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Directing Bitopic or Bivalent Molecules to D3 Receptors for the Treatment of Substance Use Disorders

Amy Hauck Newman, Ph.D. Scientific Director NIDA – IRP http://irp.drugabuse.gov/Newman.php @anewman2014 HN

O NH

HO

N Penn-Yale Addiction Center of Excellence O N Addictions Seminar Series Cl April 12, 2021 J. Med. Chem. 2020, 62(20) 9061-9078. The concept: bivalent ligands

N N N N N OH OH OH OH HO

O O O HO H O HO H NH2 N HO H H O N O OH HO H H H

(1), (-)- (2), β-Naltrexamine (3), (4),

OH HO

H H O O H H N N

O O HO OH N n N n X X Increased Binding Affinity O Increased O HN NH H H Phil Portoghese HO O O OH N N OH HO

Single monomer OBS binding Single monomer OBS binding

O X O n HN NH H H O HO O HO N OH OH N ??? Erez, M.; Takemori, A. E.; Portoghese, P. S. J. Med. Chem. 1982,

Bivalent binding pose - both dimer OBS targeted 25, 847.; Portoghese, P. S. et al., Life Sci. 1982, 31, 1283. The concept: bitopic ligands

TIPS 2013, 34, 59. Linker The design: Responsible for unique binding interactions Helps optimal binding pose and consequent conformations

Often responsible for modulating allosteric profiles and enhancing OBS SBP or ABS Y X

Primary

Often inspired by Secondary Pharmacophore classic orthosteric ligands Often inspired by allosteric Often responsible for ligands or aromatic synthons agonism or antagonism Often responsible for modulating binding affinity, selectivity, functional potency Newman, et al., J. Med. Chem. 2020, 62(20) 9061-9078 and The application: Dopamine Receptors

D1-like D2-like

D1R D5R D2R D3R D4R

-Activate G!i/o -Activate G!s/olf -Stimulate cAMP production -Inhibit cAMP production -Expressed post-synaptically -Expressed post- and pre- synaptically Why D3R?

• .Unlike D2R, D3R expression is largely localized in the mesolimbic _.brain region

• Reward experience induced by of abuse is intrinsically connected to increased dopamine levels within this pathway

• D3R blockade results in inhibition/reduction of self-

administration of nicotine, cocaine, Volkow et al. Physiol Rev (2019) , methamphetamine, and (Heidbreder and Newman, 2010) Heidbreder and Newman, Ann. N.Y. Acad. Sci. (2010) Koob et al. Science (1997)

Targeting the D3R receptor for Substance Use Disorders • Small molecule SAR has led to the discovery of highly selective D3R ligands,

• The D3R crystal structure provided a template for structure-based investigation

Advances in , Emerging Targets and Therapeutics for the Treatment of Psychostimulant Abuse, 2014, 69, 267-300 The current crisis in the United States Targeting the D3R for

J. Med. Chem. 2015, 58(15) 6195-6213.

Comfort Boateng VK4-116 is a highly selective D3R antagonist

linker

*

SP

PP

hD3R IC50=360 nM hERG IC50=43 µM But is it “druggable”?

Vivek Kumar Kumar et al, JMC, 2016, 59, 7634. Pretreatment with VK4-116 dose-dependently decreases self administration in rats and had no effect on sucrose

Zhi-Bing You, Ewa Galaj and Zheng-Xiong Xi You et al., Neuropsychopharmacology, 2019, 44, 1415. VK4-116 attenuates oxycodone induced reinstatement of drug seeking behavior Oxy 1 mg/kg

You et al., Neuropsychopharmacology 2019, 44, 1415. VK4-116 prevents dose escalation in oxycodone self administering male and female rats

Olivier George & Giordano de Guglielmo, Scripps/UCSD De Guglielmo et l. Front. Behav. Neurosci 2020, 23, 292. Pretreatment with VK4-116 inhibits acquisition of oxycodone self administration 30 Vehicle VK 5mg/kg VK 15 mg/kg With VK pretreatment Without20 VK pretreatment

10

* * * * infusions(3hr) Oxycodone 0 60 With pretreatent Without pretreatent * * * * * * * *

40

20 You et al., Neuropsychopharmacology 2019, 44, 1415. Inactive lever-presses (3 hr) lever-presses Inactive 0 1 2 3 4 5 6 7 8 9 10 11 Testing day 2 mg/kg) in theplate in hot test 2 mg/kg) antinociceptive effects lowof doses oxycodone (0.5 VK4 with Pretreatment

MPE% 100 20 40 60 80 0 0.5 -116 enhances the Oxycodone dose (mg/kg) Oxycodone VK 25 mg/kg VK 15 mg/kg VK 5mg/kg Vehicle ** 1 Youetal., ** 2 Neuropsychopharmacology 4 2019, 44, 1415. 44, 2019, - Does VK4-116 have translational potential?

