pharmaceuticals

Review An Overview of the Biological ActivityActivity ofof Pyrrolo[3,4Pyrrolo[3,4-‐c]pyridine Derivatives

1 2, Anna WWójcickaójcicka 1 and Aleksandra Redzicka 2,**

1 1 DepartmentDepartment of of Pharmaceutical Pharmaceutical Technology Technology,, Faculty Faculty of of Pharmacy, Pharmacy, Wroclaw Wroclaw Medical University, BorowskaBorowska 211A, 211A, 50 50-556‐556 Wroc Wrocław,ław, Poland; Poland; [email protected] [email protected] 2 Department of Medicinal Chemistry, Faculty of Pharmacy, Wroclaw Medical University, Borowska 211, 2 Department of Medicinal Chemistry, Faculty of Pharmacy, Wroclaw Medical University, Borowska 211, 5050-556‐556 Wroc Wrocław,ław, Poland Poland * Correspondence: [email protected] * Correspondence: [email protected]

Abstract: Pyrrolo[3,4Pyrrolo[3,4-‐cc]pyridine]pyridine is is one one of of the the six six structural structural isomers isomers of the bicyclic ring system containingcontaining a a pyrrole moiety fused to a pyridine nucleus. The broad spectrum of pharmacological propertiesproperties of pyrrolo[3,4 pyrrolo[3,4-‐c]pyridine]pyridine derivatives is is the the main reason for developing new compounds containingcontaining this this scaffold. scaffold. This This review review presents presents studies studies on on the the biological biological activity activity of of pyrrolo[3,4 pyrrolo[3,4-‐ cc]pyridines]pyridines that that have have been been reported reported in in the scientific scientific literature. Most of these derivatives have been studiedstudied as as analgesic and sedative agents. Biological Biological investigations investigations have have shown shown that that pyrrolo[3,4 pyrrolo[3,4-‐ cc]pyridines]pyridines can can be be used to to treat diseases of the nervous and immune systems. Their antidiabetic, antimycobacterial,antimycobacterial, antiviral, antiviral, and antitumor activities also have been found.

Keywords:Keywords: pyrrolo[3,4-pyrrolo[3,4‐cc]pyridine;]pyridine; pyrrolepyrrole heterocycles; heterocycles; biological biological activity; activity; structure-activity structure‐activity rela- relationships;tionships; analgesic analgesic activity; activity; azaizoindoles; azaizoindoles; anticancer anticancer activity activity



 Citation: Wójcicka, A.; Redzicka, A.

AnCitation: OverviewWójcicka, of the Biological A.; Redzicka, A. ActivityAn Overview of Pyrrolo[3,4 of the Biological‐c]pyridine 1. Introduction Introduction Derivatives.Activity of Pyrrolo[3,4- Pharmaceuticalsc]pyridine 2021, 14, Pyrrolopyridines areare heterocyclic heterocyclic compounds compounds containing containing a five-membered a five‐membered pyrrole pyrrole ring 354.Derivatives. https://doi.org/10.3390/Pharmaceuticals 2021, 14, ringfused fused to a six-membered to a six‐membered pyridine pyridine ring. They ring. are alsoThey called are azaindolesalso called or azaindoles azaizoindoles, or ph14040354354. https://doi.org/10.3390/ azaizoindoles,depending on the depending arrangement on the of arrangement nitrogen atoms of in nitrogen the bicyclic atoms structure. in the bicyclic Pyrrolopyridines structure. ph14040354 occur in six isomeric forms (Figure1). Academic Editor: Antonella Messore Pyrrolopyridines occur in six isomeric forms (Figure 1).

Academic Editor: Antonella Messore Received: 28 February 2021 H N Accepted:Received: 289 April February 2021 2021 Published:Accepted: 911 April April 2021 2021 NH Published: 11 April 2021 N Publisher’s Note: MDPI stays N N N H neutralPublisher’s with Note: regardMDPI to jurisdictional stays neutral claims in published maps and with regard to jurisdictional claims in 1H‐pyrrolo[2,3‐b]pyridine 6H‐pyrrolo[3,4‐b]pyridine 1H‐pyrrolo[3,2‐b]pyridine institutionalpublished maps affiliations. and institutional affil- iations. H N Copyright: © 2021 by the authors. NH Licensee MDPI, Basel, Switzerland. Copyright: © 2021 by the authors. N N This article is an open access article N Licensee MDPI, Basel, Switzerland. N distributed under the terms and This article is an open access article H conditions of the Creative Commons distributed under the terms and Attribution (CC BY) license conditions of the Creative Commons 1H‐pyrrolo[2,3‐c]pyridine 2H‐pyrrolo[3,4‐c]pyridine 1H‐pyrrolo[3,2‐c]pyridine (http://creativecommons.org/licenses Attribution (CC BY) license (https:// /by/4.0/). creativecommons.org/licenses/by/ Figure 1. IsomericIsomeric forms forms of pyrrolopyridines. 4.0/).

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Pharmaceuticals 2021, 14, 354. https://doi.org/10.3390/ph14040354 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2021, 14, 354 2 of 26 25

TheThe pyrrolopyridinepyrrolopyridine scaffold scaffold occurs occurs in polyheterocyclicin polyheterocyclic compounds compounds of natural of natural origin. origin.The best The known best is known the alkaloid is the camptotheca alkaloid camptotheca1, isolated from 1, isolated the Camptotheca from the acuminata Camptothecatree. acuminataAs a topoisomerase tree. As a topoisomerase I inhibitor, it has I inhibitor, found application it has found in application cancer treatment in cancer and treatment is active andagainst is active HIV-1 against [1]. Other HIV alkaloids‐1 [1]. Other containing alkaloids the containing pyrrolopyridine the pyrrolopyridine system in their system structure in theirinclude structure pumiloside include2a ,pumiloside deoxypumiloside 2a, deoxypumiloside2b isolated from 2bOphiorrhiza isolated from pumila Ophiorrhiza[2], and pumilavariolin [2], B 3a and, deoxyvariolin variolin B B3a3b, deoxyvariolinisolated from an B antarctic3b isolated sponge fromKirkpatrickia an antarctic variolosa sponge[3], Kirkpatrickiaand the antiviral variolosa alkaloid [3], and mappicin the antiviral4, which alkaloid was mappicin isolated from 4, whichMappia was foetida isolated[4], from and Mappiaanticancer foetida meriolin [4], and derivatives anticancer5a–b meriolin[3] (Figure derivatives2). 5a‐b [3] (Figure 2).

O O N O N N

1 O N H H H O HO O 2a OH N H NH2 O OH R N

3a) R= OH HO OH 3b) R= H

N N O N

H2N N H H O O 2b N OH H N N NH2 O OH 4 R N HO HO OH 5a) R= H

5b) R= OCH3 N N H FigureFigure 2. 2. AlkaloidsAlkaloids 1–51–5 containingcontaining pyrrolopyridine pyrrolopyridine scaffold. scaffold.

DueDue to their structure containing two important pharmacophores, i.e., pyrrole and pyridine, pyrrolopyridines have have been been the subject of many pharmacological studies over thethe last last hundred hundred years. years. Pyrrole Pyrrole derivatives derivatives show show a a wide wide range range of of pharmacological pharmacological activity. activity. PyrrolesPyrroles could build the structure of porphyrin rings. Many of the drugs drugs contain a pyrrole pyrrole ring,ring, forfor example,example, nootropic nootropic (Piracetam), (Piracetam), anti-inflammatory anti‐inflammatory (Indometacin), (Indometacin), statins statins (Flu- (Fluvastatin),vastatin), neuroleptics neuroleptics (Molindone), (Molindone), drugs drugs used toused treat to insomnia treat insomnia (Zopiclone), (Zopiclone), migraine migraine(Sumatriptan), (Sumatriptan), Parkinson diseaseParkinson (Procyclidine), disease (Procyclidine), and erection disorders and erection (Tadalafil). disorders Com- (Tadalafil).pounds containing Compounds a pyrrole containing ring found a pyrrole in essential ring found oils arein usedessential as anticancer oils are used agents as anticancer(5-iodotubercidin) agents (5 [5‐iodotubercidin)] and antibacterial [5] agentsand antibacterial (Cefepim, agents Doripenem, (Cefepim, Meropenem) Doripenem, [6]. Meropenem)Fostemsavir has [6]. recently Fostemsavir been approved has recently for HIV been treatment. approved Drugs for HIV containing treatment. a pyrrolopy- Drugs containingridine scaffold a pyrrolopyridine such as Vemurafenib scaffold or such Pexidartinib as Vemurafenib are used or inPexidartinib anticancer are therapy used [3in]. anticancerPyrrolo[3,2- therapyc]pyridine [3]. Pyrrolo[3,2 derivatives‐c show]pyridine inhibitory derivatives effect show against inhibitory FMS kinase effect andagainst are FMSpromising kinase candidates and are for promising anticancer candidates and antiarthritic for anticancer drug development and antiarthritic [7]. drug developmentThis review [7]. presents biologically active derivatives of the pyrrolo[3,4-c]pyridine iso- c mer.This Pyrrolo[3,4- review ]pyridinepresents biologically derivatives haveactive not derivatives been introduced of the into pyrrolo[3,4 medicine‐c so]pyridine far, but as it results from studies presented in the literature, many of them show biological activity. isomer. Pyrrolo[3,4‐c]pyridine derivatives have not been introduced into medicine so far,

Pharmaceuticals 2021, 14, 354 3 of 26

but as it results from studies presented in the literature, many of them show biological activity.

2. Biological Activity of Pyrrolo[3,4‐c]pyridine Derivatives 2.1. Antidiabetic Activity of Pyrrolo[3,4‐c]pyridine Derivatives Knutsen et al. [8] described 4‐substituted 6‐methyl‐pyrrolo[3,4‐c]pyridine‐1,3(2H)‐ dione derivatives 6 (Figure 3) that effectively reduce blood glucose levels without affecting the concentration of circulating insulin. The present compounds reduce blood glucose levels by stimulating glucose uptake into muscle and fat cells. Pharmaceuticals 2021, 14, 354 The ability of pyrrolo[3,4‐c]pyridine‐1,3(2H)‐dione derivatives 6 to stimulate the3 of 25 incorporation of glucose into lipids was tested. The maximum increase in insulin sensitivity achieved in the dose range of 0.3–100 μM of the tested compounds, normalized to the full insulin dose‐response (100%), was determined. The phenoxy substituent in the 4‐position2. Biological turned Activity out to ofsignificantly Pyrrolo[3,4- influencec]pyridine the Derivatives activity of the derivatives. While 4‐ phenoxy2.1. Antidiabetic‐6‐methyl‐ Activitypyrrolo[3,4 of Pyrrolo[3,4-c]pyridine‐c]pyridine‐1,3(2H)‐dione Derivatives derivatives increased the insulin sensitivityKnutsen of mouse et al. adipocytes [8] described by 4-substituted7.4–37.4% (Figure 6-methyl-pyrrolo[3,4- 3). The substituentc]pyridine-1,3( in the para2H )- positiondione of derivatives the phenyl6 (Figurering increased3) that effectively the activity. reduce Compounds blood glucose with 4 levels‐phenoxy without‐(6a) affecting, 4‐(4‐ iodophenoxy)the concentration‐(6b), of4‐ circulating(3,4‐dichlorophenoxy) insulin. The‐(6c present) and compounds 4‐(4‐methylphenoxy) reduce blood‐ glucose(6d) derivativeslevels by increased stimulating the glucose insulin uptakesensitivity into by muscle more and than fat 30% cells. [8].

% increase in O H insulin N R R1 sensitivity O 6a H H 31.2 6b I H 37.4 6c Cl Cl 30.0 6d CH3 H 33.3 H3C N O 6e OCH3 H 18.6 6f H COOC2H5 7.4 6

1 R

R Figure 3. 6-Methyl-pyrrolo[3,4-c]pyridine-1,3(2H)-dione derivatives 6a–f increased the insulin sensitivity. Figure 3. 6‐Methyl‐pyrrolo[3,4‐c]pyridine‐1,3(2H)‐dione derivatives 6a–f increased the insulin sensitivityThe. ability of pyrrolo[3,4-c]pyridine-1,3(2H)-dione derivatives 6 to stimulate the in- corporation of glucose into lipids was tested. The maximum increase in insulin sensitivity Due to the efficacy of the present compounds 6 to reduce the blood glucose, they may achieved in the dose range of 0.3–100 µM of the tested compounds, normalized to the full find application in the prevention and treatment of disorders involving elevated plasma insulin dose-response (100%), was determined. The phenoxy substituent in the 4-position blood glucose, such as hyperglycemia and ailments in which such a reduction of blood turned out to significantly influence the activity of the derivatives. While 4-phenoxy-6- glucosemethyl-pyrrolo[3,4- is beneficial: typec]pyridine-1,3( 1 diabetes,2H diabetes)-dione derivatives as a consequence increased of the obesity, insulin diabetic sensitivity dyslipidemia,of mouse adipocytes hypertriglyceridemia, by 7.4–37.4% (Figureinsulin3 ).resistance, The substituent impaired in the glucose para position tolerance, of the hyperlipidemia,phenyl ring increased cardiovascular the activity. diseases, Compounds and hypertension. with 4-phenoxy-( 6a), 4-(4-iodophenoxy)- (6bAldose), 4-(3,4-dichlorophenoxy)-( reductase (AR) is an6c enzyme) and 4-(4-methylphenoxy)-( present in the lens and6d) derivativesbrain that eliminates increased the excessinsulin glucose sensitivity by catalyzing by more glucose than 30% reduction [8]. to sorbitol. The accumulation of sorbitol may causeDue neuropathy to the efficacy in the of peripheral the present nerves compounds and cataracts6 to reduce in the the lens. blood AR glucose, inhibitors they reducemay secondary find application complications in the prevention caused by diabetes and treatment mellitus, of especially disorders in involving tissues where elevated glucoseplasma uptake blood is glucose,not insulin such‐dependent. as hyperglycemia and ailments in which such a reduction ofDa blood Settimo glucose et al. is [9] beneficial: synthesized type a 1series diabetes, of 6‐methylpyrrolo[3,4 diabetes as a consequence‐c]pyridine of‐1,3 obesity,‐dione dia- alkanoicbetic dyslipidemia,acid derivatives hypertriglyceridemia, 7 with potential AR insulininhibitory resistance, activity impaired(Figure 4). glucose The obtained tolerance, compoundshyperlipidemia, were evaluated cardiovascular for their diseases, ability and to hypertension.inhibit AR in vitro in the extract of rat lenses. TheAldose biological reductase data (AR) showed is an enzymethat the presentacidic derivatives in the lens proved and brain to thatbe new eliminates AR inhibitors.excess glucose The presence by catalyzing of the glucose carboxylic reduction group to at sorbitol. an adequate The accumulation distance from of sorbitol the may cause neuropathy in the peripheral nerves and cataracts in the lens. AR inhibitors reduce secondary complications caused by diabetes mellitus, especially in tissues where glucose uptake is not insulin-dependent. Da Settimo et al. [9] synthesized a series of 6-methylpyrrolo[3,4-c]pyridine-1,3-dione alkanoic acid derivatives 7 with potential AR inhibitory activity (Figure4). The obtained compounds were evaluated for their ability to inhibit AR in vitro in the extract of rat lenses. The biological data showed that the acidic derivatives proved to be new AR inhibitors. The presence of the carboxylic group at an adequate distance from the pyrrolopyridine scaffold is important for their activity. Compounds with a small chain length of carboxylic acid proved to be much more potent inhibitors than propionic or iso-propionic acid derivatives. Its corresponding ethyl ester analogs were inactive even at a higher concentration range. Among amide analogs most potent was 4-trifluoromethyl-benzylamide derivative 7c, but no more active than 7a. The inhibition potency of compounds 7a–c was similar to the Pharmaceuticals 2021, 14, 354 4 of 26

pyrrolopyridine scaffold is important for their activity. Compounds with a small chain Pharmaceuticals 2021, 14, 354 length of carboxylic acid proved to be much more potent inhibitors than propionic or4 of iso 25‐ propionic acid derivatives. Its corresponding ethyl ester analogs were inactive even at a higher concentration range. Among amide analogs most potent was 4‐trifluoromethyl‐ benzylamide derivative 7c, but no more active than 7a. The inhibition potency of −5 −6 referencecompounds Alrestatin 7a‐c was and similar Sorbinil to the at concentrations: reference Alrestatin 10 andM (70–90%) Sorbinil andat concentrations: 10 M (33–43%). 10−5 ICM50 (70–90%)(the concentration and 10−6 M at(33–43%). which a IC substance50 (the concentration exerts half of at itswhich maximal a substance inhibitory exerts effect) half valuesof its maximal for compounds inhibitory7a– ceffect)were 1.4valuesµM, for 2.5 compoundsµM, and 1.7 7µa–cM, were respectively. 1.4 μM, The 2.5 μ mostM, and active 1.7 derivativesμM, respectively.7a–b were The evaluated most active as inhibitors derivatives of glutathione 7a–b were lens evaluated depletion as in galactosemicinhibitors of rats.glutathione Glutathione lens depletiondepletion in in the galactosemic lens occurs rats. rapidly Glutathione after induction depletion of galactosemia in the lens inoccurs rats (followingrapidly after the induction galactose of diet). galactosemia None of the in testedrats (following compounds the weregalactose active diet). in maintaining None of the glutathionetested compounds levels in were the rats’active lenses, in maintaining which may glutathione be due to levels problems in the with rats’ metabolism lenses, which or ocularmay be bioavailability. due to problems with metabolism or ocular bioavailability.

