Synthesis and SAR Studies of 1H-Pyrrolo[2,3-b]pyridine-2-carboxamides as Phosphodiesterase 4B (PDE4B) Inhibitors

Abstract

Herein we report the synthesis, SAR, and biological evaluation of a series of 1H-pyrrolo[2,3-b]pyridine-2-carboxamide derivatives as selective and potent PDE4B inhibitors. Compound 11h is a PDE4B preferring inhibitor and exhibited acceptable in vitro ADME and significantly inhibited TNF-α release from macrophages exposed to pro-inflammatory stimuli (i.e., lipopolysaccharide and the synthetic bacterial lipopeptide Pam3Cys). In addition, 11h was selective against a panel of CNS receptors and represents an excellent lead for further optimization and preclinical testing in the setting of CNS diseases.

Phosphodiesterases (PDEs) are a family of enzymes that catalyze the hydrolysis of the secondary signal messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Both cAMP and cGMP are essential components for cell signaling and are known to regulate the function of airway smooth muscle, inflammatory cells, and immune cells.^1,2 PDEs in mammals have been characterized into at least 11 different families of isozymes (PDE1–PDE11) that differ in their selectivity and specificity for the hydrolysis of cAMP and cGMP.³ Among them PDE4, PDE7, and PDE8 are cAMP specific while PDE5, PDE6, and PDE9 are cGMP specific.⁴ PDE4 is expressed predominantly in inflammatory and immune cells including lymphocytes, macrophages, eosinophils, and neutrophils where it tightly regulates the intracellular concentrations of cAMP.² The critical role of cAMP in regulating inflammatory and autoimmune processes makes PDE4 an attractive target for treating inflammatory diseases such as chronic obstructive pulmonary disorder (COPD), asthma, rheumatoid arthritis, and psoriasis.²

PDE4 exists in four known isoforms (PDE4A–D), among which PDE4A, B, and D are widely expressed in the brain.⁵ Inhibition of PDE4 has been shown to have anti-inflammatory and antipsychotic effects in various preclinical models.² In addition to inflammatory processes, PDE4B has been implicated in a number of other therapeutic roles, such as cancer,⁶ Alzheimer’s disease,⁵ addiction,⁷⁻⁹ and Parkinson’s disease.¹⁰ The pan-PDE4 inhibitors such as roflumilast and apremilast have been approved for the treatment of COPD and psoriatic arthritis. Rolipram, another nonselective PDE4 inhibitor, was developed as a potential treatment for depression, but its clinical use was abandoned due to a narrow therapeutic index.¹¹ However, these drugs have been hampered by dose limiting gastrointestinal side effects, such as nausea and emesis. These side effects are hypothesized to be partially associated with the inhibition of PDE4D isoform.¹² Hence there is an urgent need for developing novel chemical scaffolds that are brain penetrant and have preferential PDE4B binding. In this paper, we describe the synthesis, docking studies, and pharmacological and biological profile of a series of 1H-pyrrolo[2,3-b]pyridine-2-carboxamide derivatives as selective and potent PDE4B inhibitors.

We first wanted to evaluate the 5,6-core structure of the previously reported PDE4B inhibitor, 1 (Figure 1) (PDE4B IC₅₀ = 0.8 μM).¹³ All of the synthesized compounds were initially evaluated for their in vitro inhibitory activity against PDE4B using rolipram as a positive control. Table 1 comprises the in vitro results of inhibitory activity against PDE4B for the title compounds under study, expressed as IC₅₀ values and (%) inhibition in parentheses. A number of variants were synthesized and evaluated in an enzymatic PDE4B and PDE4D assay (BPS Biosciences, San Diego, CA) for activity. Our aim of this initial SAR evaluation was to systematically evaluate the nitrogen atom and their placement within the 5,6-framework in order to get a better understanding of the potency contributions of these atoms. The first compound, pyrazolo[1,5-a]pyridine (2), showed some activity against PDE4B; however, there was an ∼5-fold loss of potency compared to 1 (2, IC₅₀ = 4.2 μM). Replacing the imidazole moiety with a thiophene (3, thieno[2,3-b]pyrazine) led to an inactive compound. Removing the 6-membered ring nitrogen to afford the 1H-benzo[d]imdazole 4 led to an ∼3-fold loss of activity (IC₅₀ = ∼ 2.4 μM), and further deletion of the imidazole nitrogen leaving the indole 5 led to an inactive compound. Interestingly, moving to the pyrrolo[2,3-b]pyridine, 7, led to an increase in potency (PDE4B IC₅₀ = 0.48 μM). Lastly, the N-methylated pyrrolo[3,2-b]pyridine, 6, was not active. This is an interesting result as compounds such as this with a further cyclic ring between the pyrrole nitrogen and the amide have been reported as potent PDE4B inhibitors.

Figure 1.