1 2 0 ) % (

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T i m e ( m i n ) Alex Gadiano and Rana Rais, JHU VK4-116 augments oxycodone antinociception in rhesus monkeys

[Oxy (mg/kg)] [Oxy (mg/kg)]

Matt Banks, VCU VK4-40 is a highly selective D3R partial

H * N N N Cl H O OH N

VK4-40 D3R Ki=0.36 nM D2R Ki=151 nM D2/D3=417

hD3R EC50=2.6 nM (18%) hERG IC50=1.4 µM

Kumar et al, JMC, 2016, 59, 7634. Synthesis of VK4-116 and VK4-40 enantiomers O O

O2N O2N OH a OH b OH O 85% O 80% O Cl Cl Cl 3 1 2 c 85% A A A H2N O2N e d O2N O 80% B B 80% B C C C 6(a-b) f 5(a-b) 4(a-b) 70% A B C H OCH Cl HN A 4-11(a) 3 4-11(b) H O N H Cl

N B O * O 7(a-b) 8 O R,S C N i Chiral * O 80% O O R,S resolution g 85% 8 O N 85% * g S O g N * O 85% R,S R O 9S H N O 2 * N A 9R OH N H N S 2 * N A 10(a-b) B H N R OH N 2 * N A C 10S(a-b) OH N B 60% h 10R(a-b) C B C h 60% 60% h NH H R,S N * N A NH H S O OH N Anver Shaik NH N H R * N A N * N A O OH N B O OH N (±)-VK4-116 C 11S(a-b) B (±)-VK4-40 11R(a-b) 11(a-b) (R)-VK4-116 B (S)-VK4-116 C (R)-VK4-40 C (S)-VK4-40 Chiral Preparative HPLC Shaik et al., JMC, 2019, 24, 9061. D2R and D3R binding data

[3H]-N-methylspiperone compe88ona

Compounds D2R D3R

Ki ± SEM (nM) Ki ± SEM (nM) D3/D2 (R)-VK04-116 10200 ± 1870 5.97 ± 1.19 1709 (S)-VK04-116 11600 ± 1150 33.4 ± 8.46 347 Alessandro Bonifazi VK04-116 11400 ± 3270 6.84 ± 1.18 1667 (R)-VK04-40 68.1 ± 12.3 0.245 ± 0.0915 278 (S)-VK04-40 200 ± 57.9 0.700 ± 0.286 286 Adrian Guerrero VK04-40 119 ± 11.1 0.351 ± 0.114 339 ABS01-106 27100 ± 11600 28.0 ± 5.53 968 Shaik et al., JMC, 2019, 24, 9061. ABS01-107 9740 ± 2100 89.9 ± 12.3 108 ABS01-113 124 ± 15.8 0.482 ± 0.150 257 ABS01-114 295 ± 30.1 2.09 ± 0.484 141 D2R and D3R binding data

[3H]-N-methylspiperone compe88ona

Compounds D2R D3R

Ki ± SEM (nM) Ki ± SEM (nM) D3/D2 (R)-VK04-116 10200 ± 1870 5.97 ± 1.19 1709 (S)-VK04-116 11600 ± 1150 33.4 ± 8.46 347 D3R antagonists VK04-116 11400 ± 3270 6.84 ± 1.18 1667

(R)-VK04-40 68.1 ± 12.3 0.245 ± 0.0915 278 D3R antagonist (S)-VK04-40 200 ± 57.9 0.700 ± 0.286 286 D R partial VK04-40 119 ± 11.1 0.351 ± 0.114 339 3 ABS01-106 27100 ± 11600 28.0 Data± 5.53 from NIDA968 CTDP ABS01-107 9740 ± 2100 89.9 ± 12.3 108 ABS01-113 124 ± 15.8 0.482 ± 0.150 257 ABS01-114 295 ± 30.1 2.09 ± 0.484 141 VK4-116 is highly metabolically stable in rat liver microsomes