3 R

O R 2 R = CH COOH, COOH, CH COOC H , COOC H R 2 2 2 5 2 5 R1= H, Cl HN O 2 R = H, COC6H5, COC6H4-p-Cl, COC6H4-p-F, COC6H4-p-CF3 3 R = H, CH3 1 H3C N R 7 F

O O O F O O F OO N OH N OH N OH H N O 2 H2N O HN O

H C N 3 H3C N Cl H C N Cl 7a 3 7b 7c

FigureFigure 4. 4. 6-Methyl-pyrrolo[3,4-6‐Methyl‐pyrrolo[3,4c‐c]pyridine-1,3-dione]pyridine‐1,3‐dione derivatives derivatives7 7as as aldose aldose reductase reductase inhibitors inhibitors and theand most the most active active derivatives derivatives7a–c. 7a–c.

GPR119GPR119 isis aa cannabinoidcannabinoid receptorreceptor expressed expressed predominantly predominantly in in the the incretin-releasing incretin‐releasing intestinalintestinal cellscells andand pancreatic pancreatic islet isletβ β‐-cells.cells. GPR119GPR119 regulatesregulates thethe secretionsecretion of of incretin incretin and and insulin.insulin. Therefore, new drugsdrugs actingacting onon thethe GPR119GPR119 couldcould findfind applicationapplication inin treatingtreating obesityobesity and and type type 2 2 diabetes. diabetes. YuYu etet al.al. [ 10[10]] synthesized synthesized a a series series of of N-[3-(1,3-dioxo-2,3-dihydro- N‐[3‐(1,3‐dioxo‐2,3‐dihydro1H‐1H-pyrrolo[3,4-‐pyrrolo[3,4‐ cc]pyridin-4-yloxy)phenyl]benzenesulfonamide]pyridin‐4‐yloxy)phenyl]benzenesulfonamide derivatives derivatives 88 as novel GPR119GPR119 agonists.agonists. TheyThey replaced replaced the the -O- ‐O‐ linkerlinker with with the the -S- ‐S‐ oror -NH-, ‐NH‐, as as exemplified exemplified in in9 9,, but but both both modifications modifications diddid notnot bringbring anyany benefit,benefit, only an efficacy efficacy reduction of of about about 20% 20% (Figure (Figure 55).). Scientists investigated the effect of pyrrolo[3,4-c]pyridine ring substituents on po- tency [10]. The methyl group for R1 was selected for its potential to improve metabolic stability. Biological studies have shown that the potency of derivatives 8 also depends on the R2 substituent. Activity increased for the ethyl and propyl chains as R2 compared to the methyl substituent or lack thereof, but large substituents (e.g., phenyl, cyclopentyl) lead to a significant loss of potency. Pharmacokinetic studies showed different metabolic decomposition of the tested compounds in both rat liver microsomes (RLM) depending on the substitution of the middle phenyl ring. The most active 6-F-phenyl derivative 8a exhibited an EC50 of 0.016 µM in the human GPR119 cAMP assay and good RLM stabil- ity with clearance intrinsic (CL- the liver ability to remove (metabolize) a drug without the restrictions placed on it being delivered to the liver cell by blood flow or protein binding) = 84 µL/min/mg. Compound 8a was also evaluated during in vivo experiments and exhibited 0.073 µM in the cynomolgus monkey GPR119 cAMP assay with 100% efficacy, and in human liver microsomes, the CL was 29 µL/min/mg. Derivative 8a showed good bioavailability in rats (95%) and 29% in cynomolgus monkeys following a 2.0 mg/kg PO dose. It did not bind to BSEP (bile salt export pump), did not show hPXR (the human Pharmaceuticals 2021, 14, 354 5 of 25

pregnane X receptor) activation and cytochrome P450 inhibition. Although the pharma- Pharmaceuticals 2021, 14, 354 cological properties were excellent, compound 8a showed high plasma protein binding,5 of 26

which precluded the assessment of its antidiabetic efficacy in vivo [10].

1 R CH3 Z Z O N O N O O S S NH O O NH A O

8 N 9 N Cl O 2 Cl O R CH3 Z= H, 2-F, 5-F, 6-F, 6Cl, 4-Me A = S or NH 1 H C R = Me, OMe, cPr Z= H, Me 3 R2= H, Me, cPr, iPr, Et, Ph

CH3 F O N O S NH O O

N Cl 8a O CH3 H3C FigureFigure 5.5. Pyrrolo[3,4-Pyrrolo[3,4c‐c]pyridine-1,3-dione]pyridine‐1,3‐dione derivatives derivatives8– 98–9with with 4-chloro-N-phenyl-benzenesulfonamide 4‐chloro‐N‐phenyl‐ substituentbenzenesulfonamide as GPR119 substituent agonists and as GPR119 the most agonists active compound and the most8a. active compound 8a.

2.2. AntimicrobialScientists investigated Activity of Pyrrolo[3,4-c]pyridines the effect of pyrrolo[3,4‐c]pyridine ring substituents on potencyThe [10]. growing The methyl problem group of bacterial for R1 was resistance selected prompts for its potential the search to for improve new antibacterial metabolic compounds.stability. Biological Pyrrolo[3,2-b]pyridine studies have shown derivatives that the show potency activity of derivatives against resistant 8 also depends strains of onE. colithe [R112 ],substituent. which stimulates Activity the increased search for for new the pyrrolopyridineethyl and propyl derivatives chains as R with2 compared antimicro- to bialthe methyl activity. substituent or lack thereof, but large substituents (e.g., phenyl, cyclopentyl) lead to a significant loss of potency. Pharmacokinetic studies showed different metabolic 2.2.1.decomposition Antiviral Activityof the tested of Pyrrolo[3,4- compoundsc]pyridine in both rat Derivatives liver microsomes (RLM) depending on theHIV-1 substitution integrase of (IN)the middle catalyzes phenyl the incorporationring. The most of active HIV-1 6‐F cDNA‐phenyl into derivative host DNA 8a 0 0 throughexhibited a an process EC50 of involving 0.016 μM twoin the steps: human processing GPR119 cAMP of the assay HIV DNA and good 3 ends RLM (3 stability-P) and strandwith clearance transfer intrinsic (ST). Due (CL to‐ thethe emergenceliver ability of to raltegravir remove (metabolize) (an IN inhibitor) a drug resistance,without the it isrestrictions imperative placed to develop on it being second-generation delivered to the inhibitors liver cell by that blood will flow remain or protein effective binding) against clinically= 84 μL/min/mg. significant Compound IN mutants. 8a was also evaluated during in vivo experiments and exhibitedResearchers 0.073 μM under in the Zhao cynomolgus [12] developed monkey a series GPR119 of newcAMP IN assay inhibitors with containing100% efficacy, the pyrrolo[3,4-and in humanc]pyridine liver microsomes, scaffold. Tricyclic the CL compoundswas 29 μL/min/mg.10 (N-benzyl-7-hydroxy-pyrrolo[3,4- Derivative 8a showed good cbioavailability]pyridine-1,3,6-trione in rats (95%) nucleus and condensed 29% in cynomolgus with a ring monkeys ranging following in size from a 2.0 five mg/kg to eight PO members)dose. It did (Figure not bind6) demonstrated to BSEP (bile low salt micromolar export pump), inhibitory did not potency show in hPXR IN assays (the humanin vitro (inhibitionpregnane X potency receptor) slightly activation increased and with cytochrome ring size), P450 good selectivityinhibition. (forAlthough the ST stepthe relativepharmacological to the 30-P properties step), and were reduced excellent, cytotoxicity compound (Figure 8a 6showed). These high analogs plasma10a –proteinc were lessbinding, sensitive which than precluded raltegravir the toassessment resistance of induced its antidiabetic by mutations efficacy in integrase.in vivo [10].

2.2. Antimicrobial Activity of Pyrrolo[3,4‐c]pyridines The growing problem of bacterial resistance prompts the search for new antibacterial compounds. Pyrrolo[3,2‐b]pyridine derivatives show activity against resistant strains of E. coli [11], which stimulates the search for new pyrrolopyridine derivatives with antimicrobial activity.

2.2.1. Antiviral Activity of Pyrrolo[3,4‐c]pyridine Derivatives HIV‐1 integrase (IN) catalyzes the incorporation of HIV‐1 cDNA into host DNA through a process involving two steps: processing of the HIV DNA 3′ ends (3′‐P) and strand transfer (ST). Due to the emergence of raltegravir (an IN inhibitor) resistance, it is imperative to develop second‐generation inhibitors that will remain effective against clinically significant IN mutants. Researchers under Zhao [12] developed a series of new IN inhibitors containing the pyrrolo[3,4‐c]pyridine scaffold. Tricyclic compounds 10 (N‐benzyl‐7‐hydroxy‐

PharmaceuticalsPharmaceuticals 2021 2021, ,14 14, ,354 354 66 of of 26 26

pyrrolo[3,4pyrrolo[3,4‐‐cc]pyridine]pyridine‐‐1,3,61,3,6‐‐trionetrione nucleusnucleus condensedcondensed withwith aa ringring rangingranging inin sizesize fromfrom fivefive to to eight eight members) members) (Figure (Figure 6) 6) demonstrated demonstrated low low micromolar micromolar inhibitory inhibitory potency potency in in IN IN Pharmaceuticals 2021, 14, 354 assaysassays in in vitro vitro (inhibition (inhibition potency potency slightly slightly increased increased with with ring ring size size)), ,good good selectivity selectivity6 (for of(for 25 thethe STST stepstep relativerelative toto thethe 33′‐′‐PP step),step), andand reducedreduced cytotoxicitycytotoxicity (Figure(Figure 6).6). TheseThese analogsanalogs 10a10a––cc were were less less sensitive sensitive than than raltegravir raltegravir to to resistance resistance induced induced by by mutations mutations in in integrase. integrase.

ClCl RR

OHOH OHOH OO FF OO FF OO OO NN NN NN NN AA OO OO

10a)10a) R= R= H H 10 10 AA = = 10b) R= F , , , 10b) R= F , , , 10c)10c) R= R= Cl Cl

FigureFigureFigure 6. 6. 6. Tricyclic TricyclicTricyclic compounds compounds compounds containing containing containing pyrrolo[3,4 pyrrolo[3,4 pyrrolo[3,4-‐c‐c]pyridine]pyridinec]pyridine scaffold scaffold scaffold 10 10 as as10 HIV HIVas‐ HIV-11‐1 integrase integrase integrase inhibitorsinhibitorsinhibitors and and and the the the most most most active active active compounds compounds compounds 10a 10a10a–––cc.c ..

ZhaoZhaoZhao et et et al. al. al. [13] [13] [13 also ]also also synthesized synthesized synthesized bicyclic bicyclic bicyclic compounds: compounds: compounds: 2 2‐‐(3(3 2-(3-chloro-4-fluorobenzyl)-‐‐chlorochloro‐‐44‐‐fluorobenzyl)fluorobenzyl)‐‐77‐‐ H c H H hydroxyhydroxy7-hydroxy-1‐‐11HH‐‐pyrrolo[3,4pyrrolo[3,4-pyrrolo[3,4-‐‐cc]pyridine]pyridine]pyridine-1,3,6(2‐‐1,3,6(21,3,6(2HH,5,5HH),5)‐‐trionetrione)-trione derivativesderivatives derivatives 1111 11 (Figure(Figure(Figure 7)77)) andand assessed them in biochemical IN tests that measure the inhibitory power. The potency assessedassessed them them in in biochemical biochemical IN IN tests tests that that measure measure the the inhibitory inhibitory power. power. The The potency potency of of of new compounds was similar50 (IC50 values for the ST reactions in the low micromolar newnew compounds compounds was was similar similar (IC (IC50 values values for for the the ST ST reactions reactions in in the the low low micromolar micromolar range: range: range: 6–22 µM). Methyl 3-(2-(3-chloro-4-fluorobenzyl)-7-hydroxy-5-isopropyl-1,3,6-trioxo- 6–226–22 μ μM).M). MethylMethyl 33‐‐(2(2‐‐(3(3‐‐chlorochloro‐‐44‐‐fluorobenzyl)fluorobenzyl)‐‐77‐‐hydroxyhydroxy‐‐55‐‐isopropylisopropyl‐‐1,3,61,3,6‐‐trioxotrioxo‐‐ 2,3,5,6-tetrahydro-1H-pyrrolo[3,4-c]pyridin-4-yl)propanoate 11a retained most of its in- 2,3,5,62,3,5,6‐‐tetrahydrotetrahydro‐‐11HH‐‐pyrrolo[3,4pyrrolo[3,4‐‐cc]pyridin]pyridin‐‐44‐‐yl)propanoateyl)propanoate 11a11a retainedretained mostmost ofof itsits hibitory potency against the three raltegravir-resistance mutant IN enzymes. In antiviral inhibitoryinhibitory potency potency against against the the three three raltegravir raltegravir‐‐resistanceresistance mutant mutant IN IN enzymes. enzymes. In In antiviral antiviral assays, it was approximately 200-, 20-, and 10-fold less affected than raltegravir concerning assays,assays, itit waswas approximatelyapproximately 200200‐‐, , 2020‐‐, , andand 1010‐‐foldfold lessless affectedaffected thanthan raltegravirraltegravir G140S/Q148H, Y143R, and N155H mutations, respectively [13]. concerningconcerning G140S/Q148H, G140S/Q148H, Y143R, Y143R, and and N155H N155H mutations, mutations, respectively respectively [13]. [13].

F FF F

OH OHOH OH O Cl OO ClCl O Cl O OO O N NN N N H3C N 11 N H3C N RR 2 O R2 OO CH O R CH33

11 H C 11a 11 H33C 11a OOOO 1 RR1 = = H, H, Me, Me, Ph, Ph, iPr, iPr, Bu Bu 2 2 R = Me, iBu, CH2OCOCH3, C2H4COOCH3, CH2OCOH, CH2OH R = Me, iBu, CH2OCOCH3, C2H4COOCH3, CH2OCOH, CH2OH FigureFigureFigure 7. 7.7. 2 2-(3-Chloro-4-fluorobenzyl)-7-hydroxy-12‐‐(3(3‐‐ChloroChloro‐‐44‐‐fluorobenzyl)fluorobenzyl)‐‐77‐‐hydroxyhydroxyH-pyrrolo[3,4-‐‐11HH‐‐pyrrolo[3,4pyrrolo[3,4c]pyridine-1,3,6(‐‐cc]pyridine]pyridine2H,5H‐‐1,3,6(1,3,6()-trione2H,5H2H,5H deriva-))‐‐ trionetrionetives 11 derivatives derivativesand the most 11 11 potent and and the compoundthe most most potent potent11a. compound compound 11a 11a. .

