The Influence and Manipulation of Acid/Base Properties in Drug Discovery

Vol. 27, 2018

Drug Discovery Today: Technologies

Editors-in-Chief

Kelvin Lam – Simplex Pharma Advisors, Inc., Boston, MA, USA

Henk Timmerman – Vrije Universiteit, The Netherlands

DRUG DISCOVERY

TODAY

TECHNOLOGIES Physicochemical characterisation in drug discovery

The inﬂuence and manipulation of acid/

base properties in drug discovery

David T. Manallack*, Elizabeth Yuriev, David K. Chalmers

Faculty of Pharmacy and Pharmaceutical Sciences, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade,

Parkville, VIC 3052, Australia

There is a growing awareness of the importance of acid/

Section editor:

base properties inmedicinalchemistryresearch.Inmany

Dr. György T. Balogh – Head of laboratory, honorary

ﬁ

drug classes, ionisable groups are present that make associate professor, Compound Pro ling Laboratory,

Gedeon Richter Plc.

critical interactions with the receptor and are essential

for potency. Yet the presence of these groups may cause

problems with oral bioavailability, pharmacokinetics, or

arises from the ionisation of weak to moderately-strong acids

toxicity. Manipulating pKa values during drug develop-

and bases present in the protein and drug. For example, in

ment or applying pro-drug techniques are strategies that biogenic amine G protein-coupled receptors (GPCRs), such as

can overcome potential deﬁcits in a variety of these the dopamine or serotonin receptors, an ionisable amino

group in the ligand forms a strong ionic interaction (a

areas. Knowledge of drug ionisation states coupled with

‘‘salt-bridge’’) with a negatively charged aspartic acid residue

a consideration of pH-speciﬁc cellular environments can

within the binding site [1]. Removal of either the positive

be used advantageously to target chemoresistance. As

charge from the ligand or the negatively charged residue from

modern drug research ventures into drug candidates the protein generally abolishes biological activity. Other drug

classes, for example the ‘‘statin’’ inhibitors of cholesterol

that exceed the rule of 5, such exploration requires an

biosynthesis, require an acidic functional group on the ligand

understanding of drug acid/base properties and how

in order to have sufﬁcient potency. The carboxylate group of

these factors affect ADMET characteristics.

these drugs forms a strong ionic interaction with a charged

lysine and an adjacent serine residue of HMG-CoA reductase,

replicating a similar interaction made by the substrate, HMG-

CoA [2]. Removal of the carboxylate greatly reduces potency.

Introduction

The strength of the interaction between ionised acid/base

The attraction or repulsion between electrostatic charges

pairs has important implications for drug development. In

underlies many chemical phenomena. Ionic interactions,

many drug targets, the contribution of the ionic interaction

hydrogen bonding and dispersion forces, are fundamentally

to ligand afﬁnity makes it difﬁcult (or impossible) for the

charge–charge interactions, and the interaction between a

medicinal chemistto substitutethe ionisable functional group,

drug and its protein binding site is dictated by the comple-

leading to these groups being a ‘non-negotiable’ feature of

mentarity of drug and binding site charge and shape. Fre-

many drug class chemotypes(Fig. 1). Recentreviews have given

quently, the binding of a drug to a protein target is dominated

insights into the acid/base proﬁles of drugs [3,4], biologically

by a single strong electrostatic interaction that, in most cases,

active substances, and screening compounds [5]. Importantly,

*Corresponding author: D.T. Manallack ([email protected]) the presence of ionised functional groups within drugs broadly

Drug Discovery Today: Technologies | Physicochemical characterisation in drug discovery Vol. 27, 2018

Risperidone

Atorvastatin

Vorioxetine Salbutamol

Statins Anti- GPCRs Naproxen depressants NSAIDs

Diclofenac Escitalopram Amines COOH

Anti- Steroids Neutral SO2NHR Acid microbials Fluticasone propionate Base

Sulfamethoxazole Phosphorus N-containing containing acids heterocycles (acid) Anti- (base) epileptics Osteoporosis Anti- nausea

Alendronate sodium Ondansetron Phenobarbitone

Drug Discovery Today: Technologies

Fig. 1. Examples of compound or therapeutic drug classes and associated ionisable functional groups. ``Neutral” refers to their molecular state under

physiological conditions.