Modifications of known PDE4B inhibitor 1, to new compound 11h.

Table 1. Identification of the Initial Lead Compound (1–7).

^aPDE4 assays run at BPS Biosciences, San Diego, CA, n = 2.

Having identified the pyrrolo[2,3-b]pyridine, 7, as a productive replacement, we further elaborated this ring system in order to fully vet this ring system. The analogs in Tables 2 and 3 were synthesized from common intermediates as shown in Scheme 1. The final compounds 11a–o were synthesized starting from the common intermediate 8, followed by Chan–Lam coupling to yield 9.¹⁴ Next, saponification of the ester led to the acid, 10, which was then reacted with a variety of amines under standard coupling conditions (T3P, DIPEA) to yield the final compounds 11a–o.¹⁵ The second set of targets, 14a–w, were synthesized in a similar fashion, but with a reversal of the steps. Initial saponification of the ester in 8, followed by amide coupling (T3P, DIPEA), led to the penultimate compound 13. Final targets, 14a–w, were isolated after Chan–Lam coupling (Cu(OAc)₂, pyridine).¹⁴

Table 2. SAR with Variations at the R₁ Group, 11a–o.

^aPDE4 assays run at BPS Biosciences, San Diego, CA, n = 2.

Table 3. SAR with Variations at Both R and R₁ Group (14a–w).

^aPDE4 assays run at BPS Biosciences, San Diego, CA, n = 2.

Scheme 1. Synthesis of the Final Target Compounds.

Reagents and conditions: (a) Cu(OAc)₂, ArB(OH)₂, pyridine, CH₂Cl₂, rt, 12 h; (b) NaOH, MeOH·H₂O; (c) T3P, NHR₁R₂, DIPEA, DMF, rt, 30 min to 4 h.

In Table 2, we explored the substitution effects of the amide portion of the 1H-pyrrolo[2,3-b]pyridine series of compounds by holding the 3,4-dichlorophenyl northern portion constant. It can be seen from the IC₅₀ values that most of the 1H-pyrrolo[2,3-b]pyridine derivatives showed moderate to good inhibition against PDE4B (IC₅₀ ranges from 0.11–1.1 μM, % inhibition of 99–20% at 10 μM). Careful analysis of the SAR (entries 11a–o) reveals the importance of ring size and extent of hydrophobicity on the overall activity and selectivity against PDE4B over PDE4D. Our first analogs explored secondary amides with small aliphatic cyclic or methyl furan groups. The cyclopropyl, 11a, and 3-fluorocyclobutane, 11c, both retained activity against PDE4B (IC₅₀ = 0.63 and 0.6 μM, respectively) and were selective for PDE4D (<50% inhibition at 10 μM, the term selective is applied to compound <50% inhibition). The cyclobutyl, 11b, difluorocyclobutyl, 11d, as well as the methylfuran, 11e, were all much less active or inactive. Moving to a tertiary amide (cyclic amide) was a more productive substitution pattern. By replacing the NH-cyclobutyl in 11b with the direct azetidine, 11f, an ∼10-fold increase in activity against PDE4B (IC₅₀ = 0.11 μM) was observed. This increase in PDE4B activity was also accompanied by an increase in PDE4D activity (IC₅₀ = 0.45 μM). Addition of a single fluorine (3-fluoroazetidine, 11g) or a difluorine (3,3-difluoroazetidine, 11h) also produced potent compounds with varying degrees of selectivity against PDE4D. The monofluoro, 11g, showed some selectivity against PDE4D (PDE4B IC₅₀ = 0.23 μM and 77% inhibition of PDE4D at 10 μM), whereas the difluoroazetidine was ∼6-fold selective for PDE4B (PDE4B IC₅₀ = 0.14 μM vs PDE4D IC₅₀ = 0.88 μM). Elongation of the tertiary amide by incorporation of a 3-trifluoromethyl group also produced an active compound (11i, PDE4B IC₅₀ = 0.43 μM), but with only an ∼2-fold selectivity versus PDE4D. Further elaboration of the amide space with a series of spirocyclic rings (entries 11j–m) only exhibited moderate to weak inhibition against PDE4B. Introduction of a basic nitrogen in the spirocyclic ring (entries 11n–o) lead to a significant loss of activity against PDE4B and PDE4D. This may be due to lower lipophilic binding compared to 11j and unfavorable interactions due to steric hindrance in the catalytic site of the enzyme. In this series, we were able to identify a novel scaffold 11h bearing a 3,3-difluoroazetidine ring, which exhibited higher inhibitory activity (0.14 μM) and 6-fold selectivity over PDE4D than its corresponding analogs. This compound was comparable in activity to the standard compound, rolipram, and was more selective versus PDE4D.