TheTheThe biologicalbiological biological datadata data suggestsuggest thatthat thethe pyrrolo[3,4 pyrrolo[3,4-pyrrolo[3,4‐‐cc]pyridine]pyridine]pyridine-1,3,6-trione‐‐1,3,61,3,6‐‐trionetrione scaffoldscaffold scaffold cancan bebebe used used used to to to create create create compounds compounds compounds insensitive insensitive insensitive to to to mutations mutations mutations of of of HIV HIV HIV-1‐‐11 integrase integrase integrase that that that have have have a a a real real potentialpotentialpotential in in in AIDS AIDS AIDS treatment. treatment. treatment. ResearchersResearchersResearchers led led led by by by Liu Liu Liu [14] [14] [14 ]synthesized synthesized synthesized a a aseries series series of of of 7 7‐ 7-hydroxy-1,3-dioxo-2,3-dihydro-‐hydroxyhydroxy‐‐1,31,3‐‐dioxodioxo‐‐2,32,3‐‐dihydrodihydro‐‐ 111HHH‐‐pyrrolo[3,4-pyrrolo[3,4-pyrrolo[3,4‐‐cc]pyridinec]pyridine]pyridine-4-carboxylates‐‐44‐‐carboxylatescarboxylates 12a 12a12a‐‐i–i (Figure i(Figure(Figure 8) 8)8 )and and evaluated evaluated their their anti anti-HIV-1anti‐‐HIVHIV‐‐11 activity.activity.activity. Most Most Most of of of the the the obtained obtained compounds compounds compounds showed showed showed moderate moderate moderate activity activity activity in in in inhibiting inhibiting inhibiting HIV HIV HIV-1‐‐11 replication.replication.replication. It It It turned turned turned out out out that that that the the the ester ester ester substituent substituent substituent at at at position position position 4 4 4 has has has a a a significant significant significant influence influence influence on the activity of derivatives. The distance between the pyrrolopyridine scaffold and the phenyl ring proved to be equally important. Derivatives in which R = ethyl, R1= OEt 2 and R = Ph, 4-FPh, 4-MePh or indol-3-yl exhibited potent activity (EC50 <10 µM). The most active ethyl 2-(4-fluorophenethyl)-7-hydroxy-1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4- c]pyridine-4-carboxylate 12j showed significant anti-HIV-1 activity (EC50 = 1.65 µM) and in vitro therapeutic index TI = 7.98 [14]. Pharmaceuticals 2021, 14, 354 7 of 26

on the activity of derivatives. The distance between the pyrrolopyridine scaffold and the phenyl ring proved to be equally important. Derivatives in which R = ethyl, R1= OEt and R2= Ph, 4‐FPh, 4‐MePh or indol‐3‐yl exhibited potent activity (EC50 <10 μM). The most

Pharmaceuticals 2021, 14, 354 active ethyl 2‐(4‐fluorophenethyl)‐7‐hydroxy‐1,3‐dioxo‐2,3‐dihydro‐1H‐pyrrolo[3,47 of 25 ‐ c]pyridine‐4‐carboxylate 12j showed significant anti‐HIV‐1 activity (EC50 = 1.65 μM) and in vitro therapeutic index TI = 7.98 [14].

Figure 8. 7-Hydroxy-1,3-dioxo-2,3-dihydro-17‐Hydroxy‐1,3‐dioxo‐2,3‐dihydro‐H1-pyrrolo[3,4-H‐pyrrolo[3,4c]pyridine-4-carboxylate‐c]pyridine‐4‐carboxylate derivatives derivatives12 12 and the mostmost activeactive compound compound12j 12j. . Respiratory syncytial virus (RSV), also called human orthopneumovirus, causes infec- Respiratory syncytial virus (RSV), also called human orthopneumovirus, causes tions of the respiratory tract. Bond et al. [15] synthesized 9b-(4-chlorophenyl)-1,2,3,9b- infectionstetrahydro-5 ofH -imidazo[1’,2’:1,2]pyrrolo[3,4-the respiratory tract. Bondc ]pyridin-5-oneet al. [15] synthesized derivatives 139b‐(Figure(4‐chlorophenyl)9). The ‐ 1,2,3,9bobtained‐tetrahydro compounds‐5H were‐imidazo[1’,2’:1,2]pyrrolo[3,4 evaluated for their activity‐c]pyridin against‐ RSV5‐one in a cytopathicderivatives ef- 13 (Figurefect assay. 9). The The most obtained potent compounds derivatives were alsoevaluated tested forfor thetheir presence activity of against alpha-1-acid RSV in a cytopathicglycoprotein effect to evaluate assay. theThe binding most potent effect to derivatives a human serum were protein. also tested The effectfor the of presence the sub- of alphastituent‐1‐ Racid was glycoprotein also investigated. to evaluate Furan, pyrazole,the binding pyrrole, effect and to a pyridine human derivativesserum protein. were The effectmost active of the against substituent RSV and R was showed also goodinvestigated. protein binding Furan, properties.pyrazole, pyrrole, The S-enantiomer and pyridine derivativesof the tested were compounds most active turned against out to be RSV active. and Derivatives showed good combining protein excellent binding anti-RSV properties. Theactivity S‐enantiomer with good of protein the tested binding compounds properties turned were out tested to be for active. RSV cross-strain Derivatives activity, combining excellentsolubility, anti lipophilicity,‐RSV activity and pharmacokinetic with good protein properties binding in rats.properties All compounds were tested exhibited for RSV crossgood‐ activitystrain activity, against the solubility, three RSV lipophilicity, strains, acceptable and pharmacokinetic solubility, and low properties lipophilicity. in rats. The All compoundspyridine derivative exhibited13a goodshowed activity excellent against bioavailability the three RSV and astrains, terrific acceptable pharmacokinetic solubility, and(PK) low profile lipophilicity. at high exposure. The pyridine Virological derivative studies showed 13a showed that derivative excellent13a bioavailabilityis a promising and a Pharmaceuticals 2021, 14, 354 direct and selective RSV fusion inhibitor and has been selected for preclinical studies8 as of 26 a terrific pharmacokinetic (PK) profile at high exposure. Virological studies showed that candidate for RSV infection treatment [15]. derivative 13a is a promising direct and selective RSV fusion inhibitor and has been selected for preclinical studies as a candidate for RSV Oinfection treatment [15]. O

N N N N N N N R O O Cl Cl 13 13a

FigureFigure 9.9. Imidazo[1’,2’:1,2]pyrrolo[3,4-Imidazo[1’,2’:1,2]pyrrolo[3,4‐cc]pyridin-5-one]pyridin‐5‐one derivatives derivatives13 13with with anti-RSV anti‐RSV activity activity and and the candidatethe candidate for preclinical for preclinical studies studies13a. 13a.

2.2.2. Antimycobacterial Activity of Pyrrolo[3,4‐c]pyridine Derivatives Mycobacterium tuberculosis (Mtb) is a species of pathogenic bacteria with a waxy coating on the surface of cells, mainly due to mycolic acid presence. Isoniazid, one of the most effective anti‐tuberculosis drugs, affects the synthesis of mycolic acids. It inhibits enoyl‐acyl carrier protein reductase (InhA), an enzyme from Mycobacterium tuberculosis, which is one of the key enzymes involved in the type II fatty acid biosynthesis pathway (FAS II) system. Ethionamide also exerts an antimycobacterial activity by inhibiting InhA. Deraeve et al. [16] synthesized pyrrolo[3,4‐c]pyridine‐3‐one derivatives 14 as new inhibitors of the InhA enzyme (Figure 10). The obtained compounds were screened in vitro at 100 and 30 μM, and the InhA inhibition percentage, MIC (minimum inhibitory concentration), and IC50 were determined against M. tuberculosis. The substitution of the phenyl ring was investigated. Unsubstituted or meta derivatives were most active. The chain substitution at the ortho position abolished this activity. Replacing the methyl or sulfur linker with oxygen or an NH group resulted in a decreased activity. On the other hand, dodecane derivative 14a is not an effective inhibitor of InhA (4% at 100 μM dose), proving that the presence of the electronegative atom is important for binding affinity. The most active derivatives 14a‐d exhibited MIC <25 μM and IC50 values in the range of 4.5–9.5 μM [11]. Derivative 14e obtained by Delaine et al. [17] inhibited 91% of InhA activity at 100 μM and 40% at 20 μM. Compound 14e exhibited higher growth inhibition of M. smegmatis than isoniazid but was less active against M. tuberculosis.

CH3 R n C12H25 R = CH , O, S, NH 2 2 1 1 R R = H, CH , C H R 3 12 25 N R2= H, C H O 12 25 H n = 7, 11, 17 N O HO 14 O N + 14e 1 2 N 14a) R=CH2, n=11, R =R =H 14b) R=S, n=11, R1=R2=H O 1 2 14c) R = H, n = 0, R =H, R = C12H25 1 2 H C O 14d) R = H, n = 0, R = C12H25, R = H 3

Figure 10. 1‐Phenyl‐pyrrolo[3,4‐c]pyridine‐3‐one derivatives 14a‐e as InhA enzyme inhibitors.

Pharmaceuticals 2021, 14, 354 8 of 26

O O

N N N N N N N R O O Cl Cl 13 13a

Pharmaceuticals 2021, 14, 354 8 of 25 Figure 9. Imidazo[1’,2’:1,2]pyrrolo[3,4‐c]pyridin‐5‐one derivatives 13 with anti‐RSV activity and the candidate for preclinical studies 13a.

2.2.2.2.2.2. Antimycobacterial Antimycobacterial Activity Activity of of Pyrrolo[3,4 Pyrrolo[3,4-‐c]pyridinec]pyridine Derivatives MycobacteriumMycobacterium tuberculosistuberculosis(Mtb) (Mtb) is is a species a species of pathogenic of pathogenic bacteria bacteria with a with waxy a coating waxy coatingon the surfaceon the surface of cells, of mainly cells, mainly due to due mycolic to mycolic acid presence. acid presence. Isoniazid, Isoniazid, one of one the of most the mosteffective effective anti-tuberculosis anti‐tuberculosis drugs, drugs, affects theaffects synthesis the synthesis of mycolic of acids. mycolic It inhibits acids. enoyl-acylIt inhibits enoylcarrier‐acyl protein carrier reductase protein (InhA), reductase an enzyme (InhA), froman enzymeMycobacterium from Mycobacterium tuberculosis, whichtuberculosis is one, whichof the keyis one enzymes of the key involved enzymes in the involved type II fatty in the acid type biosynthesis II fatty acid pathway biosynthesis (FAS II) pathway system. (FASEthionamide II) system. also Ethionamide exerts an antimycobacterial also exerts an antimycobacterial activity by inhibiting activity InhA. by inhibiting InhA. DeraeveDeraeve et et al. al. [16] [16] synthesized pyrrolo[3,4 pyrrolo[3,4-‐cc]pyridine]pyridine-3-one‐3‐one derivatives derivatives 1414 asas new new inhibitorsinhibitors of of the the InhA InhA enzyme enzyme (Figure (Figure 10). 10 ).The The obtained obtained compounds compounds were were screened screened in vitroin vitro at 100at 100 and and 30 30 μM,µM, and and the the InhA InhA inhibition inhibition percentage, percentage, MIC MIC (minimum (minimum inhibitory inhibitory concentration),concentration), and and IC IC5050 werewere determined determined against against M.M. tuberculosis tuberculosis.. The The substitution substitution of of the the phenylphenyl ring ring was investigated. Unsubstituted Unsubstituted or or meta meta derivatives were most active. The chainchain substitution substitution at at the the ortho ortho position abolished abolished this this activity. Replacing Replacing the the methyl methyl or sulfursulfur linker linker with with oxygen oxygen or or an an NH NH group group resulted resulted in in a a decreased activity. activity. On the other hand,hand, dodecane dodecane derivative derivative 14a14a isis not not an an effective effective inhibitor inhibitor of of InhA InhA (4% (4% at at 100 100 μµMM dose), dose), provingproving that that the the presence presence of of the the electronegative electronegative atom atom is is important important for for binding binding affinity. affinity. 14a-d µ TheThe most most active active derivatives derivatives 14a‐d exhibitedexhibited MIC MIC <25 <25 μMM and and IC IC50 50valuesvalues in inthe the range range of 4.5–9.5of 4.5–9.5 μMµ M[[11].11 Derivative]. Derivative 14e14e obtainedobtained by by Delaine Delaine et et al. al. [17] [17 ]inhibited inhibited 91% 91% of of InhA InhA activityactivity at 100 μµM and 40%40% atat 20 20µ μM.M. Compound Compound14e 14eexhibited exhibited higher higher growth growth inhibition inhibition of ofM. M. smegmatis smegmatisthan than isoniazid isoniazid but but was was less less active active against againstM. M. tuberculosis tuberculosis. .

CH3 R n C12H25 R = CH , O, S, NH 2 2 1 1 R R = H, CH , C H R 3 12 25 N R2= H, C H O 12 25 H n = 7, 11, 17 N O HO 14 O N + 14e 1 2 N 14a) R=CH2, n=11, R =R =H 14b) R=S, n=11, R1=R2=H O 1 2 14c) R = H, n = 0, R =H, R = C12H25 1 2 H C O 14d) R = H, n = 0, R = C12H25, R = H 3

FigureFigure 10. 10. 11-Phenyl-pyrrolo[3,4-‐Phenyl‐pyrrolo[3,4‐cc]pyridine]pyridine-3-one‐3‐one derivatives derivatives 14a14a‐–ee asas InhA InhA enzyme enzyme inhibitors. inhibitors.

Westhuyzen et al. [18,19] synthesized a series of 7-amino-2-benzyl-4-methyl-1,3-dioxo- 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine derivatives 15–16 (Figure 11) and assessed their an- timycobacterial activity. Nitriles and amides 15 were not active with MIC90 (the lowest con- centration of the drug inhibiting the growth of over 90% of the bacterial population) above 160 µM, while the esters exhibited good activity in vitro (MIC90 <0.15 µM). The moderately active carboxylic acid (MIC90 = 3.13 µM) showed significantly improved solubility and mi- crosomal metabolic stability. Compounds 16 with the 1,2,4-oxadiazole moiety instead of the ester group showed better in vitro activity against Mtb and metabolic stability, but the 1,3,4- oxadiazole derivative exhibited a lower activity. The 3-hydroxy analog 15a exhibited good activity in vitro and improved solubility. Removal of the methyl group from the pyridine ring resulted in a significant loss of activity. All tested compounds were not toxic to VERO cells. The most active, stable and highly soluble 7-amino-2-(3-chlorobenzyl)-4-methyl-6-(3- methyl-1,2,4-oxadiazol-5-yl)-1H-pyrrolo[3,4-c]pyridine-1,3-dione 16 was evaluated using an in vivo pharmacokinetic study. Mouse PK studies showed low plasma exposure and high clearance compared to good metabolic stability in liver microsomes. Pharmaceuticals 2021, 14, 354 9 of 26