Passage across cells [permeability, absorption and distribu-

impacts drug behaviour within the body. Acidic and basic

tion]

characteristics, in combination with whole molecule lipophi-

Toxicity [e.g. hERG channel blockade, phospholipidosis]

licity, affect drug behaviour in a number of broad areas:

Pharmacokinetics [clearance, plasma and tissue binding,

Molecular interactions with the target macromolecule metabolism]

42 www.drugdiscoverytoday.com

Vol. 27, 2018 Drug Discovery Today: Technologies | Physicochemical characterisation in drug discovery

Formulation [dissolution within the GI tract, stability], and

However, in an increasing number of cases, alternative com-

Environmental impact

pound classes or binding modes can be identiﬁed that can

In addition to the important role of ionisable groups in remove the requirement for the acid or base to be present. A

forming drug-receptor interactions, drug acidic and basic case in point is found with non-steroidal anti-inﬂammatory

groups impinge on many aspects of drug behaviour; quite drugs (NSAIDs). NSAIDs bind to the cyclooxygenase (COX)

often,unfavourably. Charged,polargroupscanadverselyaffect enzyme, which performs a key step in the inﬂammation

drug solubility, oral absorption and distribution, protein plas- pathway. While all early NSAIDs contained a carboxylic acid

ma binding, and can introduce toxicity through off-target group that was thought to make an essential interaction with

effects such as interactions with the hERG ion channel. To Arg 120 in the COX catalytic site (e.g. ibuprofen, Fig. 2),

address these issues, there is often a need to modulate the acid/ research into alternative chemotypes began in the 1960s. At

base behaviour of the candidate compounds during the drug the time, NSAID drugs that contained a carboxylic acid

development process. A variety of approaches are possible; needed multiple daily doses due to rapid metabolism and

there is scope to use pro-drugs, manipulate the pKa values excretion. Stringent design criteria were used to ﬁnd agents

through structural changes, utilise bioisosteres with improved that could be dosed once a day. From this research the oxicam

properties, exploit pH microenvironments for better drug tar- class of NSAIDs emerged in the late 1970s, where Pﬁzer

geting, or take advantage of active transport mechanisms. In speciﬁcally sought to ﬁnd potent COX inhibitors without a

larger compounds, ionisable groups generally have detrimen- carboxylic acid group (e.g. piroxicam, Fig. 2). The oxicam

tal effects on drug properties, for example causing low perme- class of compounds still contains the weakly acidic enolate

ability. This mini-review explores some of the above issues, group (Fig. 2) and binds to the same location as other NSAIDs

providing medicinal chemistry examples with additional com- but the enolate does not interact directly with Arg 120 [6].

mentary on less druggable targets. While piroxicam also contains a weak pyridine base (p a

2.33), this particular group would only be ionised at very

Strategies to deal with problematic acidic or basic low pH values. The development of selective COX-2 inhibi-

functional groups in small molecules tors exposed further structural classes that lack acidic func-

As discussed above, strong ionic interactions are empirically tionalities (e.g. celecoxib, Fig. 2). This illustration serves to

essential and non-removable features of many drug classes. demonstrate that ionisable groups can be replaced but success

Ibuprofen Piroxicam Celecoxib

Compound 15

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Fig. 2. Inhibitors of cyclooxygenase, a key enzyme in inﬂammation pathways: ibuprofen (pKa 4.9) is ionised at physiological pH; piroxicam and celecoxib are

weaker acids (pKa values 5.1 and 10.7, respectively). The enolate group of piroxicam is highlighted with a dashed red box. Compound 15 (Liu et al. [7]) binds

to an allosteric site on the b2 adrenergic receptor and functions as an antagonist.

www.drugdiscoverytoday.com 43

Drug Discovery Today: Technologies | Physicochemical characterisation in drug discovery Vol. 27, 2018

often necessitates a large body of knowledge about the bind- idine analogues, or a piperazine group was used to reduce

ing requirements of the target to be gathered. hERG afﬁnity [11]. The lower pKa values of basic compounds