V K 4 -1 1 6

1 0 5 ) ( ±)V K 4 -1 1 6 %

( 9 0

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n 3 0 e c r 1 5 e P 0 0 3 0 6 0 Shaik et al., JMC, 2019, 24, 9061. T im e (m in ) VK4-40 is highly metabolically stable in rat liver microsomes

V K 4 -4 0

1 0 5 ) ( ±)V K 4 -4 0 %

( 9 0

g (R ) V K 4 -4 0

n 7 5 i

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n 3 0 e c r 1 5 e P 0 0 3 0 6 0 Shaik et al., JMC, 2019, 24, 9061. T im e (m in ) R-and S-VK4-116 are both orally available and highly brain penetrant

R V K 4 - 1 1 6 S V K 4 - 1 1 6 ) 2 5 0 0 ) 1 5 0 0 g g / / P la s m a ( 1 0 m g / k g ) l l o o B r a i n ( 1 0 m g / k g ) B r a i n ( 1 0 m g / k g ) m 2 0 0 0 m p p

P la s m a ( 1 0 m g / k g ) r r 1 0 0 0 o o

1 5 0 0 L L m m / / l l o 1 0 0 0 o 5 0 0 m m p p ( (

5 0 0 c c n n o o C 0 C 0 0 2 4 6 8 1 0 0 2 4 6 8 1 0

T i m e ( h r ) T i m e ( h r )

Plasma (10mg/kg) Brain (10mg/kg) Plasma (10mg/kg) Brain (10mg/kg) Area= 2201 8412 Area= 1068 4360

Shaik et al., JMC, 2019, 24, 9061. R-and S-VK4-40 are both orally available and highly brain penetrant

R V K 4 - 4 0 S V K 4 - 4 0 ) 5 0 0 0 g )

/ P la s m a ( 1 0 m g / k g ) 5 0 0 0 l g

/ P la s m a ( 1 0 m g / k g ) o l

B r a i n ( 1 0 m g / k g ) o

m 4 0 0 0 B r a i n ( 1 0 m g / k g ) p m 4 0 0 0

p r

o r 3 0 0 0 o L 3 0 0 0 L m / l m / o

2 0 0 0 l

o 2 0 0 0 m p m (

1 0 0 0 p ( c 1 0 0 0 n c o n o C 0

C 0 0 2 4 6 8 1 0 0 2 4 6 8 1 0 T i m e ( h r ) T i m e ( h r ) Plasma (10mg/kg) Brain (10mg/kg) Plasma (10mg/kg) Brain (10mg/kg) Area= 1985 21609 Area= 2678 16665

The brain to plasma ratios were similar for each enantiomeric pair, but VK4-40 demonstrated higher ratios overall. Shaik et al., JMC, 2019, 24, 9061. R-VK4-40 reduces oxycodone self administration

100 Baseline R-VK4-40 Pretreatment (10 mg/kg, i.p.) R-VK4-40 Pretreatment (20 mg/kg, i.p.) 80

60

Infusions 40

20

0 3.125e-3 6e-3 0.0125 0.025 0.05 Oxycodone Dose (mg/kg)

Chloe Jordan Jordan et al., 2019, 158, 107609. R-VK4-40 augments oxycodone-induced analgesia

100 Vehicle R-VK4-40 Pretreatment (10 mg/kg, i.p.)

50 * ^ 0 %MPE

-50

-100 0.5 1 2 4 Oxycodone Dose (mg/kg)

Jordan et al., Neuropharmacology 2019, 158, 107609. Earlier generations of D3R antagonists increased blood pressure, especially in the presence of cocaine

Appel & Acri, 2016; 2017 Neither R-VK4-40 nor R-VK4-116 exacerbate cocaine’s cardiovascular effects

150 500 A +Vehicle B (n=8/group) +Cocaine 10 mg/kg 140 450 *** 130 ** 400 120 ++ 350 +++ 110 ++ Heart Rate (BPM) Rate Heart 300 ++ 100 +++ BloodPressure Hg)(mm 90 250 V 3 10 20 V 3 10 20 R-VK4-40 Dose (mg/kg) R-VK4-40 Dose (mg/kg)