Pharmaceuticals 2021, 14, 354 9 of 26

Westhuyzen et al. [18,19] synthesized a series of 7‐amino‐2‐benzyl‐4‐methyl‐1,3‐ dioxo‐2,3‐dihydro‐1H‐pyrrolo[3,4‐c]pyridine derivatives 15–16 (Figure 11) and assessed Westhuyzen et al. [18,19] synthesized a series of 7‐amino‐2‐benzyl‐4‐methyl‐1,3‐ their antimycobacterial activity. Nitriles and amides 15 were not active with MIC90 (the dioxo‐2,3‐dihydro‐1H‐pyrrolo[3,4‐c]pyridine derivatives 15–16 (Figure 11) and assessed lowest concentration of the drug inhibiting the growth of over 90% of the bacterial their antimycobacterial activity. Nitriles and amides 15 were not active with MIC90 (the population) above 160 μM, while the esters exhibited good activity in vitro (MIC90 <0.15 lowest concentration of the drug inhibiting the growth of over 90% of the bacterial μM). The moderately active carboxylic acid (MIC90 = 3.13 μM) showed significantly population) above 160 μM, while the esters exhibited good activity in vitro (MIC90 <0.15 improved solubility and microsomal metabolic stability. Compounds 16 with the 1,2,4‐ μM). The moderately active carboxylic acid (MIC90 = 3.13 μM) showed significantly oxadiazole moiety instead of the ester group showed better in vitro activity against Mtb improved solubility and microsomal metabolic stability. Compounds 16 with the 1,2,4‐ and metabolic stability, but the 1,3,4‐oxadiazole derivative exhibited a lower activity. The oxadiazole moiety instead of the ester group showed better in vitro activity against Mtb 3‐hydroxy analog 15a exhibited good activity in vitro and improved solubility. Removal and metabolic stability, but the 1,3,4‐oxadiazole derivative exhibited a lower activity. The of the methyl group from the pyridine ring resulted in a significant loss of activity. All 3‐hydroxy analog 15a exhibited good activity in vitro and improved solubility. Removal tested compounds were not toxic to VERO cells. The most active, stable and highly soluble of the methyl group from the pyridine ring resulted in a significant loss of activity. All 7‐amino‐2‐(3‐chlorobenzyl)‐4‐methyl‐6‐(3‐methyl‐1,2,4‐oxadiazol‐5‐yl)‐1H‐pyrrolo[3,4‐ tested compounds were not toxic to VERO cells. The most active, stable and highly soluble c]pyridine‐1,3‐dione 16 was evaluated using an in vivo pharmacokinetic study. Mouse PK 7‐amino‐2‐(3‐chlorobenzyl)‐4‐methyl‐6‐(3‐methyl‐1,2,4‐oxadiazol‐5‐yl)‐1H‐pyrrolo[3,4‐ studies showed low plasma exposure and high clearance compared to good metabolic c]pyridine‐1,3‐dione 16 was evaluated using an in vivo pharmacokinetic study. Mouse PK Pharmaceuticals 2021, 14, 354 stability in liver microsomes. 9 of 25 studies showed low plasma exposure and high clearance compared to good metabolic stability in liver microsomes. N NH2 O NH2 O 1 O R R N NH2 O NH2 O 1 N O R N R N N N N R N N N N O CH O CH R 15 3 16 3 O R= COOEt,CH COOMe,O COOPr, CH 3 161 3 CONHR',15 COOH, CN for R = CF3, R = H R= COOEt, COOMe, COOPr, 1 for R1 = CH3: R = F, Cl, Br, OCH3, CONHR', COOH, CN for R = CF3, R = H H3C O NH2 CF , OCF , OCHF , SO CH , SCF , O for 3R1 = CH3 : R = F,2 Cl, 2Br, 3OCH , 3 CH OH, N(CH3 ) , p-OPh-4-F, CN3 H C O NH CF2, OCF , OCHF3 2 , SO CH , SCF , 3 O 2 O 3 3 2 2 3 3 N CH2OH, N(CH3)2, p-OPh-4-F, CN O N N N OH CH3 15a OH CH3 15a Figure 11. 7‐Amino‐2‐benzyl‐4‐methyl‐pyrrolo[3,4‐c]pyridine‐1,3‐dione derivatives 15–16 with an antimycobacterial activity and the most active compound 15a. Figure 11. 7-Amino-2-benzyl-4-methyl-pyrrolo[3,4-7‐Amino‐2‐benzyl‐4‐methyl‐pyrrolo[3,4‐cc]pyridine]pyridine-1,3-dione‐1,3‐dione derivatives derivatives 15–1615–16 withwith an an antimycobacterial activity and the most active compound 15a. 2.2.3.antimycobacterial Antibacterial activity and Antifungaland the most Activity active compound of Pyrrolo[3,4 15a. ‐c]pyridine Derivatives 2.2.3. Antibacterial and Antifungal Activity of Pyrrolo[3,4-c]pyridine Derivatives 2.2.3.Mannich Antibacterial bases and 17a Antifungal‐b (Figure Activity12) of 4of‐methyl Pyrrolo[3,4‐6‐phenylpyrrolo[3,4‐c]pyridine Derivatives‐c]pyridine ‐1,3‐ dioneMannich obtained bases by Wojcicka17a–b (Figure et al. 12[20]) of exhibited 4-methyl-6-phenylpyrrolo[3,4- moderate activity againstc]pyridine-1,3-dione Staphylococcus Mannich bases 17a‐b (Figure 12) of 4‐methyl‐6‐phenylpyrrolo[3,4‐c]pyridine‐1,3‐ aureus.obtained Compound by Wojcicka 17a et al.also [20 reduced] exhibited the moderate growth of activity Candida against albicansStaphylococcus in a statistically aureus. dione obtained by Wojcicka et al. [20] exhibited moderate activity against Staphylococcus significantCompound manner.17a also reduced the growth of Candida albicans in a statistically significant manner. aureus. Compound 17a also reduced the growth of Candida albicans in a statistically significant manner.

NH O O NO N Cl N NH O O NO N O Cl N O

O O N N

N17a 17bN

Figure 12. Mannich17a bases 17a–b displayed activity17b against S. aureus and C. albicans. Figure 12. Mannich bases 17a‐b displayed activity against S. aureus and C. albicans. 2.3.Figure Anticancer 12. Mannich Activity bases of 17a Pyrrolo[3,4-c]pyridine‐b displayed activity against Derivatives S. aureus and C. albicans. In a study by Kalai et al. [21], 3,5-bis(4-fluorobenzylidene)-1-(1,1,3,3-tetramethyl-1,2- dihydropyrrolo[3,4-c]pyridin-6-yl)piperidin-4-on N-oxide 18 was synthesized (Figure 13). The antitumor activity of the obtained compound was assessed by measuring its cytotoxic-

ity against ovarian and breast cancer cell lines and noncancerous cardiac cell lines. Tested compound 18 exhibited moderate cytotoxicity against ovarian cancer cells and limited toxicity toward breast cancer and healthy cardiac cell lines [21]. Pharmaceuticals 2021, 14, 354 10 of 26

Pharmaceuticals 2021, 14, 354 10 of 26

2.3. Anticancer Activity of Pyrrolo[3,4‐c]pyridine Derivatives In a study by Kalai et al. [21], 3,5‐bis(4‐fluorobenzylidene)‐1‐(1,1,3,3‐tetramethyl‐1,2‐ dihydropyrrolo[3,42.3. Anticancer Activity‐c]pyridin of Pyrrolo[3,4‐6‐yl)piperidin‐c]pyridine‐4‐ onDerivatives N‐oxide 18 was synthesized (Figure 13). The antitumorIn a study byactivity Kalai etof al. the [21], obtained 3,5‐bis(4 ‐compoundfluorobenzylidene) was assessed‐1‐(1,1,3,3 by‐ tetramethylmeasuring‐ 1,2its‐ cytotoxicitydihydropyrrolo[3,4 against‐ covarian]pyridin and‐6‐yl)piperidin breast cancer‐4‐on cell N ‐linesoxide and 18 was noncancerous synthesized cardiac (Figure cell13). lines.The antitumorTested compound activity 18of exhibitedthe obtained moderate compound cytotoxicity was againstassessed ovarian by measuring cancer cells its Pharmaceuticals 2021, 14, 354 andcytotoxicity limited toxicity against toward ovarian breast and breastcancer cancerand healthy cell lines cardiac and cell noncancerous lines [21]. cardiac10 of cell 25 lines. Tested compound 18 exhibited moderate cytotoxicity against ovarian cancer cells and limited toxicity towardO breast cancer and healthy cardiac cell lines [21].

O

F N F N F N F CH N 3 18 CH3 CH N 3 18 - CH3 H3C CH3 O N Figure 13. N‐oxide 3,5‐bis(4‐fluorobenzylidene)- ‐1‐(1,1,3,3‐tetramethyl‐1,2‐dihydropyrrolo[3,4‐ H3C CH O c]pyridin‐6‐yl)piperidin‐4‐on 18. 3 FigureFigure 13. 13.N-oxide N‐oxide 3,5-bis(4-fluorobenzylidene)-1-(1,1,3,3-tetramethyl-1,2-dihydropyrrolo[3,4- 3,5‐bis(4‐fluorobenzylidene)‐1‐(1,1,3,3‐tetramethyl‐1,2‐dihydropyrrolo[3,4c]pyridin-6-‐ yl)piperidin-4-onc]pyridinNicotinamide‐6‐yl)piperidin18. phosphoribosyltransferase‐4‐on 18. (NAMPT) catalyzes the condensation of nicotinamide (NAM) with 5‐phosphoribosyl‐1‐pyrophosphate to nicotinamide mononucleotideNicotinamideNicotinamide (NMN), phosphoribosyltransferase phosphoribosyltransferase which is the first step (NAMPT) in(NAMPT) NAD+ catalyzes biosynthesis. catalyzes the condensation the Blocking condensation ofNAMPT nicoti- of namideactivitynicotinamide (NAM)may impair with(NAM) cell 5-phosphoribosyl-1-pyrophosphate growth.with Thus,5‐phosphoribosyl NAMPT inhibition‐1‐pyrophosphate to is nicotinamide one of the antito mononucleotide‐ cancernicotinamide therapy (NMN),strategies.mononucleotide which is the (NMN), first step which in NAD+ is the biosynthesis.first step in NAD+ Blocking biosynthesis. NAMPT activity Blocking may NAMPT impair cellactivity growth.Dragovich may Thus, impair et NAMPT al. cell [22] growth. inhibition synthesized Thus, is NAMPT one 4‐ ofsulfonylobenzyl the inhibition anti-cancer is‐ one therapyderivatives of the strategies. anti of‐cancer pyrrolo[3,4 therapy‐ cstrategies.]pyridineDragovich‐ 2‐carboxamide et al. [22] synthesized 19 (Figure 4-sulfonylobenzyl- 14) and evaluated derivatives them as potent of pyrrolo[3,4- NAMPT cinhibitors.]pyridine- 2-carboxamideDerivativesDragovich that19 (Figureshowedet al. [22] 14 strong) andsynthesized evaluatedactivity against4 them‐sulfonylobenzyl as NAMPT, potent NAMPT good‐ derivatives in inhibitors. vitro stability of Derivativespyrrolo[3,4 against‐ thathumanc]pyridine showed liver‐2 ‐microsomes, strongcarboxamide activity and 19 against (Figure sufficient NAMPT, 14) water and evaluated good solubilityin vitro them werestability as referred potent against NAMPTfor further human inhibitors. studies. liver microsomes,ManyDerivatives of these andthat compounds sufficientshowed strong water exhibited solubility activity minimal against were referred inhibition NAMPT, for furthergoodof the in cytochrome studies. vitro stability Many P450 of against these 3A4 compoundsisoformhuman liverand exhibited severalmicrosomes, CYP minimal isoforms and inhibitionsufficient (2D6, water of1A2, the 2C19), solubility cytochrome but werewere P450 referredstrong 3A4 isoform CYP2C9 for further and inhibitors. severalstudies. CYPTheMany isoformsbinding of these (2D6,of compoundsthese 1A2, derivatives 2C19), exhibited but wereto mouseminimal strong plasma CYP2C9inhibition protein inhibitors. of the was cytochrome The typically binding moderateP450 of these 3A4 derivatives(approximatelyisoform and to several mouse 90% CYP plasmabound). isoforms protein Molecule (2D6, was 19a typically1A2, also 2C19), showed moderate but antiproliferativewere (approximately strong CYP2C9 effects 90% inhibitors. bound).against MoleculeseveralThe binding human19a alsoof tumor these showed cell derivatives antiproliferativelines. Based to onmouse that effects data,plasma against compound protein several was19a human wastypically tumor accepted cellmoderate for lines. in Basedvivo(approximately mouse on that PK data, and 90% xenograft compound bound). efficacyMolecule19a was studies. 19a accepted also The showed 19a for derivativein vivoantiproliferativemouse showed PK satisfactory and effects xenograft against PK efficacypropertiesseveral studies.human in mice tumor The and19a cellwasderivative lines. effective Based showed in theon thatPC satisfactory‐ 3data, mouse compound xenograft PK properties 19a model. was in acceptedDerivative mice and for was 19a in effectiveexhibitedvivo mouse in good the PK PC-3 bioavailabilityand mouse xenograft xenograft efficacyand plasma model. studies. exposure Derivative The 19a after 19aderivative oralexhibited administration showed good satisfactory bioavailability to female PK andNCRproperties plasma mice [22].in exposure mice and after was oral effective administration in the PC to‐3 female mouse NCR xenograft mice [model.22]. Derivative 19a exhibited good bioavailability and plasma exposure after oral administration to female NH2 NCR mice [22].O O

N N NH N NH NH2 O O N R N S S N N NH O N NH O 19O 19a O N R N S S R= O O 19O 19a O N , N , , , N , N N CH R= 3 CH3 N NH2 O O H3CN , N , , , N , N N CH FigureN 14.NH 4‐Sulfonylobenzyl3 ‐ derivativesCH3 of pyrrolo[3,4O ‐c]pyridineO ‐2‐carboxamide 19 as potent Figure 14. 2 4-Sulfonylobenzyl- derivatives of pyrrolo[3,4-c]pyridine-2-carboxamide 19 as potent inhibitors of NAMPT and the mostH3C active compound 19a. inhibitors of NAMPT and the most active compound 19a. Figure 14. 4‐Sulfonylobenzyl‐ derivatives of pyrrolo[3,4‐c]pyridine‐2‐carboxamide 19 as potent inhibitorsWojcicka of NAMPT et al. [20 and] synthesized the most active a series compound of 4-methyl-6-phenylpyrrolo[3,4- 19a. c]pyridine-1,3- dione derivatives by modifying of the substituent at position 2 of the pyrrolo[3,4 c]pyridine scaffold (Figure 15). N-alkil-4-methyl-6-phenyl-1H-pyrrolo[3,4-c]pyridine-1,3-diones 20a–s were obtained and screened for their antitumor activity in vitro. Mannich bases 20g–s with an IC50 value in the range of 19–29 µg/mL were the most active. Pharmaceuticals 2021, 14, 354 11 of 26

Pharmaceuticals 2021, 14, 354 11 of 26

Wojcicka et al. [20] synthesized a series of 4‐methyl‐6‐phenylpyrrolo[3,4‐c]pyridine‐ 1,3‐dione derivatives by modifying of the substituent at position 2 of the pyrrolo[3,4 c]pyridineWojcicka scaffold et al. [20](Figure synthesized 15). N‐alkil a series‐4‐methyl of 4‐‐6methyl‐phenyl‐6‐‐1phenylpyrrolo[3,4H‐pyrrolo[3,4‐c]pyridine‐c]pyridine‐1,3‐‐ 1,3diones‐dione 20a derivatives‐s were obtained by modifying and screened of the for substituent their antitumor at position activity 2 inof vitro.the pyrrolo[3,4 Mannich Pharmaceuticals 2021, 14, 354 cbases]pyridine 20g‐ sscaffold with an (Figure IC50 value 15). in N the‐alkil range‐4‐methyl of 19–29‐6‐ μphenylg/mL‐ were1H‐pyrrolo[3,4 the most active.‐c]pyridine 11‐ of1,3 25‐ diones 20a‐s were obtained and screened for their antitumor activity in vitro. Mannich O bases 20g‐s with an IC50 value in the range of 19–29 μg/mL were the most active. N a) R = CH2COOCH3 m) R = R O O b) R = CH2COOC2H5 O N N a)c) R = CH COCCOOCHH m) R = 2 6 5 3 n) R = N R d) R = CH O O O b) R = CH23COOC2H5 O N e) R = CH2CONHC2H5 c) R = CH2COC6H5 N n) R = N d)f) RR == CHCH2CONHCH 2C6H5 o) R = O 3 N g) R = CH N(C H ) O CH3 e) R = CH2CONHC4 9 22H5 N h) R = CH2NHC4H9 o) R = N 20 f) R = CH2CONHCH2C6H5 p) R = N N i) R = CH NHC H OC H g) R = CH2N(C 6H 5) 2 5 CH3 2 4 9 2 N j) R = CH2NHCH2-4-ClC6H5 h) R = CH2NHC4H9 20 p) R = NN k) R = CH2NHC6H5 r) R = i) R = CH2NHC6H5OC2H5 l) R = N j) R = CH2NHCH2-4-ClC6H5 N N k) R = CH2NHC6H5 r) R = s) R = N l) R = O N