Based on the above results, we further evaluated a combination of aryl and amide substituents on the 1H-pyrrolo[2,3-b]pyridine ring system to develop more potent and selective PDE4B inhibitors. The aryl groups were chosen based on the activity in the originally reported series.¹³ In entries 14a–c, when R was 3-chloro-4-methylphenyl, increasing the size of the amide led to a decrease in activity by ∼2–20 fold, similar to that seen previously. However, moving to the 3-fluoroazetidine, 14d, did not produce the same improvement in potency as above, but this seemed to be an anomaly. When evaluating other R groups (4-chloro-3,5-difluorophenyl, 14f–i, 4-cyano-3,5-difluorophenyl, 14j–o, 4-chloro-3-fluorophenyl, 14s–v), PDE4B activity was improved by using the 3-fluoroazetidine tertiary amide. In order to fully investigate the effect of R substitution on the core ring, we explored a series of heteroaryl groups while keeping R₁(3-fluoroazetidine) constant as shown in entries 14w–z,bb, Table 3. These heteroaryl substituents indicate that substituted pyridyl (entries 14x and 14z) retains activity against PDE4B (IC₅₀ = 0.49 and 0.98 μM, respectively); although the activity is less than that of 11h. Interestingly, the 3-fluoro-4-methoxyphenyl group, 14y, lost activity against PDE4B; however, the activity could be regained by the addition of an additional fluorine (3,5-difluoro-4-methoxyphenyl, 14bb, IC₅₀ = 0.41 μM). This compound also showed ∼15-fold selectivity versus PDE4D. To our surprise, the 3,3-difluoroazetidine moiety (i.e., 11h) that provided the best compound previously, was not a productive moiety in this new group of compounds (e.g., 14e and others). Lastly, a methylene spacer was introduced between the phenyl group and the pyrrole (benzyl), and this change led to an inactive compound (14w).

In order to gain an understanding of the structural basis for the SAR observed, we also studied the binding of the synthesized inhibitors through computational modeling. The 3D structure of the PDE4B was taken from a Protein Data Bank entry (PDB: 4X0F) having the ligand rolipram in place. Docking studies were performed using Maestro 11.0 (Schrödinger, Small-Molecule Drug Discovery Suite 2018-3) software using PDE4B in complex with (R)-(−)-rolipram as the starting point. The optimized ligands were docked using Glide software in Maestro 11.0. The resulting structures were then analyzed using pose viewer to identify specific contacts between ligand and receptors. As shown in Figure 2 (top), the primary SAR study and docking results suggested that the entry 11h core ring shows π–π stacking interactions with residues Phe618 and Tyr405. The azetidine ring extends into the catalytic domain to block the access of cAMP thereby laying the foundation for PDE4B inhibition. The docking results were also consistent with the observed SAR studies. Smaller groups like cyclopropyl/difluoroazetidine occupy the smaller pocket in the catalytic domain while larger spirocyclic groups at this position lead to steric hindrance and therefore unfavorable interactions leading to significant loss of PDE4B inhibitory activity.

Figure 2.

Predicted binding mode of pyrrolo[2,3-b]pyridine inhibitor 11h in the active site of PDE4B structure (PDB code 4X0F).

Since PDE4 inhibitors have well-documented anti-inflammatory properties,² we evaluated the ability of these compounds to inhibit macrophage pro-inflammatory activity by measuring the production of the classical pro-inflammatory cytokine TNF-α (Figure 3). Two well-characterized pro-inflammatory stimuli were tested, namely lipopolysaccharide (LPS) and a synthetic lipopeptide (Pam3Cys) that represent prototypical bacterial ligands for Toll-like receptors 4 and 2, respectively (TLR4 and TLR2).¹⁶ All of the test compounds inhibited TNF-α production by mouse bone marrow-derived macrophages to the same extent or better than the well-characterized PDE4 inhibitor rolipram (Figure 3). A dose-dependent response was observed with regard to LPS-induced TNF-α release, where higher concentrations were less effective across the group of compounds (Figure 3A), a phenomenon which has been reported with other PDE4 inhibitors.¹⁷ A similar dose–response was observed with some compounds when macrophages were stimulated with Pam3Cys (Figure 3B); nevertheless, cytokine production was significantly reduced across the compound class. None of the compounds elicited TNF-α production when tested alone (data not shown). It is noted that the compounds are all equally active in this assay, and this is an effect we are currently investigating. Collectively, these findings support the biological action of the target compounds, which model the anti-inflammatory actions of the well-characterized PDE4 inhibitor rolipram.¹⁸

Figure 3.