42 1500 C D Chuck Schindler C) 40 ** o 1000 * 38

500

Activity (counts)Activity 36 Temperature ( *** ++ ++ +++ +++ 0 34 V 3 10 20 V 3 10 20 R-VK4-40 Dose (mg/kg) R-VK4-40 Dose (mg/kg) Chloe Jordan Jordan et al., JPET, 2019, 371, 602. R-VK4-40 and R-VK4-116 attenuate oxycodone’s cardiovascular effects

150 A +Vehicle 500 B (n=8/group) +Oxycodone 1 mg/kg 140 450 130 *** * 400 120 + ++ 350 ++ 110 ** ++ ++ (BPM) Rate Heart 300 100 +++** BloodPressure Hg)(mm 90 250 V 3 10 20 V 3 10 20 R-VK4-40 Dose (mg/kg) R-VK4-40 Dose (mg/kg)

1500 C 42 D

C) 40 o 1000 ** *** 38 ** ++ +++ 500

Activity (counts)Activity 36 Temperature ( +++**

0 34 V 3 10 20 V 3 10 20 R-VK4-40 Dose (mg/kg) R-VK4-40 Dose (mg/kg) Jordan et al., JPET, 2019, 371, 602. SUMMARY-1

• VK4-116 and VK 4-40 are highly selective bitopic D3R antagonists/partial agonists • Enantiomers have been synthesized; binding, metabolism and PK studies are complete • They do not bind to opioid (µ, d, k) receptors, thus the mechanism appears to be through D3R blockade • Significant and promising behavioral data have been collected on the antagonists (±)VK4-116 and R-VK4-40 in rats. Behavioral evaluation of a new set of 3-F analogues is underway • Neither R-VK4-40 nor R-VK4-116 adversely affect peripheral biometrics or cardiovascular effects of oxycodone OR cocaine. • Further development is underway to advance our lead molecules to clinical trials

Newman, A. H., Kumar V., Shaik, A. Dopamine D3 receptor selective antagonists/partial agonists; method of making and uses thereof. Int. Patent Application filed 3-8-2017. Newman, A. H., Kumar V., Shaik, A. Dopamine D3 receptor selective antagonists/partial agonists and uses thereof. Provisional Patent Application filed 10-7-2018. The challenge: Dopamine Receptors

D1-like D2-like

D1R D5R D2R D3R D4R

-Activate G!i/o -Activate G!s/olf -Stimulate cAMP production -Inhibit cAMP production -Expressed post-synaptically -Expressed post- and pre- synaptically Why D3R Agonists?

• Therapeutic Agents: Potentially useful in treating locomotor associated diseases: • Parkinson’s Disease, Restless Leg Syndrome • Pharmacological Tools: Develop selective ligands that allow pharmacological studies of D3R function.

Commonly used and in vivo:

H N N O N HO N H2N S N N H

Pramipexole Quinpirole (+)-PD128,907 D3R Ki = 1.32 nM D3R Ki = 24 nM D3R Ki = 1.69 nM D2R Ki = 11.1 nM D2R Ki = 4.8 nM D2R Ki = 20.5 nM D2R/D3R = 8.41 D2R/D3R = 0.2 D2R/D3R = 12.1 Can we apply the bitopic concept to D3R agonists? D3R Bitopic Agonists: Design Primary Pharmacophore Selection:

O O HO N N N

O H2N (+)-PD128,907 PF592,379 hD2R Ki = 20.5 nM hD2R Ki = 1740 nM hD3R Ki = 1.69 nM hD3R Ki = 185 nM D2R/D3R = 12.1 D2R/D3R = 9.41 Battiti et al, ACSMCL 2020, 11, 1956.