N N

s) R = N O Figure 15. N‐alkil‐4‐methyl‐6‐phenyl‐1H‐pyrrolo[3,4‐c]pyridine‐1,3‐dione derivatives 20a‐s. Figure 15. N-alkil-4-methyl-6-phenyl-1H-pyrrolo[3,4-c]pyridine-1,3-dione derivatives 20a–s. Phosphoinositide 3‐kinases (PI3Ks) are a family of intracellular lipid signaling FigureenzymesPhosphoinositide 15. Nthat‐alkil catalyze‐4‐methyl the 3-kinases‐6 ‐phosphorylationphenyl‐ (PI3Ks)1H‐pyrrolo[3,4 are of athe‐ familyc]pyridine hydroxyl of‐ intracellular1,3 group‐dione in derivatives the lipid 3‐position signaling 20a‐s. of the en- zymesphosphatidylinositol that catalyze the ring. phosphorylation PI3Ks can control of the many hydroxyl vital cellular group in processes the 3-position such as of growth, the phos- phatidylinositolproliferation,Phosphoinositide and ring. survival. PI3Ks 3‐kinases canTherefore, control (PI3Ks) manythe areinhibition vital a family cellular of PI3Ksof processes intracellular can be such used as lipid growth,in anticancer signaling prolif- enzymeseration,therapy. and that survival. catalyze Therefore,the phosphorylation the inhibition of the of PI3Ks hydroxyl can group be used in in theanticancer 3‐position therapy. of the phosphatidylinositolCollierCollier et et al. al. [ 23[23]] ring. synthesized synthesized PI3Ks can 4-methyl-2-[1-(2,2,2,-trifluoroethyl)-1 4 control‐methyl many‐2‐[1‐(2,2,2, vital cellular‐trifluoroethyl) processesH‐1-pyrazol-4-yl]-1H such‐pyrazol as growth,‐4‐yl]H‐ - proliferation,pyrrolo[3,4-1H‐pyrrolo[3,4c]pyridine-3,6-dione and‐c]pyridine survival.‐3,6 Therefore,‐dione derivatives derivatives the inhibition21 (Figure 21 (Figure of16 ).PI3Ks The 16). substitution can The be substitution used of in the anticancer carbonyl of the therapy.groupcarbonyl in the group 6-position in the 6 with‐position a 5,6-dimethoxypyridin-3-yl with a 5,6‐dimethoxypyridin ring led‐3‐yl to ring derivative led to derivative21a, which showed21a, Collierwhich high etshowed PI3K al. [23]γ affinity high synthesized PI3K andγ inhibitedaffinity 4‐methyl theand‐2 monocyte ‐[1inhibited‐(2,2,2,‐ chemoattractant trifluoroethyl)the monocyte‐ 1chemoattractant protein-1H‐pyrazol MCP-1‐4‐yl] ‐ 1inducedproteinH‐pyrrolo[3,4‐1 chemotaxis MCP‐‐c1]pyridine induced of THP-1 ‐chemotaxis3,6‐dione cells (IC derivatives of50 THP= 270‐1 nM). cells 21 (Figure(IC50 = 270 16). nM). The substitution of the carbonyl group in the 6‐position with a 5,6‐dimethoxypyridin‐3‐yl ring led to derivative CH 21a, which showedO high PI3K3 γ affinity and inhibited Othe monocyteCH3 chemoattractant protein‐1 MCP‐1 induced chemotaxisR of THP‐1 cells (IC50 = 270 nM). N N N N N CH N CH N O 3 N O 3 RO N F N N NF N N F N F N F NF O 21 O 21a N F F O CH3 H3C F F F O FigureFigure 16. 4‐4-Methyl-2-[1-(2,2,2,-trifluoroethyl)-1Methyl‐2‐[1‐(2,2,2,‐trifluoroethyl)F‐1H‐pyrazolH-pyrazol-4-yl]-1‐4‐yl]‐1H‐pyrrolo[3,4H-pyrrolo[3,4-‐c]pyridinc]pyridin-3-one‐3‐one 21 21a derivativesderivatives 2121 andand compoundcompound 21a21a as the potent inhibitor of PI3Ks. PI3Ks. O CH3 H3C FigureSpleenSpleen 16. 4‐Methyl tyrosinetyrosine‐2‐[1 kinase‐kinase(2,2,2,‐ (SYK) trifluoroethyl)(SYK) is is a a non-receptor non‐1H‐‐pyrazolreceptor‐ cytoplasmic4 ‐yl]cytoplasmic‐1H‐pyrrolo[3,4 kinase kinase‐c]pyridin that that is ais‐3 key ‐onea key me- derivativesdiatormediator for for various21 andvarious compound inflammatory inflammatory 21a as cells.the cells. potent Therefore Therefore inhibitor its its of inhibition inhibition PI3Ks. isis an essential therapeutic therapeutic targettarget in in bothboth neoplasticneoplastic and inflammatoryinflammatory diseases. FLT3 FLT3 (fms (fms like like tyrosine tyrosine kinase kinase 3) 3) is is a a cytokinecytokineSpleen receptorreceptor tyrosine expressedexpressed kinase onon(SYK) thethe surfaceis a non of‐ manyreceptor hematopoietic cytoplasmic progenitor progenitor kinase that cells. cells. is a key mediatorLam for et al.various [24] prepared inflammatory 6-[(2-aminocyclohexyl)amino]-1,2-dihydro-3 cells. Therefore its inhibition is an essentialH-pyrrolo[3,4- therapeutic targetc]pyridin-3-one in both neoplastic derivatives and 22inflammatoryas SYK inhibitors diseases. (Figure FLT3 (fms17). like The tyrosine 4-(3-methylanilino)- kinase 3) is a cytokinesubstituted receptor compounds expressed exhibited on the surface good enzymatic of many hematopoietic and cellular potency. progenitor It turnedcells. out that the 7-fluoro substitution had a significant effect on the activity. Replacing methy- laniline with a methylpyrazole ring increased the activity of the 4-fluoro derivative. The methylpyrazole derivative 22a showed the best in vitro profile and was selective for kinases (it was potent toward both SYK and FLT3). Compound 22a showed inhibition toward an SYK-dependent and FLT3-dependent cell line in a cell proliferation assay. It demonstrated strong tumor growth inhibition (TGI) after 20 days of treatment. This derivative blocked PharmaceuticalsPharmaceuticals 20212021,, 1414,, 354354 1212 ofof 2626

LamLam etet al.al. [24][24] preparedprepared 66‐‐[(2[(2‐‐aminocyclohexyl)amino]aminocyclohexyl)amino]‐‐1,21,2‐‐dihydrodihydro‐‐33HH‐‐pyrrolo[3,4pyrrolo[3,4‐‐ cc]pyridin]pyridin‐‐33‐‐oneone derivativesderivatives 2222 asas SYKSYK inhibitorsinhibitors (Figure(Figure 17).17). TheThe 44‐‐(3(3‐‐methylanilino)methylanilino)‐‐ substitutedsubstituted compoundscompounds exhibitedexhibited goodgood enzymaticenzymatic andand cellularcellular potency.potency. ItIt turnedturned outout thatthat thethe 77‐‐fluorofluoro substitutionsubstitution hadhad aa significantsignificant effecteffect onon thethe activity.activity. ReplacingReplacing methylanilinemethylaniline withwith aa methylpyrazolemethylpyrazole ringring increasedincreased thethe activityactivity ofof thethe 44‐‐fluorofluoro derivative.derivative. TheThe Pharmaceuticals 2021, 14, 354 12 of 25 methylpyrazolemethylpyrazole derivativederivative 22a22a showedshowed thethe bestbest inin vitrovitro profileprofile andand waswas selectiveselective forfor kinaseskinases (it(it waswas potentpotent towardtoward bothboth SYKSYK andand FLT3).FLT3). CompoundCompound 22a22a showedshowed inhibitioninhibition towardtoward anan SYKSYK‐‐dependentdependent andand FLT3FLT3‐‐dependentdependent cellcell lineline inin aa cellcell proliferationproliferation assay.assay. ItIt demonstratedanti-IgDdemonstrated stimulated strongstrong expression tumortumor growthgrowth of CD86 inhibitioninhibition in murine (TGI)(TGI) peripheral afterafter 2020 B cellsdaysdaysin ofof vivo treatment.treatment.. Compound ThisThis derivative22aderivativenamed blockedblocked TAK-659 antianti has‐‐IgDIgD been stimulatedstimulated used in expression clinicalexpression trials ofof CD86 toCD86 treat inin advanced murinemurine peripheralperipheral solid tumors BB cellscells and inin vivo.lymphomavivo. CompoundCompound malignancies 22a22a namednamed [24]. TAKTAK‐‐659659 hashas beenbeen usedused inin clinicalclinical trialstrials toto treattreat advancedadvanced solidsolid tumorstumors andand lymphomalymphoma malignanciesmalignancies [24].[24]. R R FF

HN NHNH2 NH HN NH 2 HHNN NH22 NH NHNH N O N NN O OO HHNN

NN NN 22a22a 2222 N CH3 N CH3 TAK-659 NH TAK-659 H C NH H33C RR == F,F, H,H, Cl,Cl, Me,Me, CN,CN,

Figure 17. 6‐[(2‐Aminocyclohexyl)amino]‐1,2‐dihydro‐3H‐pyrrolo[3,4‐c]pyridin‐3‐one derivatives Figure 17. 66-[(2-Aminocyclohexyl)amino]-1,2-dihydro-3‐[(2‐Aminocyclohexyl)amino]‐1,2‐dihydro‐3HH-pyrrolo[3,4-‐pyrrolo[3,4c‐c]pyridin-3-one]pyridin‐3‐one derivatives derivatives22 22 as SYK inhibitors and compound 22a used in clinical trials. 22as SYKas SYK inhibitors inhibitors and and compound compound22a 22aused used in clinicalin clinical trials. trials.

2.4.2.4. AntiAnti Anti-Inflammatory‐‐InflammatoryInflammatory Activity Activity of of Pyrrolo[3,4 Pyrrolo[3,4-c]pyridinePyrrolo[3,4‐‐c]pyridinec]pyridine Derivatives Derivatives MatrixMatrix metalloproteinases metalloproteinases (MMPs) (MMPs) are are zinc zinc-dependentzinc‐‐dependentdependent proteolytic proteolytic enzymes enzymes involved involved inin the the degradation and regeneration of of the the extracellular extracellular matrix.matrix. High High levels levels of of MMPs MMPs may may causecause a a varietyvariety ofof pathologies,pathologies, includingincluding metastaticmetastatic cancer cancer and and arthritis,arthritis, andand thereforetherefore inhibitioninhibition of of these these enzymes enzymes is is considered considered anan importantimportant therapeutictherapeutic target.target. CholletChollet etetet al. al.al. [ 25[25][25]] synthesized synthesizedsynthesized 5-(1,3-dioxo-1,3-dihydro-2 55‐‐(1,3(1,3‐‐dioxodioxo‐‐1,31,3‐‐dihydrodihydroH‐‐22-pyrrolo[3,4-HH‐‐pyrrolo[3,4pyrrolo[3,4c]pyridin-2-yl)-‐‐cc]pyridin]pyridin‐‐22‐‐ yl)2-{[(4’-chloro[1,1’-biphenyl]-4-yl)sulfanyl]methyl}-yl)‐‐22‐‐{[(4’{[(4’‐‐chloro[1,1’chloro[1,1’‐‐biphenyl]biphenyl]‐‐44‐‐yl)sulfanyl]methyl}yl)sulfanyl]methyl}N-hydroxypentanamide‐‐NN‐‐hydroxypentanamidehydroxypentanamide23 (2323Figure (Figure(Figure 18) 50 50 50 18)as18) an asas an inhibitoran inhibitorinhibitor of ofof MMPs MMPsMMPs with withwith IC ICIC5050= == 33 nM nM forfor MMPMMP MMP-2,‐‐2,2, ICIC IC5050 == 1212= nM 12nM nM forfor MMP forMMP MMP-9‐‐99 andand andICIC50

=IC= 848450 nMnM= 84 forfor nM MMPMMP for MMP-13.‐‐13.13.

OO ONH N ONH N OHOH NN SS

OO

2323 Cl Cl FigureFigure 18. 18. 555-(1,3-Dioxo-1,3-dihydro-2‐‐(1,3(1,3‐‐DioxoDioxo‐‐1,31,3‐‐dihydrodihydro‐‐22HHH‐‐pyrrolo[3,4pyrrolo[3,4-pyrrolo[3,4-‐‐cc]pyridin]pyridinc]pyridin-2-yl)-2-{[(4’-chloro[1,1’-biphenyl]-4-‐‐22‐‐yl)yl)‐‐22‐‐{[(4’{[(4’‐‐chloro[1,1’chloro[1,1’‐‐biphenyl]biphenyl]‐‐44‐‐ yl)sulfanyl]methyl}yl)sulfanyl]methyl}-yl)sulfanyl]methyl}‐‐NN‐-hydroxypentanamide‐hydroxypentanamidehydroxypentanamide 2323..

SondhiSondhi etetet al. al. [al.26 ][26] synthesized[26] synthesizedsynthesized 2-((tetrahydrofuran-2-yl)methyl)-2 22‐‐((tetrahydrofuran((tetrahydrofuran‐‐22‐‐yl)methyl)yl)methyl)H-pyrrolo[3,4-‐‐22HH‐‐pyrrolo[3,4pyrrolo[3,4c]pyridine-‐‐ c1,3-dionec]pyridine]pyridine24‐‐1,31,3(Figure‐‐dionedione 192424) and(Figure(Figure evaluated 19)19) andand it for evaluatedevaluated anti-inflammatory itit forfor antianti‐ activity.‐inflammatoryinflammatory Compound activity.activity.24 CompoundshowedCompound 26% 2424 activity showedshowed at 26%26% a dose activityactivity of 50 mg/kgatat aa dosedose per ofof os. 5050 mg/kgmg/kg perper os.os.

PharmaceuticalsPharmaceuticals 20212021, 14, 14, 354, 354 1313 of of 26 25 Pharmaceuticals 2021, 14, 354 13 of 26

O O

N N N N O O O O 24 24

FigureFigure 19. 19. 2‐((Tetrahydrofuran2-((Tetrahydrofuran-2-yl)methyl)-2‐2‐yl)methyl)‐2HH‐pyrrolo[3,4-pyrrolo[3,4-‐c]pyridinec]pyridine-1,3-dione‐1,3‐dione 2424. . Figure 19. 2‐((Tetrahydrofuran‐2‐yl)methyl)‐2H‐pyrrolo[3,4‐c]pyridine‐1,3‐dione 24. 2.5.2.5. Sedative Sedative Activity Activity of ofPyrrolo[3,4 Pyrrolo[3,4-c]pyridine‐c]pyridine Derivatives Derivatives 2.5. Sedative Activity of Pyrrolo[3,4‐c]pyridine Derivatives WhileWhile conducting conducting studies onon pyrrolo[3,4- pyrrolo[3,4c]pyridines‐c]pyridines to obtainto obtain new anxiolyticnew anxiolytic deriva- derivativestivesWhile of the of buspirone conductingthe buspirone type, studies type,Sladowska’s´ Śonladowska’s pyrrolo[3,4 team team noticed‐c]pyridines noticed that that theseto theseobtain compounds compounds new anxiolytic are oftenare oftennotderivatives activenot active in of this the in regard. buspironethis regard. Instead, type, Instead, they Śladowska’s show they other show team pharmacological other noticed pharmacological that these effects, compounds in effects, particular, inare particular,sedativeoften not [sedative 27active] and analgesicin [27] this and regard. analgesic activity Instead, [27 activity–29 ].they The [27–29]. analgesicshow Theother activityanalgesic pharmacological of activity the new of pyrrolo[3,4- effects,the new in pyrrolo[3,4cparticular,]pyridines‐c ]pyridinessedative is described [27] is described inand the analgesic following in the activity section.following [27–29]. section. The analgesic activity of the new pyrrolo[3,4TakingTaking ‐intoc into]pyridines accountaccount is the thedescribed results results of in previous ofthe previous following work work [section.28,29 ],[28,29], in 1994, in the 1994, above the researchers above researchersobtainedTaking a obtained series into ofaccount a nine series new theof N-substitutednine results new ofN ‐previoussubstituted piperazinalkyl work piperazinalkyl derivatives[28,29], in derivatives of1994, 6-methyl-2-(1- the ofabove 6‐ methylpiperidine)-pyridine-1,3-dioneresearchers‐2‐(1‐piperidine) obtained a‐pyridine series of (Figure‐ 1,3nine‐dione new 20)[ (Figure N27‐substituted]. 20) [27]. piperazinalkyl derivatives of 6‐ methyl‐2‐(1‐piperidine)‐pyridine‐1,3‐dione (Figure 20) [27]. R R N N O O H3C N H3C N

N n=1,3,4 N N n=1,3,4 N R R O CH3 N O CH3 N C6H5 o‐chlorophenylC6H5 o‐chlorophenyl pyrimidynyl pyrimidynyl FigureFigure 20. 20. GeneralGeneral structure structure pyrrolo[3,4 pyrrolo[3,4-‐c]pyridinesc]pyridines [27]. [27 ]. Figure 20. General structure pyrrolo[3,4‐c]pyridines [27]. SeveralSeveral pharmacological pharmacological studies studies were were performed performed with with regard regard to to the the newly newly obtained obtained structures,structures,Several including including pharmacological testing testing for: for: studies were performed with regard to the newly obtained structures, including testing for: ● • acuteacute toxicity toxicity in in mice; mice; •● acute toxicity in mice; ● spontaneousspontaneous locomotor locomotor activity activity in in mice; mice; •● pantetrazol-inducedspontaneous locomotor seizures activity in mice;in mice; ● pantetrazol‐induced seizures in mice; •● anxiolyticpantetrazol properties‐induced seizures in a four-plate in mice; test in mice; ● • anxiolyticamphetamine-induced properties in a motor four‐plate hyperactivity test in mice; in mice; ● anxiolytic properties in a four‐plate test in mice; ● • amphetaminepain reactivity‐induced in a writhing motor hyperactivity syndrome test in in mice; mice. ● amphetamine‐induced motor hyperactivity in mice; ● painThe reactivity test results in showeda writhing that syndrome out of the test tested in mice. series of compounds, derivatives 25 and 26● (Figurepain reactivity 21) significantly in a writhing decreased syndrome locomotor test in activity mice. during a one-hour observation. The test results showed that out of the tested series of compounds, derivatives 25 and 26 (FigureThe 21)test significantly results showed decreased that out locomotor of the tested activity series during of compounds, a one‐hour derivatives observation. 25 and 26 (Figure 21) significantly decreased locomotor activity during a one‐hour observation.