Alternatively, the requirement for a charged group in the also increased oral absorption without affecting potency

primary binding site can be circumvented by identifying signiﬁcantly [11]. In a study of anti-malarial drugs, Dong

alternative, functional binding pockets or allosteric sites. et al. [12] found that higher p a values were associated with

In GPCRs that recognise biogenic amines, designing drugs higher metabolic stability. While high basicity is usually

for orthosteric sites can pose selectivity problems and most regarded as undesirable, in this case, metabolic stability

receptors have an absolute requirement for a basic amino was a fundamental need for single dose anti-malarial treat-

group. A recent crystal structure of the b2 adrenergic receptor ment. Routine investigation of compounds with varied pKa

has revealed an allosteric site at the cytoplasmic end of this values as part of normal SAR practices [3] is certainly war-

GPCR with a bound allosteric ligand that lacks acidic and ranted, given the impact this parameter has on important

basic groups (compound 15, Fig. 2) [7]. While not a drug, biopharmaceutical properties such as toxicity and pharma-

compound 15 heralds the promise of ﬁnding neutral ligands cokinetics.

that can avoid orthosteric sites. Essential ionisable groups that produce undesirable

In cases where an essential acidic or basic functional group ADMET properties can be ameliorated using pro-drug strate-

cannot be removed, the adverse inﬂuence of the group can be gies. Of the small molecule drugs approved by the FDA since

modulated by ﬁne-tuning its structure to modify its basicity. 2010, just over 10% (19 substances) are pro-drugs and, annu-

Reduction of amine pKa values has been used widely to avoid ally, this proportion is not diminishing [13]. Of these recently

toxicity problems such as hERG channel blockade [8–10]. approved pro-drugs, nine were developed to improve perme-

Charifson and Walters [3] describe many examples where pKa ation through increased lipophilicity by masking an acidic or

values were modiﬁed to correct toxicity or pharmacokinetics basic group (e.g. tenofovir alafenamide). Other purposes for

issues. In most cases, the pKa of a base was lowered, which pro-drugging were to increase solubility (e.g. isavuconazo-

correspondingly raises compound lipophilicity by increasing nium sulfate), exploit transporter mechanisms (e.g. droxi-

the proportion of the drug in a neutral state at physiological dopa) and to modify or manipulate metabolic and

pH. In a study of spleen tyrosine kinase inhibitors, modiﬁca- pharmacokinetic proﬁles (e.g. azilsartan medoximil and ari-

tion of a piperidine moiety to morpholine, ﬂuorinated piper- piprazole lauroxil) (Fig. 3) [13]. Most structural changes were

Tenofovir alafenamide Isavuconazonium sulfate

Droxidopa Azilsartan medoximil Aripiprazole lauroxil

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Fig. 3. Example pro-drug structures that have been approved for use since 2010. The promoiety is coloured red.

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Vol. 27, 2018 Drug Discovery Today: Technologies | Physicochemical characterisation in drug discovery

introduced either to mask hydroxyl groups or an acidic 614.1 Da, logP 4.4) while venetoclax blocks the Bcl-2 protein

functionality. and is used to treat chronic lymphocytic leukemia, and it

clearly breaks the Ro5 (MW 867.3 Da, logP > 5). Both com-

Acid-base properties and membrane permeability of pounds inhibit PPIs and have been recently approved by the

larger compounds FDA (Fig. 4). Venetoclax and liﬁtegrast were developed using

Drug developers are no longer limiting themselves to com- rational, structure-informed design, with careful attention

pounds that meet the Ro5, at least in part because many new being paid to biopharmaceutical properties during develop-

and attractive targets involve the inhibition of protein–pro- ment.

tein interactions (PPIs). In most cases, interacting proteins Nonpeptide macrocycles are an important class of com-

have a large contact area that can range from 350 to 9500 A˚ pounds with useful biological activities. Interestingly, al-

[14] and compounds that disrupt these interactions inevita- though they exceed the Ro5 and contain a variety of polar

bly need to cover a signiﬁcant fraction of the binding site. In acid/base groups macrocycles often demonstrate oral bio-

the past few years, an increasing number of compounds that availability [15]. These properties have been investigated

exceed a molecular weight of 500 Da or go beyond the rule of by Kihlberg and co-workers who examined the permeability

