon atoms (C₃-C₄); alkyl can be substituted with 1 or 2 substituents selected from -CN, -OH, halo, and -NHCOCH₃

12. alkylene is a bivalent linear saturated hydrocarbon having 1 to 4 carbon atoms (C₁-C₄) or a branched bivalent saturated hydrocarbon having 3 to 4 carbon atoms (C₃-C₄)

13. alkoxy is a linear O-Linked hydrocarbon of between 1 and 3 carbon atoms (C₁-C₃) or a branched O-Linked hydrocarbon of between 3 and 4 carbon atoms (C₃-C₄); alkoxy may optionally be substituted with 1 or 2 substituents independently selected from -OH, F, -CF₃, -CN, and N(R₁₂)₂

14. halo is I, Br, Cl, or F

15. heterocyclyl is a 4-, 5-, or 6-, membered carbon-containing nonaromatic ring containing one or two ring members that are selected from N, NR₁₆, and O; the heterocyclyl can be substituted with 1, 2, 3, or 4 substituents selected from oxo, alkyl, alkoxy, -OH, -CF₃, halo, -CN, -COOR₁₂, and -CONR₁₄R₁₅

16. R₁₄ and R₁₅ can be selected from alkyl and H

17. R₁₆ is selected from alkyl and H

4. BIOLOGICAL ASSAY

FXIIa inhibitory activity in vitro was determined using standard published methods [67–69]. Human FXIIa was incubated at 25°C with a fluorogenic substrate and several concentrations of the test molecule. Residual enzyme activity was determined by measuring the change in absorbance at λ₄₁₀ nm, and the IC₅₀ value for the test molecule was determined. Selectivity against factor XIa was determined similarly.

5. BIOLOGICAL DATA

The potency of the inhibitors against both factor XIIa and factor XIa was provided as IC₅₀ values. Table 1 shows representative examples:

Table 1.

Inhibition profiles of key structures against human FXIIa.

Inhibitor	FXIIa IC50 (nM)	FXIa IC50 (nM)
18.04	< 1,000	> 40,000
18.12	< 1,000	33,1000
18.01	1,000-3,000	36,400
18.210	1,000-3,000	NA
18.13	3,000-10,000	NA
18.213	3,000-10,000	NA
18.02	10,000-40,000	NA
18.208	10,000-40,000	NA

CONCLUSION

FXIIa is being targeted for developing effective anticoagulants that are not associated with bleeding risk and many other diseases. The preparation of FXIIa inhibitors of 6-(arylaminomethyl) isoquinolines derivatives has been described. Key inhibitors were evaluated for their ability to inhibit FXIIa. The claimed inhibitors’ potential and pharmacokinetics are yet to be determined in appropriate animal models.

LIST OF ABBREVIATIONS

ACEI

Angiotensin-Converting Enzyme Inhibitors

AMD

Age-Related Macular Degeneration

ARB

Angiotensin Il Receptor Blocker

BK

Bradykinin

DIC

Disseminated Intravascular Coagulation

DME

Diabetic Macular Edema

HAE

Hereditary Angioedema

KKS

Kallikrein Kinin System

NPY

Neuropeptide Y

PARs

Proteinase-Activated Receptors

tPA

Tissue Plasminogen Activator

VEGF

Vascular Endothelial Growth Factor