Pharmaceuticals 2021, 14, 354 14 of 25 Pharmaceuticals 2021, 14, 354 14 of 26 Pharmaceuticals 2021, 14, 354 14 of 26

O NN O NN H3C H3C H3C H3C N N N N O N N O N N N N N N N O N N N O N N N N O O

25 26 25 26 Figure 21. Derivatives of pyrrolo[3,4‐c]pyridines 25 and 26 with significantly sedative activity [27]. FigureFigure 21. 21.Derivatives Derivatives of of pyrrolo[3,4- pyrrolo[3,4c‐]pyridinesc]pyridines25 25and and26 26with with significantly significantly sedative sedative activity activity [27 [27].]. Compound 25 showed this effect at doses of 1/10 and 1/20 LD50, 26 at doses of 1/10, CompoundCompound 25 25 showed showed this this effect effect at at doses doses of of 1/10 1/10 and and 1/20 1/20 LD LD5050,, 26 26 at at doses doses of of 1/10, 1/10, 1/20, 1/40, and even 1/80 of LD50 [27]. In the other tests mentioned above, the activity of 1/20,1/20, 1/40,1/40, and even 1/801/80 of LD50 [27].[27]. In In the other teststests mentionedmentioned above,above, the the activity activity of of compounds was not as promising. None of the compounds tested at doses of 1/10 LD50 compoundscompounds was was not not as as promising. promising. None None of of the the compounds compounds tested tested at at doses doses of of 1/10 1/10 LD LD50 showed antinociceptive activity in the writhing test [27]. 50 showedshowed antinociceptive antinociceptive activity activity in in the the writhing writhing test test [ 27[27].]. A year later, the same scientists obtained another series of compounds derived from AA year year later, later, the the same same scientists scientists obtained obtained another another series series of of compounds compounds derived derived from from pyrrolo[3,4‐c]pyridines [30]. This time, they studied the influence of other amino‐ and pyrrolo[3,4-pyrrolo[3,4c‐]pyridinesc]pyridines [30 [30].]. This This time, time, they they studied studied the influencethe influence of other of amino-other amino and alkyl-‐ and alkyl‐(aryl‐)aminoalkyl(hydroxyalkyl) substituents on the activity of 3,4‐ (aryl-)aminoalkyl(hydroxyalkyl)alkyl‐(aryl‐)aminoalkyl(hydroxyalkyl) substituents substituents on the activity on of 3,4-pyridinedicarboximide.the activity of 3,4‐ pyridinedicarboximide. They synthesized fifteen differently substituted derivatives, of Theypyridinedicarboximide. synthesized fifteen They differently synthesized substituted fifteen derivatives, differently ofsubstituted which only derivatives, compound of which only compound 27 (Figure 22) deserved attention. Only compound 27 significantly 27which(Figure only 22 compound) deserved 27 attention. (Figure Only22) deserved compound attention. 27 significantly Only compound depressed 27 significantly locomotor depressed locomotor activity at doses of 1/10 and 1/20 LDmin. [30]. activitydepressed at doses locomotor of 1/10 activity and 1/20 at doses LDmin of.[ 1/1030]. and 1/20 LDmin. [30].

H3C H3C O O N OH N N OH N N N N N N N O O 27 27

Figure 22. 2‐[2‐hydroxy‐3‐(4‐phenyl‐1‐piperazinyl)propyl]‐4‐piperidino‐6‐methyl‐1H‐pyrrolo[3,4‐ FigureFigure 22. 22.2-[2-hydroxy-3-(4-phenyl-1-piperazinyl)propyl]-4-piperidino-6-methyl-1 2‐[2‐hydroxy‐3‐(4‐phenyl‐1‐piperazinyl)propyl]‐4‐piperidino‐6‐methyl‐1H‐-pyrrolo[3,4-pyrrolo[3,4‐ c]pyridine‐1,3(2H)‐dione 27. c]pyridine-1,3(2c]pyridine‐1,3(2HH)-dione)‐dione27 27. . TheThe strongstrong pharmacological pharmacological activity activity of compoundof compound27 encouraged 27 encouraged scientists scientists to intro- to The strong pharmacological activity of compound 27 encouraged scientists to duceintroduce further further modifications. modifications. They obtained They obtained a series ofa series compounds of compounds in which the in piperidinewhich the introduce further modifications. They obtained a series of compounds in which the grouppiperidine present group at position present 2 wasat position replaced 2 with was pyrrolidine replaced with or morpholine. pyrrolidine Phenylpiperazine or morpholine. piperidine group present at position 2 was replaced with pyrrolidine or morpholine. orPhenylpiperazine pyrimidinylpiperazine or pyrimidinylpiperazine was retained as an amine was residueretained of theas an pharmacophore. amine residue The of link the Phenylpiperazine or pyrimidinylpiperazine was retained as an amine residue of the betweenpharmacophore. the core and The the link pharmacophore between the fragment core and was the a propyl,pharmacophore butyl, or 2-hydroxypropylfragment was a pharmacophore. The link between the core and the pharmacophore fragment was a chainpropyl, [31 butyl,]. or 2‐hydroxypropyl chain [31]. propyl, butyl, or 2‐hydroxypropyl chain [31]. ThisThis time,time, allall thethe testedtested substancessubstances significantlysignificantly inhibitedinhibited thethe spontaneousspontaneous locomotorlocomotor This time, all the tested substances significantly inhibited the spontaneous locomotor activityactivity ofof micemice duringduring aa one-hourone‐hour observation.observation. CompoundsCompounds 2828 andand 2929 showedshowed thethe most most activity of mice during a one‐hour observation. Compounds 28 and 29 showed the most potentpotent sedative sedative effects effects (Figure (Figure 23 23).). These These compounds compounds were were active active up up to to doses doses of of 1/160 1/160 LD LD5050 potent sedative effects (Figure 23). These compounds were active up to doses of 1/160 LD50 ((2828)) andand 1/801/80 LD 50 ((2929).). (28) and 1/80 LD50 (29).

Pharmaceuticals 2021, 14, 354 15 of 25 PharmaceuticalsPharmaceuticals 2021 2021, 14, 14, 354, 354 1515 of of 26 26

H C H C H3 C H3C 3 NN OO OO N N N OHOH NN N OHOH NN NN NN NN NN NN ClCl N OO O

2828 2929

FigureFigureFigure 23. 23.23. Pyrrolo[3,4 Pyrrolo[3,4Pyrrolo[3,4-‐‐cc]pyridines]pyridinesc]pyridines 282828 andandand 29 29significantlysignificantly inhibited inhibited thethe spontaneousspontaneous the spontaneous locomotor locomotor locomotor activityactivity in in mice. mice. activity in mice.

TheTheThe results resultsresults indicate indicateindicate that that replacing replacing thethe piperidine piperidine ring ring atat at positionposition position 22 2 ofof of the the the pyrrolo[3,4 pyrrolo[3,4 pyrrolo[3,4-‐‐ c]c]c]pyridinepyridinepyridine scaffold scaffold scaffold with with other other pharmacophore pharmacophore substituents doesdoes notnot substantiallysubstantially alter alter the the pharmacologicalpharmacological action action profile profileprofile [31]. [[31].31]. ContinuingContinuingContinuing the the research,research, research, thethe the scientistsscientists scientists synthesized synthesized thethe the nextnext next seriesseries series ofof compounds ofcompounds compounds in in whichinwhich which thethe the piperidinepiperidine piperidine groupgroup group waswas was replacedreplaced replaced by by an an alkoxy alkoxy groupgroup group [32].[32]. [32]. TheThe The structuresstructures structures ofof compoundscompoundscompounds 30–34 3030–34–34 are are shown shown in in Figure Figure 24.24.

Figure 24. New derivatives of 2,3‐dihydro‐4‐methoxy(ethoxy)‐6‐methyl‐1,3‐dioxo‐1H‐pyrrolo[3,4‐ FigureFigure 24. 24. NewNew derivatives derivatives of of 2,3 2,3-dihydro-4-methoxy(ethoxy)-6-methyl-1,3-dioxo-1‐dihydro‐4‐methoxy(ethoxy)‐6‐methyl‐1,3‐dioxo‐1HH‐pyrrolo[3,4-pyrrolo[3,4-‐ c]pyridine 30a‐e [32]. cc]pyridine]pyridine 30a30a‐–ee [32].[32]. Compounds 30c, 30d, and 30e significantly suppressed the spontaneous locomotor CompoundsCompounds 30c,30c, 30d30d,, and 30e significantlysignificantly suppressed the spontaneous locomotor activity of mice during a 30 min observation up to a dose of 12.5mg/kg. Imide 31 was activityactivity of of mice during a 30 minmin observationobservation upup toto aa dosedose ofof 12.5mg/kg. 12.5mg/kg. Imide 31 was active at a dose of 200 mg/kg. All of the studied compounds (30a–e) exhibited analgesic activeactive at a dose of 200 mg/kg.mg/kg. All All of of the the studied compounds ( 30a–e30a–e) exhibited analgesic activity [32]. It is described in the following section. activityactivity [32]. [32]. It It is is described described in in the the following following section. section. In the next publication [33], Śladowska’s´ team described the synthesis and properties InIn the the next next publication publication [33], [33], Śladowska’sSladowska’s team team described described the the synthesis synthesis and and properties proper- tiesof of2‐(4 2-(4-substituted)butyl‐substituted)butyl derivatives derivatives of of some some 2,3-dihydro-1,3-dioxo-12,3‐dihydro‐1,3‐dioxo‐1HH‐pyrrolo[3,4-pyrrolo[3,4-‐ ofc]pyridines. 2‐(4‐substituted)butyl New derivatives derivatives have an alkoxyof some group 2,3 ‐atdihydro position‐1,3 2,‐dioxo butyl‐ 1linker,H‐pyrrolo[3,4 and an ‐ cc]pyridines.]pyridines. New derivativesderivatives have have an an alkoxy alkoxy group group at position at position 2, butyl 2, butyl linker, linker, and an and amine an residueamine residue (arylpiperazines, (arylpiperazines, or 1,2,3,4-tetrahydroisoquinoline or 1,2,3,4‐tetrahydroisoquinoline substituent) substituent) instead instead of a piper- of aminea piperazinyl residue (arylpiperazines,group. All tested or compounds 1,2,3,4‐tetrahydroisoquinoline significantly suppressed substituent) the spontaneous instead of aazinyl piperazinyl group. Allgroup. tested All compounds tested compounds significantly significantly suppressed suppressed the spontaneous the spontaneous locomotor activitylocomotor of miceactivity during of amice 30-min during observation. a 30‐min While observation. 2-[(4-phenyl-1-piperazinyl)butyl]-4- While 2‐[(4‐phenyl‐1‐ locomotorpiperazinyl)butyl] activity‐ 4‐ofmethoxy mice ‐6during‐methyl ‐1aH ‐30pyrrolo[3,4‐min observation.‐c]pyridine ‐While1,3(2H )‐2dione‐[(4‐phenyl 31‐1 ‐ piperazinyl)butyl]methoxy-6-methyl-1‐4‐Hmethoxy-pyrrolo[3,4-‐6‐methylc]pyridine-1,3(2‐1H‐pyrrolo[3,4H)-dione‐c]pyridine 31 (Figure‐1,3(2 25H),)‐dione given in doses31 (Figure 25), given in doses of 1/20, 1/40 and 1/80 LD50, inhibited spontaneous locomotor of 1/20, 1/40 and 1/80 LD50, inhibited spontaneous locomotor activity in mice by 86 (Figure 25), given in doses of 1/20, 1/40 and 1/80 LD50, inhibited spontaneous locomotor (activityp < 0.02), in 67 mice (p < by 0.05), 86 (p and < 0.02), 47%, 67 respectively. (p < 0.05), and 47%, respectively. activity in mice by 86 (p < 0.02), 67 (p < 0.05), and 47%, respectively.

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O NN O NN H3C H3C N N N N O O O H3C O H3C 31 31

Figure 25. 2‐[(4‐phenyl‐1‐piperazinyl)butyl]‐4‐methoxy‐6‐methyl‐1H‐pyrrolo[3,4‐c]pyridine‐ Figure 25. 2-[(4-phenyl-1-piperazinyl)butyl]-4-methoxy-6-methyl-1H-pyrrolo[3,4-c]pyridine-1,3(2H)- 1,3(2FigureH)‐ 25.dione 2‐[(4 31.‐phenyl ‐1‐piperazinyl)butyl]‐4‐methoxy‐6‐methyl‐1H‐pyrrolo[3,4‐c]pyridine‐ dione1,3(2H31)‐.dione 31. The other compounds in in this this series series significantly significantly reduced reduced locomotor locomotor activity activity in in mice, mice, by 87–The82% other when compounds administered in atthis 1/20 series LD50 significantly. However, when reduced administered locomotor at activity doses of in 1/40 mice, by 87–82% when administered at 1/20 LD50. However, when administered at doses of 1/40 andby 87 1/80LD–82% when50, they administered decreased spontaneous at 1/20 LD50 . locomotorHowever, whenactivity administered by 59–47% atand doses 26– 13%,of 1/40 and 1/80LD50, they decreased spontaneous locomotor activity by 59–47% and 26–13%, and 1/80LD50, they decreased spontaneous locomotor activity by 59–47% and 26–13%, respectivelyrespectively [[33].33]. respectively [33]. InIn 2009, researchers reported [34] [34] the the synthesis synthesis and and pharmacological pharmacological results results of of novel novel In 2009, researchers reported [34] the synthesis and pharmacological results of novel 11HH-pyrrolo[3,4-‐pyrrolo[3,4‐cc]pyridine-1,3(2]pyridine‐1,3(2H)-diones.)‐diones. These These compounds compounds were were obtained obtained by by modifying modifying 1H‐pyrrolo[3,4‐c]pyridine‐1,3(2H)‐diones. These compounds were obtained by modifying previouslypreviously describeddescribed structures [32,35,36]. [32,35,36]. The The modifications modifications consisted consisted of of the the following: following: ●previously described structures [32,35,36]. The modifications consisted of the following: • replacementreplacement of of the the phenyl phenyl ring ring at atthe the N‐4 N-4 position position of piperazine of piperazine with benzyl with benzyl or or ● benzhydrylbenzhydrylreplacement groups; of the phenyl ring at the N‐4 position of piperazine with benzyl or •● replacementreplacementbenzhydryl ofgroups; N-substitutedN‐substituted piperazines by other cyclic amines amines (morpholine, (morpholine, piperi- ● dine,piperidine,replacement or pyrrolidine). or ofpyrrolidine). N‐substituted piperazines by other cyclic amines (morpholine, Thepiperidine, authors oraimed pyrrolidine). was to investigate if if and and how how the the performed performed structural structural changes changes wouldwouldThe affect authors the the toxicity, toxicity, aimed analgesic,was analgesic, to investigate and and sedative sedative if and effects how effects of the the of performed tested the tested compounds structural compounds [34]. changes The [34 ]. Theresultswould results affectindicated indicated the toxicity, that that all analgesic, all tested tested andpyrrolo[3,4 pyrrolo[3,4- sedative‐c effects]pyridinec]pyridine-1,3(2 of ‐the1,3(2 testedHH)‐diones)-diones compounds significantly significantly [34]. The suppressedsuppressedresults indicated the spontaneous that all locomotor tested pyrrolo[3,4 activity activity of of ‐micec mice]pyridine during during‐1,3(2 a a 30 30-minH‐min)‐diones observation. observation. significantly suppressedThe mostmost the potent potent spontaneous effect effect (ED (ED locomotor5050= = 12.0312.03 mg/kg) activitymg/kg) was wasof mice producedproduced during by by a compound 30compound‐min observation.32 32(Figure (Figure 26 ). Moreover,26). Moreover,The most compound compoundpotent32 effectwas 32 (ED notwas50 toxic not= 12.03 toxic (LD 50mg/kg) (LD>200050 >2000was mg/kg) produced mg/kg) [34]. [34]. The by compoundThe antinociceptive antinociceptive 32 (Figure activ- ityactivity26). of Moreover, the of pyrrolo[3,4- the pyrrolo[3,4 compoundc]pyridines‐c]pyridines 32 was derivatives not derivatives toxic described (LD 50described >2000 in this mg/kg) in publication this [34].publication The is reportedantinociceptive is reported in the followinginactivity the following of section. the pyrrolo[3,4 section. ‐c]pyridines derivatives described in this publication is reported in the following section.