5 (bRo5) [15] have reached the market. bRo5 compounds of non-peptidic macrocycles in relation to their utility for

exceed more than one of Lipinski’s criteria: MW < 500 Da, drug discovery [15,16]. Kihlberg et al. assembled a library of

logP < 5, number of H-bond donors <5 and number of H- 200 non-peptidic macrocycles and explored the relationships

bond acceptors <10. Liﬁtegrast inhibits the binding of the between their overall molecular properties and cell perme-

integrin LFA-1 to ICAM-1 and is a treatment of dry eye ability [16]. In general, they found that the presence of an

syndrome, and represents a large MW compound (MW ionised acidic or basic functional group was detrimental to

Lifitegrast. PPI target: LFA-1/ICAM-1 Venetoclax. PPI target: Bcl-2 family

7 BL3020-1

Drug Discovery Today: Technologies

Fig. 4. PPI-inhibitors liﬁtegrast, which disrupts LFA-1 and ICAM-1 interactions and venetoclax, which targets the Bcl-2 family. The structures of compound

7 (Over et al. [16]) and BL3020-1 are also shown. Dashed lines illustrate hydrogen bonds.

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Drug Discovery Today: Technologies | Physicochemical characterisation in drug discovery Vol. 27, 2018

cell permeability [15]. Curiously, tertiary amines in the pres- these changes result in the local extracellular pH dropping as

ence of a phenyl ring demonstrated enhanced cell perme- more protons are pumped out of cells [21,22]. In some cases,

ation via a synergistic effect. Structural analysis of these chemoresistance to anti-cancer drugs can be directly attrib-

particular compounds also demonstrated that, in some cases, uted to the more acidic extracellular environment around

charged groups could be masked through intramolecular cancer cells. This resistance to drug treatment is explained by

hydrogen bonding or shielded by aromatic rings. This mask- ‘ion trapping’, where the lower pH surrounding cancer cells

ing behaviour was evident in compound 7 (Fig. 4) and the results in weakly basic drugs being more highly ionised than

shielding of polar groups in general was found to be a driver of at physiological pH and thus reducing the effective drug

permeability [16]. The concept of shielding to reduce polarity lipophilicity and making it more difﬁcult for the ionised drug

and thus assist membrane passage could be explored more to cross membranes.

extensively in bRo5 macrocycles [17]. In a similar manner to low pH conditions around cancer

Cyclic peptides are also a focus of drug discovery scientists cells, weak bases that enter cells can be ‘deactivated’ by

looking to develop new therapeutic agents [18]. Cyclic pep- trapping within acidic vesicles within the cells [23]. To illus-

tides of more than a few residues break the Ro5. General trate the effect that pH change has on ionisation states,

factors that inﬂuence oral bioavailability of cyclic peptides consider the anti-cancer drug sunitinib, which is 99.4%

include the well-known properties such as hydrogen bonding ionised at a pH of 6.7 (i.e. the pH in the range found in

capacity, lipophilicity, molecular weight and compound ﬂex- the extracellular environment of a tumour) compared to

iblity. In addition, the charge state inﬂuences permeability 97.3% at physiological pH. This subtle difference is sufﬁcient

largely from a polarity/lipophilicity point of view. Namely, to trap sunitinib within lysosomes [24]. As a general conse-

cell penetration by a compound is hampered by excessive quence, knowledge of the environment outside and within

polarity, which is not compatible with the lipophilic nature certain intracellular compartments of cancer cells can inform

of membranes. Likewise, the permeability of very hydropho- drug design to better target this condition. The design of less

bic compounds is detrimentally affected by either poor solu- basic substances that still have a sufﬁcient fraction of com-

bility or a tendency to get stuck in lipophilic environments. pound in the unionised state at pH values in the range 6.5–

Encouragingly, Pauletti et al. observed that a net positive 7.1, may help attenuate chemoresistance.

charge using hydrophilic peptides enhanced their ability to Alternatively, drugs that counteract the ability of cancer

permeate an artiﬁcial membrane [19]. For example, the cyclic cells to pump protons out of cells by targeting proteins such