Target compounds inhibit macrophage pro-inflammatory activity. Bone marrow-derived macrophages were unstimulated (−) or pretreated with the indicated concentrations of the well-characterized PDE4 inhibitor rolipram or target compounds (1 or 10 μM) for 30 min prior to LPS (100 ng/mL; A) or Pam3Cys (10 μg/mL; B) stimulation. After a 24 h treatment period, conditioned medium was collected to quantitate TNF-α expression by ELISA. Results are reported as the mean values (±SD) from 3 independent replicates for each treatment and were repeated in two separate experiments. *, p < 0.05; **, p < 0.01; #, p < 0.0001; One-way ANOVA with Dunnett’s multiple comparisons posthoc analysis.

Finally, having identified new PDE4 inhibitors, we next evaluated select compounds in a battery of Tier 1 in vitro DMPK assays (Table 4). A number of compounds evaluated displayed low (Q_H < 25%) clearance in both human and mouse microsomes (14x, 14z, 14bb), and several other compounds displayed moderate clearance (14i, 14j) in microsomes (Q_H < 50%). In addition to showing low clearance, both pyridine containing compounds (14x, 14z) displayed an excellent free fraction profile. In fact, most of the compounds evaluated had free fraction >3% in both species, with the exceptions of 11a and 14a in mice plasma (<1%).

Table 4. In Vitro DMPK of Selected Compounds.

	Intrinsic Clearance (mL/min/kg)a,b
	Human		Mouse		Plasma Protein Binding (%fu)a,c
Cmpd	CLINT	CLHEP	CLINT	CLHEP	Human	Mouse
1	29.6	12.3	149.5	56.2	8.5	5.6
11a	178.6	18.8	157.4	57.3	2.6	0.6
11f	194.3	19.0	1213.7	83.8	3.9	3.0
11h	97.1	17.3	100.0	47.4	3.8	2.7
11i	295.6	19.6	1142.5	83.5	2.3	2.2
14a	172.5	18.7	551.4	77.4	1.1	0.6
14i	33.3	12.5	104	48.2	11.3	5.1
14j	31.5	12.6	<49.5	<31.9	23.5	9.6
14x	<23.1	<11	133.7	53.8	4.9	6.6
14z	<23.1	<11	<49.5	<31.9	47.4	39.3
14bb	<23.1	<11	<49.5	<31.9	30.2	13.3

Stability in Human Hepatocyte S9 Fractions
	t1/2 (min)	CLHEP (mL/min/kg)	ER
11h	49.4	12.6	0.63

^aIn vitro DMPK studies performed at Q2 Solutions, Indianapolis, IN.

^bIntrinsic and predicted hepatic clearance based on experiments in liver microsomes.

^c%f_u = % fraction unbound.

Lastly, we profiled 11h against a wider panel of PDE isoforms (see Supporting Information Table 1) in a radioligand binding assay for % inhibition at 10 μM. 11h was selective against all of the isoforms tested (<50% inhibition), except PDE3B (87% inhibiton). In addition, 11h was also tested in the NIMH psychoactive drug screening program (PDSP), which tests compounds against a wide panel of targets (see Supporting Information Table 2).¹⁹ In this panel, 11h was not active (<50% inhibition) against all receptors except 5-HT_2C (59% inhibition) and σ2 (70% inhibition). However, follow-up IC₅₀ determinations showed 11h was only weakly active (5-HT_2c, IC₅₀ > 10 μM and σ2, IC₅₀ = 7.8 μM).

In summary, a scaffold-hopping experiment identified a novel 1H-pyrrolo[2,3-b]pyridine-2-carboxamide series of PDE4B inhibitors. The compounds display a range of PDE4B inhibition and selectivity versus PDE4D. In addition, we have shown using molecular modeling how these compounds interact with the enzyme. We have also shown that our compounds are equipotent with rolipram in cellular assays inhibiting macrophage pro-inflammatory cytokine activity. Finally, compounds also show good in vitro PK profiles. Further optimization and in vivo activity are ongoing and will be reported in due course.

Glossary

Abbreviations

PDE4

phosphodiesterase 4

K_p

total brain:plasma ratio

T3P

propylphosphonic anhydride

DIPEA

diisopropylethyl amine

DMF

dimethylformamide

AUC

area under the curve

TNF

tumor necrosis factor

ELISA

enzyme-linked immunosorbent assay

PK

pharmacokinetics

DMPK

drug metabolism and pharmacokinetics

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00369.

• General methods for the synthesis and characterization of all compounds, methods for the cellular assay, and selectivity data (PDF)

Author Contributions

C.R.H. and T.K. drafted and corrected the manuscript with input from all authors. A.K.V., C.D.A., and S.S. performed the chemical synthesis, and C.R.H. oversaw the medicinal chemistry and target selection and interpreted the biological data. A.K.V. performed the molecular modeling. A.L.A. and C.E.H. performed the macrophage cellular assays, and T.K. oversaw the experiments. S.K. and N.G. performed the PK studies. Y.A. oversaw all of the PK experiments.

C.R.H. thanks the UNMC College of Pharmacy for start-up funds.

The authors declare no competing financial interest.