O HO N H3C O HO N

H3C N N N N O O H3C O O 32

H3C Figure 26. 2-[2-hydroxy-3-(4-benzyl-1-piperidinyl)propyl]-4,6-dimethyl-132 H-pyrrolo[3,4-c]pyridine- Figure 26. 2‐[2‐hydroxy‐3‐(4‐benzyl‐1‐piperidinyl)propyl]‐4,6‐dimethyl ‐1H‐pyrrolo[3,4‐c]pyridine‐ 1,3(21,3(2HH)-dione)‐dione 32.. Figure 26. 2‐[2‐hydroxy‐3‐(4‐benzyl‐1‐piperidinyl)propyl]‐4,6‐dimethyl‐1H‐pyrrolo[3,4‐c]pyridine‐ 1,3(2InInH) ‐ 2020,dione2020, Szkatuła 32Szkatu. ł eta al.et [al.37 ][37] published published a paper a reportingpaper reporting the synthesis the synthesis and pharmaco- and logicalpharmacological results of novelresults 1H -pyrrolo[3,4-of novel 1Hc‐]pyridine-1,3(2pyrrolo[3,4‐c]pyridineH)-dione‐1,3(2 derivativesH)‐dione with derivatives potential sedativeIn and2020, analgesic Szkatuła activity. et al. The[37] structures published of a new paper derivatives reporting33a –the33h synthesisare shown and in Figurepharmacological 27[37]. results of novel 1H‐pyrrolo[3,4‐c]pyridine‐1,3(2H)‐dione derivatives

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Pharmaceuticals 2021, 14, 354 17 of 25 with potential sedative and analgesic activity. The structures of new derivatives 33a–33h are shown in Figure 27 [37].

O

H3C amine N N n

O O R 33 Compd. R n amine Compd. R n amine

33a CH3 4 33e C2H5 2 HON HN N

H CO 3 33b CH3 2 33f CH3 1 HN N HN N

Cl 33c C2H5 2 33g C2H5 1 HN N HN N

Cl 33d CH3 2 33h CH3 1 HN N HN N

H CO 3 Cl

FigureFigure 27. 27. NewNew derivatives derivatives of of 1 1HH‐-pyrrolo[3,4-pyrrolo[3,4‐cc]pyridine-1,3(2]pyridine‐1,3(2H)-dioneH)‐dione33a 33a–33h–33h with with a sedative a sedative activity activity (and an(and analgesic an analgesic activity). activity). All new compounds 33a–h inhibited the locomotor activity in mice, and the two mostAll active new compounds (33b, 33d) 33a–h also extended inhibited the the durationlocomotor of activity thiopental in mice, anesthesia. and the two Biological most activestudies (33b, concerning 33d) also imides extended33b theand duration33d were of supplemented thiopental anesthesia. with the determination Biological studies of the concerningeffect of intraperitoneal imides 33b and administration 33d were supplemented of tested compounds with the ondetermination the duration of of the thiopental- effect of inducedintraperitoneal sleep. The administration mechanism and of extenttested of compounds blood-brain on barrier the duration crossing wereof thiopental not defined.‐ inducedHowever, sleep. the observedThe mechanism sedative and effect extent may indicateof blood good‐brain penetration barrier crossing of the compoundswere not defined.in question However, into the the central observed nervous sedative system, effect which may hasindicate not been good proven penetration either [of37 ].the As compoundsmentioned in earlier, question the into authors the central also described nervous the system, analgesic which activity has not of been the new proven derivatives. either [37].Their As results mentioned are presented earlier, the in theauthors section also Analgesic described activity. the analgesic activity of the new derivatives. Their results are presented in the section Analgesic activity. 2.6. Analgesic Activity of Pyrrolo[3,4-c]pyridine Derivatives 2.6. AnalgesicAs mentioned Activity of above, Pyrrolo[3,4Sladowska’s´ ‐c]pyridine team Derivatives initiated work on pyrrolo[3,4-c]pyridine derivativesAs mentioned to synthesize above, Ś buspirone-inspiredladowska’s team compounds.initiated work As on a result pyrrolo[3,4 of pharmacological‐c]pyridine derivativesstudies, these to synthesize compounds buspirone were found‐inspired not compounds. to have the intendedAs a result anxiolytic of pharmacological effect but to studies,exhibit these other compounds pharmacological were effects.found not The to first have structure the intended that inspired anxiolytic the authors effect but to work to exhibiton analgesic other pharmacological pyrrolo[3,4-c]pyridine effects. derivatives The first structure was the that non-toxic inspired compound the authors27 to[27 work,30,31 ]. onThe analgesic researchers pyrrolo[3,4 decided‐c]pyridine to modify derivatives the structure was of this the compoundnon‐toxic compound to obtain new 27 [27,30,31]. pyrrolo[3,4- Thec]pyridine researchers derivatives decided with to modify potential the analgesic structure activity. of this These compound modifications to obtain consisted new mainly of replacing the pyridine present in the structure of 27 with another pharmacophore group. At position 2, the pyridine ring was replaced by morpholine, pyrrolidine, 4-

Pharmaceuticals 2021, 14, 354 18 of 25

methylpiperazine, or a chlorine atom. The analgesic effect of compounds was determined in two behaviorally different tests: phenylbenzoquinone-induced writhing syndrome test and hot-plate (thermal analgesic stimulus) test in mice. Unfortunately, most of the imides tested did not show any analgesic effect during testing [31]. Continuing their study, researchers observed that replacement of the piperidine group by an alkoxy one gave non-toxic substances with powerful analgesic properties [32]. The structures of compounds 30a–30e are presented in Figure 24. The analgesic action of the new 1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione derivatives 30a–30e was investigated on mice using the hot-plate and writhing tests. In both tests (writhing and hot-plate tests), all structures (30a–30e) tested showed analgesic activity superior to that of the reference acetylsalicylic acid (ASA). In the writhing test, imide 30a, containing a methoxy group at position 2 of the pyridine ring and an unsubstituted phenyl at the N-4 position of piperazine, proved to be the most active com- pound. Replacement of the methoxy group with an ethoxy group (imide 30b) contributed to an almost fourfold decrease in analgesic activity. Similarly, the activity of imide 30c with a methoxy group in the pyridine ring was higher than that of its ethoxy analog 30e. This result indicates that the potency of the analgesic effect in the writhing test is influenced by the type of alkoxy group at position 2 of the pyridine ring. The authors also observed that the introduction of trifluoromethyl or methoxy groups to the phenyl substituent caused a significant inhibition of spontaneous locomotor activity in mice. As it is known, analgesics can act centrally, peripherally, or both centrally and peripherally. Opi- oid analgesics, like morphine, base their analgesic effects mainly on a central mechanism, whereas non-steroidal anti-inflammatory drugs, like ASA, on a peripheral mechanism [38]. However, this division is not unambiguous as salicylates exert their analgesic effects also partly through the central mechanism [39]. Since the structures studied were active in both pharmacological tests, they may show analgesic effects both in the central and peripheral mechanism. However, as suggested by the authors, elucidation of the exact mechanism requires further pharmacological studies [32]. Continuing the research in 2005, Sladowska’s´ team designed another series of com- pounds with potential analgesic activity [35]. To this end, the researchers further modified the structure of 1H-pyrrolo[3,4-c]pyridine-1,3(2H)-diones 30a and 30b by: • elimination of the hydroxyl group from the alkyl chain; • introduction of a methoxy group at position 2 of the phenyl ring on N-4-piperazine; • replacement of the phenyl ring with a 2-pyrimidine ring; • replacement of the N-aryl(heteroaryl) piperazine group with tetrahydroisoquinoline and morpholine substituents; • shortening of the alkyl chain at the nitrogen atom to C-1. Considering the above rationale, they synthesized the corresponding 1H-pyrrolo[3,4- c]pyridine-1,3(2H)-diones (Figure 28) to obtain compounds with potent analgesic activity [35]. Pharmaceuticals 2021, 14, 354 19 of 25 Pharmaceuticals 2021, 14, 354 19 of 26

Serie I Serie II H3C H3C

O 2 O 1 R N R N N N N N Am RO O CH O O 3 34 35 Compd. R R1 R2 Compd. Am

34a CH3 H C6H5 35a HNN

HNN 34b C2H5 H C6H5 35b

H CO 3

34c CH3 H 2‐OCH3C6H4 35c HN

34d C2H5 H 2‐OCH3C6H4 35d HN O

34e C2H5 OH pyrimidinyl

FigureFigure 28. 28. DerivativesDerivatives ofof 1H-pyrrolo[3,4-‐pyrrolo[3,4‐c]pyridine]pyridine-1,3(2‐1,3(2HH))-diones‐diones 3434 andand 3535. .

TheThe analgesicanalgesic activity of compounds 34 34 and and 35 35 was was tested tested in in two two assays: assays: hot hot-plate‐plate andand writhingwrithing tests. In In the the writhing writhing syndrome syndrome test, test, all all tested tested substances, substances, i.e., i.e., 3434 andand 3535, , showedshowed strongstrong analgesic activity. The strongest strongest effect effect was was produced produced by by compounds compounds 34a 34a andand 34c34c,, whichwhich werewere effectiveeffective up toto aa dosedose ofof 0.780.78 mg/kg.mg/kg. The The most most active active compound compound in in thisthis testtest waswas imideimide 34c, containing an o o-methoxyphenyl‐methoxyphenyl substituent substituent at at the the N N-4‐4 position position of of piperazinepiperazine andand aa methoxy group at position 2 2 of of the the pyridine pyridine ring. ring. None None of of the the structures structures obtainedobtained was more more active active than than the the leading leading structure structure 30a 30a in this in this test. test. This This indicates indicates that the that themost most preferable preferable modification modification involves involves removing removing a hydroxyl a hydroxyl group at group the β propyl at the positionβ propyl positionand introducing and introducing of a methoxy of a methoxy group group at the at ortho the ortho position position of the of the phenyl phenyl ring. ring. Only Only compoundcompound 35a35a (the(the most toxic imide) imide) was was more more active active in in the the hot hot-plate‐plate test test than than compound compound 30a30a.. Compound 35a showed significantsignificant activity up to a dose of 9 mg/kg in in this this test. test. In In the the casecase ofof 4-ethoxy4‐ethoxy derivatives,derivatives, no increase in analgesic activities activities was was observed observed concerning concerning imideimide 30b30b.. The pharmacological results results obtained obtained indicate indicate that that most most of of the the structures structures studiedstudied showshow very interesting analgesic properties. properties. The The results results of of preliminary preliminary radioligand radioligand bindingbinding studiesstudies suggest suggest that that these these compounds compounds show show a weak a affinityweak affinity for µ-opioid for μ‐ receptors,opioid whichreceptors, probably which playprobably a role play in theira role mechanism in their mechanism of action. of However,action. However, the authors the authors do not explaindo not explain the mechanism the mechanism of action of ofaction the obtainedof the obtained pyrrolo[3,4- pyrrolo[3,4c]pyridine‐c]pyridine derivatives derivatives [35]. [35]. In 2006, the researchers obtained another series of compounds. This time, modifi- cationsIn to2006, model the structure researchers30a involvedobtained introducing another series chlorine of andcompounds. fluorine atomsThis time, to the phenylmodifications ring at to the model N-4 position structure of 30a piperazine. involved Additionally, introducing chlorine in several and cases, fluorine the OH atoms group to wasthe phenyl removed ring from at the linkerN‐4 position [36]. The of analgesicpiperazine. activity Additionally, of the compounds in several obtainedcases, the in OH this studygroup was was measured removed usingfrom thethe writhing linker [36]. syndrome The analgesic and hot-plate activity tests. of Allthe testedcompounds imides showedobtained significant in this study antinociceptive was measured activity using the in phenylbenzoquinone-induced writhing syndrome and hot‐plate writhing tests. All test, and the ED50 values ranged from 3.51 to 16.04 mg/kg [36].

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Pharmaceuticals 2021, 14, 354 20 of 25

tested imides showed significant antinociceptive activity in phenylbenzoquinone‐ induced writhing test, and the ED50 values ranged from 3.51 to 16.04 mg/kg [36]. Importantly,Importantly, these these compounds compounds were were non-toxic. non‐toxic. However, However, the analgesic the analgesic activity activity (tested in(tested both in the both hot-plate the hot and‐plate writhing and writhing syndrome syndrome tests) tests) of the of compounds the compounds in this in this series series was lowerwas lower than than the model the model structure structure30a previously 30a previously adopted adopted by the by authors the authors [34]. [34]. InIn 2020, Dziubina Dziubina et et al. al. [40] [40 published] published a paper a paper that thataimed aimed to investigate to investigate the potential the po- tentialanalgesic, analgesic, antiedematous antiedematous (anti‐inflammatory) (anti-inflammatory) and antiallodynic and antiallodynic activities activitiesusing two using 1H‐ twopyrrolo[3,4 1H-pyrrolo[3,4-‐c]pyridinec]pyridine-1,3(2‐1,3(2H)‐dioneH )-dionederivatives derivatives 36 and36 37and (Figure37 (Figure 29) 29 in) in various various experimentalexperimental modelsmodels ofof pain.pain.