+ +

peptide BL3020-1 (Fig. 4), which mimics a-melanocyte stim- as the Na /H exchanger (NHE) or other transporters, could

ulating hormone, was found to have reasonable oral bioavail- be used to change the tumour pH environment [25]. The use

ability. Its absorption appeared to be partly achieved via an of proton pump inhibitors has therefore been proposed as a

active transport mechanism. Certainly this absorption mech- way to offset the pH difference between the acidic external

anism is of interest and may have potential utility particularly and alkaline intracellular environment of cancer cells [23].

where a charged guanidine is present. Usefully, co-treatment with omeprazole has been found to

While some research groups have begun to characterise the avoid some aspects of chemoresistance to certain cancer

biopharmaceutical properties of bRo5 compounds there is drugs [26].

more to be learned about the way that their proﬁles affect

bioavailability and other ADMET properties of bRo5 com- Conclusions

pound classes. While not all PPI inhibitors break the Ro5, the Consideration of molecular acid/base properties is critical in

consensus is that future discovery efforts will increasingly drug development. The acid/base properties of biologically

look to larger substances in order to disrupt the binding of active compounds control their membrane permeability,

one protein to another. Accordingly, special consideration distribution within the body, biological activities, and meta-

must be made of the permeability of these large compounds, bolic fate. As many of the examples above show, the ability to

which will be driven by factors such as lipophilicity and ‘tune’, or even eliminate, acid/base groups is, in many cases,

charge states. the key to developing successful drugs. Overall, our under-

standing of the ways that the acid/base properties of drugs

Harnessing drug pKa for tumour targeting affect the behaviour within the body is growing. However, as

Better understanding of the pH of cellular microenviron- drug developers seek to disrupt protein–protein interactions

ments is another factor inﬂuencing drug discovery research. it is likely that larger bRo5 substances will be needed. At

For example, pH differences occur within tumours where present, there is lack of information concerning tolerable

rapid growth leads to areas of hypoxia and necrotic tissue. general and acid/base properties of bRo5 compounds and

Under low oxygen conditions, tumour cells alter their me- further research is required to understand which chemical

tabolism, resulting in an accumulation of lactate and in- scaffolds and molecular properties will afford the highest

creased amino acid and glucose metabolism [20]. Together, probability of success in this domain.

46 www.drugdiscoverytoday.com

Vol. 27, 2018 Drug Discovery Today: Technologies | Physicochemical characterisation in drug discovery

References [13] Rautio J, Karkkainen J, Sloan KB. Prodrugs - recent approvals and a glimpse

of the pipeline. Eur J Pharm Sci 2017;109:146–61.

[1] Chien EY, Liu W, Zhao Q, Katritch V, Han GW, Hanson MA, et al.

[14] Sowmya G, Breen EJ, Ranganathan S. Linking structural features of protein

Structure of the human dopamine D3 receptor in complex with a D2/D3

complexes and biological function. Protein Sci 2015;24:1486–94.

selective antagonist. Science 2010;330:1091–5.

[15] Matsson P, Doak BC, Over B, Kihlberg J. Cell permeability beyond the rule

[2] Istvan ES, Deisenhofer J. Structural mechanism for statin inhibition of

of 5. Adv Drug Deliv Rev 2016;101:42–61.

HMG-CoA reductase. Science 2001;292:1160–4.

[16] Over B, Matsson P, Tyrchan C, Artursson P, Doak BC, Foley MA, et al.

[3] Charifson PS, Walters WP. Acidic and basic drugs in medicinal chemistry:

Structural and conformational determinants of macrocycle cell

a perspective. J Med Chem 2014;57:9701–17.

permeability. Nat Chem Biol 2016;12:1065–74.

[4] Manallack DT, Prankerd RJ, Yuriev E, Oprea TI, Chalmers DK. The

[17] Whitty A, Zhong M, Viarengo L, Beglov D, Hall DR, Vajda S. Quantifying

signiﬁcance of acid/base properties in drug discovery. Chem Soc Rev

the chameleonic properties of macrocycles and other high-molecular-

2013;42:485–96.

weight drugs. Drug Discov Today 2016;21:712–7.