H3C H3C O O O O O O H3C H3C N N N N N N O N N O O O H3C H3C O

H3C 36 37

FigureFigure 29. 29. DerivativesDerivatives of 1H-pyrrolo[3,4-‐pyrrolo[3,4‐c]pyridine]pyridine-1,3(2‐1,3(2HH))-diones‐diones 3636 andand 3737. . The authors performed a number of pharmacological tests, including the hot‐plate The authors performed a number of pharmacological tests, including the hot-plate test, formalin test, capsaicin test, and oxaliplatin‐induced allodynia test. The hot‐plate test test, formalin test, capsaicin test, and oxaliplatin-induced allodynia test. The hot-plate test is commonly used to evaluate centrally acting analgesics, and nociceptive responses in is commonly used to evaluate centrally acting analgesics, and nociceptive responses in this this test are of supraspinal origin. In the experiment conducted, only 36 at a dose of 20 test are of supraspinal origin. In the experiment conducted, only 36 at a dose of 20 mg/kg mg/kg significantly increased the latency time of the pain response. However, as the significantly increased the latency time of the pain response. However, as the authors authors suggest, its effect seems to be due to sedation (compound 36 at the highest suggest, its effect seems to be due to sedation (compound 36 at the highest analgesic dose analgesic dose significantly decreased locomotor activity) rather than antinociceptive significantly decreased locomotor activity) rather than antinociceptive action [40]. action [40]. The formalin test is used to identify tonic inflammatory pain. It assumes two phases The formalin test is used to identify tonic inflammatory pain. It assumes two phases of stimulus perception – the first, lasting about 5 min after administration of an irritant of stimulus perception – the first, lasting about 5 min after administration of an irritant (formalin), manifested by licking, shaking, biting of the paw by the animal, and the second, (formalin), manifested by licking, shaking, biting of the paw by the animal, and the following several minutes, by tonic pain. The first phase results from the conduction of second, following several minutes, by tonic pain. The first phase results from the the pain stimulus along the C-fiber, whereas the second phase is the effect of increasing conduction of the pain stimulus along the C‐fiber, whereas the second phase is the effect inflammation and central sensitization in response to a peripheral stimulus. In this test of increasing inflammation and central sensitization in response to a peripheral stimulus. compounds 36 and 37 (5–20 mg/kg) showed antinociceptive activity in both phases, but – itIn was this more test compounds pronounced 36 in and the second37 (5 20 phase mg/kg) of theshowed test. Formalin-inducedantinociceptive activity pain involvesin both numerousphases, but channels, it was more receptors, pronounced and signaling in the second pathways; phase therefore, of the test. the authors Formalin verified‐induced the involvementpain involves of numerous opioidergic, channels, adenosinergic receptors, and and nitrergic signaling systems pathways; in the analgesic therefore, effects the ofauthors compounds verified36 theand involvement37 in the model of opioidergic, of tonic pain. adenosinergic The antinociceptive and nitrergic effect systems of both in compoundsthe analgesic was effects not reversed of compounds by systemic 36 and injection 37 in of the naloxone, model suggestingof tonic pain. an opioid- The independentantinociceptive analgesic effect of effect both of compounds the test substances. was not Next,reversed the effectsby systemic of the injection compounds of onnaloxone, the adenosinergic suggesting system an opioid were‐independent tested. In this analgesic test, caffeine effect of (10 the mg/kg), test substances. which by Next, itself hasthe effects no effect, of the reversed compounds the effect on the of adenosinergic compound 37 systemon formalin-induced were tested. In this pain test, responses caffeine in(10 both mg/kg), phases. which Moreover, by itself thehas antinociceptiveno effect, reversed effects the effect of 37 ofwere compound blocked 37 by on DPCPX formalin (1,3-‐ dipropyl-8-cyclopentyl-xanthine),induced pain responses in both phases. an adenosine Moreover, A1 the antagonist, antinociceptive confirming effects that of they 37 were were mediatedblocked by by DPCPX adenosine (1,3 A1‐dipropyl receptors.‐8‐cyclopentyl As for the‐xanthine), effects of thesean adenosine compounds A1 antagonist, on the nitr- ergicconfirming system that (nitric they oxide were is mediated involved by in adenosine the analgesic A1 receptors. effect of many As for drugs the effects with differentof these analgesiacompounds mechanisms), on the nitrergic L-NAME system (n (nitricω-nitro-L-arginine oxide is involved methyl in the ester analgesic hydrochloride) effect of admin-many istrationdrugs with reduced different the analgesia antinociceptive mechanisms), effect of L compound‐NAME (nω‐36nitroonly‐L in‐arginine behaviors methyl associated ester withhydrochloride) the second administration phase. L-NAME reduced administration the antinociceptive did not affect effect the of compound antinociceptive 36 only effect in ofbehaviors compound associated55. The with possible the second analgesic phase. effect L‐NAME of compounds administration36 and did37 onnot neurogenic affect the pain was investigated using a capsaicin-induced pain model in mice. Capsaicin induces an immediate response at the application site, called neurogenic inflammation, which results

Pharmaceuticals 2021, 14, 354 21 of 25

from the activation of transient receptor potential vanilloid-1 and the release of mediators substance P and glutamate. Both compounds 36 and 37 reduced capsaicin-induced pain behaviors in a dose-dependent manner. In the next step of the study, the authors tested the potential anti-allodynic efficacy of the tested compounds 36 and 37 in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN) pain induced by oxaliplatin, an anticancer drug commonly used in the model of neuropathy in humans. A single adminis- tration of oxaliplatin in animals (mice and rats) induces a painful peripheral neuropathy with associated mechanical allodynia. It is characterized by two phases: an early phase that develops soon after cytostatic administration (several hours after administration), and a late phase that occurs after several days [41]. The results of compounds 36 and 37 showed that as little as one dose of oxaliplatin decreased the threshold of pain sensi- tivity to mechanical stimuli, and tactile allodynia was observed as early as three hours after oxaliplatin injection. The observed effect of oxaliplatin was durable, as it was also found seven days after its administration. Test compounds 36 and 37 significantly reduced mechanical hypersensitivity at doses of 5 and 10 mg/kg. In the late phase of allodynia, both compounds 36 and 37 were significantly but slightly less effective than in the early phase of the test. The researchers also conducted in vitro studies. In this study, a bio- chemical assay was performed to determine the effect of the tested compounds on COX-2 levels. The COX-2 enzyme plays an essential role in the inflammatory response and its expression is induced after exposure of macrophages to LPS (lipopolysaccharide) or other pro-inflammatory stimuli. The RAW 264.7 cell line was used for this study. Compounds 36 and 37 significantly reduced COX-2 levels in LPS-stimulated cells. This result suggests at least partial anti-inflammatory properties of the tested compounds. The obtained pharmacological results showed that compounds 36 and 37 exhibits broad-spectrum activity in several pain models (neurogenic pain, inflammatory pain, and chemotherapy-induced peripheral neuropathic pain) [40]. Krzyzak˙ et al. [42] synthesized a novel N-substituted 1H-pyrrolo[3,4-c]pyridine- 1,3(2H)-diones derivatives 38–39. The compounds were tested for their inhibitory activity against COX-1/ COX-2 and BSA (bovine serum albumin) interaction. The researchers obtained two series of compounds (Figure 30). Series I—Mannich bases with an arylpiper- azine moiety 38a–38c. Series II—structures with a two-carbon linker and a cyclic amine 39a, 39b (Figure 30)[42]. In vitro, COX-1 and COX-2 inhibition assays were performed, and pharmacological results showed that all compounds 38–39 have the potential to inhibit both enzymes. It should be noted that COX-1 is involved in the synthesis of prostaglandins responsible for maintaining normal body function in the kidneys, intestines, and other organs, while COX-2 is an isoform that plays a major role in inflammation and associated pain [43,44]. In the study presented by the researchers, the COX-1 enzyme is inhibited more effectively (than the reference meloxicam) by compounds 38a and 39a, while COX-2 is inhibited more strongly than the reference drug by all compounds (except 38c). The highest selectivity towards the COX-2 enzyme was shown by structure 39a, for which the COX selectivity ratio (IC50(COX-2)/IC50(COX-1)) is 0.55 (for meloxicam—0.71) [42]. The interaction with BSA was investigated by fluorescence spectroscopy and circular dichroism measurement. To understand the binding interaction of compounds 38–39 in the active site of COX and BSA, a molecular docking study was performed [42]. The obtained experimental and molecular docking results confirmed that the main interaction forces between the studied 38–39 structures and BSA are hydrogen bonding and van der Waals forces [42]. Pharmaceuticals 2021, 14, 354 22 of 25 Pharmaceuticals 2021, 14, 354 22 of 26

Serie I Serie II R O H C H3C 3 N R O N n H3C N N O OCH 3 N N O

O 39 38 Compd. R Compd. n R 38a H 39a 2 HN

38b o‐CH3 39b 2 HON

38c m‐CF3

Figure 30. Derivatives of 1 H‐-pyrrolo[3,4-pyrrolo[3,4‐c]pyridine-1,3(2]pyridine‐1,3(2H)-diones)‐diones 38 and 39.

InAs vitro mentioned, COX‐1 in and the COX previous‐2 inhibition section, assays compounds were performed,33a–33h (Figure and pharmacological27) presented in- resultsteresting showed antinociceptive that all compounds activity in addition 38–39 have to the the strong potential sedative to inhibit activity both that enzymes. has already It shouldbeen described be noted [ 37that]. CompoundsCOX‐1 is involved 33a–33h in (exceptthe synthesis33c) were of prostaglandins not toxic (LD50 responsible>2000mg/kg). for maintainingThe analgesic normal activity body of new function compounds in the waskidneys, studied intestines, using the and hot-plate other organs, test and while the COXwrithing‐2 is test.an isoform All tested that 3,4-pyridinedicarboximide plays a major role in inflammation33a–33h andwere associated active in thepain writhing [43,44]. Intest the (ED study50 = presented 3.25–19.2 by mg/kg), the researchers, and their the analgesic COX‐1 activities enzyme is in inhibited this study more exceeded effectively the (thaneffect the of the reference reference meloxicam) ASA (ED50 by= compounds 39.15 mg/kg). 38a In and addition, 39a, while the pharmacologicalCOX‐2 is inhibited activity more stronglyof the two than imides the 33breference(ED50 =drug 3.25 by mg/kg all compounds and 33d (ED (except50 = 3.67mg/kg) 38c). The washighest comparable selectivity to towardsthat of morphine the COX‐ used2 enzyme as a second was shown reference by structure drug (ED 39a50 =, for 2.44 which mg/kg). the However,COX selectivity in the ratiocase of(IC the50(COX hot-plate‐2)/IC50 test,(COX the‐1)) analgesic is 0.55 (for effects meloxicam—0.71) observed for the [42]. tested structures did not reachThe statistical interaction significance with BSA [37 was]. investigated by fluorescence spectroscopy and circular Pharmaceuticals 2021, 14, 354 23 of 26 dichroismBased measurement. on the obtained To results understand and taking the binding into account interaction the results of compounds obtained in previous38–39 in thestudies active [31 site,33 –of35 COX], the and authors BSA, aimed a molecular to determine docking the study relationship was performed between [42]. the structure and biologicalThe obtained activities experimental in the group and molecular of N-substituted docking 1H results-pyrrolo[3,4- confirmedc]pyridine-1,3(2 that the mainH)- interactiondiones.diones. InIn their forces results, between thethe researchersthe studied considered considered38–39 structures the the effecteffect and ofof BSA thethe following followingare hydrogen modificationsmodifications bonding andonon pharmacologicalpharmacological van der Waals forces activityactivity [42]. (Figure(Figure 31 31):): (I) Asan(I) alkoxymentionedan alkoxy substituent in substituentthe previous in the pyridinein thesection, pyridine ring; compounds ring; 33a–33h (Figure 27) presented interesting(II) the(II) length antinociceptivethe length of the alkylof the link;activity alkyl link; in addition to the strong sedative activity that has already(III) the been type ofdescribed amino residue. [37]. Compounds 33a–33h (except 33c) were not toxic (LD50 (III) the type of amino residue. >2000mg/kg). The analgesic activity of new compounds was studied using the hot‐plate test and the writhing test. All tested 3,4‐pyridinedicarboximide 33a–33h were active in the H3C writhing test (ED50 = 3.25–19.2 mg/kg), and their analgesic activities in this study exceeded the effect of the referenceO ASA (ED50 = 39.15 mg/kg). In addition, the pharmacological N activity of the two imidesOH 33b (ED N50 = 3.25 mg/kg and 33d (ED50 = 3.67mg/kg) was comparable to thatN of morphineN used as a second reference drug (ED50 = 2.44 mg/kg). However,R O in the case of the hot‐plate test, the analgesic effects observed for the tested structures didO not reach statistical significance [37]. I II III Based on the obtained results and taking into account the results obtained in previous studies [31,33–35], the authors aimed to determine the relationship between the structure FigureFigure 31.31. StructureStructure ofof imides. imides. (I)(I) 3,4-pyridinedicarboximide 3,4‐pyridinedicarboximide (R=CH (R=CH3,C, C2H 5), (II) linker, (III)(III) typetype ofof and biological activities in the group of N‐substituted 1H‐pyrrolo[3,43 2 5 ‐c]pyridine‐1,3(2H)‐ aminoamino residueresidue [ [37].37].

The potency of the analgesic effect of the studied compounds is decisively influenced by the type of alkoxy substituent in the pyridine ring (I). Compounds substituted with methoxy group in this position are more active than their ethoxy analogues. In most cases, a phenylpiperazine moiety was introduced as the amine residue (III). The most active compounds had an unsubstituted phenyl ring in this grouping. Introduction of alkyl substituents at the ortho or meta position (o‐OCH3, m‐CF3) or halogen atoms was not preferred. Replacement of the phenylpiperazine moieties with another cyclic amine (morpholine, tetrahydroisoquinoline) also led to attenuation of the analgesic activity. The length of the alkyl bond between the pyrrole ring (II) and the amine residue (III) also affects the analgesic potency. Removal of the hydroxyl group weakens the pharmacological effect. Shortening of this linker to 2 or 1 carbon atom also causes a decrease in activity, whereas lengthening the linker to 4 carbon atoms (butyl derivatives) did not increase the potency [37].

3. Conclusions Pyrrolo[3,4‐c]pyridines are biheterocyclic compounds containing a pyrrole ring condensed with a pyridine ring in their structure. The pharmacophore groups, which determine the biological properties of pyrrolopyridines, are most often attached to the pyrrole ring. The review of collected literature shows that carbonyl substituents in the pyrrole ring significantly impact the activity of pyrrolopyridines. The imide structure of such structures determines their physicochemical properties. Imide‐based compounds are often neutral and hydrophobic, which determines their ability to penetrate biological membranes [45,46]. Pyrrolo[3,4‐c]pyridine derivatives have not been introduced into medicine so far, however, as it results from the studies presented in the literature, they exhibit various pharmacological activities. Derivatives with analgesic and sedative properties are by far the largest group [27–37,40,42]. Their antidiabetic [8–10], antiviral [12–15], antimycobacterial [16–19], and anticancer [20–24] activities have also been described. They can also be used in diagnostics as chemosensors to image Fe3+ in living HepG2 cells [47]. The diverse profile of biological activity may be an incentive to continue research into this group of compounds.

Author Contributions: Conceptualization, A.W. and A.R.; writing—original draft preparation, A.W. and A.R.; writing—review and editing, A.W. and A.R.; visualization, A.W. and A.R.; supervision, A.W. and A.R. All authors have read and agreed to the published version of the manuscript.

Pharmaceuticals 2021, 14, 354 23 of 25

The potency of the analgesic effect of the studied compounds is decisively influenced by the type of alkoxy substituent in the pyridine ring (I). Compounds substituted with methoxy group in this position are more active than their ethoxy analogues. In most cases, a phenylpiperazine moiety was introduced as the amine residue (III). The most active compounds had an unsubstituted phenyl ring in this grouping. Introduc- tion of alkyl substituents at the ortho or meta position (o-OCH3, m-CF3) or halogen atoms was not preferred. Replacement of the phenylpiperazine moieties with another cyclic amine (morpholine, tetrahydroisoquinoline) also led to attenuation of the analgesic activity. The length of the alkyl bond between the pyrrole ring (II) and the amine residue (III) also affects the analgesic potency. Removal of the hydroxyl group weakens the pharmacological effect. Shortening of this linker to 2 or 1 carbon atom also causes a decrease in activity, whereas lengthening the linker to 4 carbon atoms (butyl derivatives) did not increase the potency [37].

3. Conclusions Pyrrolo[3,4-c]pyridines are biheterocyclic compounds containing a pyrrole ring con- densed with a pyridine ring in their structure. The pharmacophore groups, which deter- mine the biological properties of pyrrolopyridines, are most often attached to the pyrrole ring. The review of collected literature shows that carbonyl substituents in the pyrrole ring significantly impact the activity of pyrrolopyridines. The imide structure of such structures determines their physicochemical properties. Imide-based compounds are often neutral and hydrophobic, which determines their ability to penetrate biological membranes [45,46]. Pyrrolo[3,4-c]pyridine derivatives have not been introduced into medicine so far, however, as it results from the studies presented in the literature, they exhibit various pharmaco- logical activities. Derivatives with analgesic and sedative properties are by far the largest group [27–37,40,42]. Their antidiabetic [8–10], antiviral [12–15], antimycobacterial [16–19], and anticancer [20–24] activities have also been described. They can also be used in diag- nostics as chemosensors to image Fe3+ in living HepG2 cells [47]. The diverse profile of biological activity may be an incentive to continue research into this group of compounds.

Author Contributions: Conceptualization, A.W. and A.R.; writing—original draft preparation, A.W. and A.R.; writing—review and editing, A.W. and A.R.; visualization, A.W. and A.R.; supervision, A.W. and A.R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Ministry of Health subvention according to the numbers SUB.D070.21.094 and SUB.D140.19.006 from the IT Simple system of Wroclaw Medical University. Conflicts of Interest: The authors declare no conflict of interest.

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