[5] Manallack DT, Prankerd RJ, Nassta GC, Ursu O, Oprea TI, Chalmers DK. A

[18] Nielsen DS, Shepherd NE, Xu W, Lucke AJ, Stoermer MJ, Fairlie DP. Orally

chemogenomic analysis of ionization constants - implications for drug

absorbed cyclic peptides. Chem Rev 2017;117:8094–128.

discovery. ChemMedChem 2013;8:242–55.

[19] Pauletti GM, Okumu FW, Borchardt RT. Effect of size and charge on the

[6] Xu S, Hermanson DJ, Banerjee S, Ghebreselasie K, Clayton GM, Garavito

passive diffusion of peptides across Caco-2 cell monolayers via the

RM, et al. Oxicams bind in a novel mode to the cyclooxygenase active site

paracellular pathway. Pharm Res 1997;14:164–8.

via a two-water-mediated H-bonding network. J Biol Chem

[20] Chen Z, Lu W, Garcia-Prieto C, Huang P. The Warburg effect and its cancer

2014;289:6799–808.

therapeutic implications. J Bioenerg Biomembr 2007;39:267–74.

[7] Liu X, Ahn S, Kahsai AW, Meng KC, Latorraca NR, Pani B, et al. Mechanism

[21] Tredan O, Galmarini CM, Patel K, Tannock IF. Drug resistance and the

of intracellular allosteric beta2AR antagonist revealed by X-ray crystal

solid tumor microenvironment. J Natl Cancer Inst 2007;99:1441–54.

structure. Nature 2017;548:480–4.

[22] Izumi H, Torigoe T, Ishiguchi H, Uramoto H, Yoshida Y, Tanabe M, et al.

[8] Fish LR, Gilligan MT, Humphries AC, Ivarsson M, Ladduwahetty T,

Cellular pH regulators: potentially promising molecular targets for cancer

Merchant KJ, et al. 4-Fluorosulfonylpiperidines: selective 5-HT2A ligands

chemotherapy. Cancer Treat Rev 2003;29:541–9.

for the treatment of insomnia. Bioorg Med Chem Lett 2005;15:3665–9.

[23] Taylor S, Spugnini EP, Assaraf YG, Azzarito T, Rauch C, Fais S.

[9] Ravula SB, Yu J, Tran JA, Arellano M, Tucci FC, Moree WJ, et al. Lead

Microenvironment acidity as a major determinant of tumor

optimization of 2-(piperidin-3-yl)-1H-benzimidazoles: identiﬁcation of 2-

chemoresistance: proton pump inhibitors (PPIs) as a novel therapeutic

morpholin- and 2-thiomorpholin-2-yl-1H-benzimidazoles as selective

approach. Drug Resist Updat 2015;23:69–78.

and CNS penetrating H(1)-antihistamines for insomnia. Bioorg Med

[24] Gotink KJ, Broxterman HJ, Labots M, de Haas RR, Dekker H, Honeywell RJ,

Chem Lett 2012;22:421–6.

et al. Lysosomal sequestration of sunitinib: a novel mechanism of drug

[10] Morgenthaler M, Schweizer E, Hoffmann-Roder A, Benini F, Martin RE,

resistance. Clin Cancer Res 2011;17:7337–46.

Jaeschke G, et al. Predicting and tuning physicochemical properties in

[25] Spugnini EP, Sonveaux P, Stock C, Perez-Sayans M, De Milito A, Avnet S,

lead optimization: amine basicities. ChemMedChem 2007;2:1100–15.

et al. Proton channels and exchangers in cancer. Biochim Biophys Acta

[11] Garton NS, Barker MD, Davis RP, Douault C, Hooper-Greenhill E, Jones E,

2015;1848:2715–26.

et al. Optimisation of a novel series of potent and orally bioavailable

[26] Luciani F, Spada M, De Milito A, Molinari A, Rivoltini L, Montinaro A,

azanaphthyridine SYK inhibitors. Bioorg Med Chem Lett 2016;26:4606–

et al. Effect of proton pump inhibitor pretreatment on resistance of solid

12.

tumors to cytotoxic drugs. J Natl Cancer Inst 2004;96:1702–13.

[12] Dong Y, Wang X, Kamaraj S, Bulbule VJ, Chiu FC, Chollet J, et al.

Structure-activity relationship of the antimalarial ozonide artefenomel

(OZ439). J Med Chem 2017;60:2654–68.