Alessandro Bonifazi, Francisco Battiti & Sophie Cemaj O O N N N N

H2N H2N PF-592379 PF-592379 hD2R Ki = 1740 nM hD2R Ki = 1740 nM hD3R Ki = 185 nM hD3R Ki = 185 nM D2/D3 = 9.41 D2/D3 = 9.41

O O O O NH NH NH N NH N DN3R Bitopic Agonists: PF592,379N Based 5 H2N O H2N H2N HN2N (2R,5S)-nor-PF-592379N 2 (2S,5S)-nor-PF-592379 (2R,5S)-nor-PF-592379 (2S,5S)-nor-PF-592379 hD2R Ki = 7100H 2nMN hD2R Ki = 2340 nM hD2R Ki = 7100 nM hD3R Ki = 1520 nMPF-592379 hD2R Ki = 2340 nM hD3R Ki = 424 nM hD3R Ki = 1520 nM hD2R Ki = 1740hD3R nM Ki = 424 nM D2/D3 = 4.67 hD3R Ki = 185 nM D2/D3 = 5.52 D2/D3 = 4.67 D2/D3 = 9.41 D2/D3 = 5.52

O O NH NH N N

H2N H2N O(2R,5S)-nor-PF-592379 O (2S,5S)-nor-PF-592379 O O O O hD2R Ki = 7100 nM O hD2R Ki = 2340 nM O hD3R Ki N= 1520 nM hD3R Ki = 424 nM N N D2/D3 = 4.67 N D2/D3 = 5.52 N N N N N N N N H H H H H2N H2N H2N H2N O(2R,5S)-AB04-87O O O (2RS,5S)-AB04-88 (2R,5S)-AB04-87 H (2R,5S)-AB04-88 H hD2RN Ki = 5220N nM N N hD2R Ki = 134 nM hD2R Ki = 5220 nM N N hD2RN Ki = 134 nMN hD3R Ki = H6470 nM H hD3R Ki = 5.96 nM hD3R Ki = 6470 nM H2N H2NhD3R Ki = 5.96 nM D2/D3 = 0.807 D2/D3 = 0.807 D2/D3 = 22.5 D2/D3 = 22.5 Battiti Cemaj, Guerrero et al. JMC, 2019 62, 6287 Linker: more than just a link

O N H N N H N N 2 O H FOB02-04 hD3R Ki = 2.84 nM hD2R Ki = 106 nM D2/D3 = 37.3

O (S) O (S) (S) (R) N H N H N (S) N N (S) N (R) (S) H N N H N N 2 O H 2 O H FOB02-04A FOB02-04B hD3R Ki = 1.85 nM hD3R Ki = 282 nM hD2R Ki = 87.8 nM hD2R Ki = 831 nM D2/D3 = 47.5 D2/D3 = 2.95 Best Compound in Series Javier Garcia Nafria, BIFI Battiti Cemaj, Guerrero et al. JMC, 2019 62, 6287 SUMMARY-2 • Bitopic agonists based on PD128,907 and PF 592,379 have been synthesized • We discovered that the 2S,5S- stereochemistry in the PF primary pharmacophore coupled with the privileged 2-indoleamide as the secondary pharmacophore and a chiral cyclopropyl linker gave the bitopic analogue, FOB02-04 - the most D3R-selective full agonist reported to date (EC50=4.15 nM; 78%) • Chirality in the primary pharmacophore and the linker matter! • A cryo-EM structure would be the first to use a bitopic agonist Our latest foray into bivalent molecules:

Novel Dual-Target Mu Opioid (MOR) and Dopamine D3 Receptor (D3R) Ligands as Potential Non-Addictive Pharmacotherapeutics for Pain Management Alessandro Bonifazi,†* Francisco O. Battiti,† Julie Sanchez,‡ Saheem Zaidi,⊥ Eric Bow,§ Mariia Makarova,§ Jenny Lam,†# Rana Rais,# Kenner Rice,§ Vsevolod Katritch,⊥ Meritxell Canals,‡ J. Robert Lane,‡ Amy Hauck Newman†*

Seva Katritch & Saheem Zaidi, USC R1 C Designing bivalent MOR/D R Z R2 3 O N N A molecules for the treatment X n B of pain and OUD X

• Based on SAR at both MOR and D3R, Z R1 n Y R2 C we have synthesized a large series of X Targeting MOR O N N Targeting D3R novel bivalent and bitopic ligands A X • In collaboration with the Rice group, B

we have incorporated novel MOR X Ar agonists with both primary and A Z B secondary for D3R C Y • Several lead compounds have been discovered with nM affinities for both MOR and D3R • The active molecules are all bivalent Chiral Resolution (as opposed to bitopic)

Alessandro Bonifazi & Francisco Battiti Novel MOR-D3R ligands range in

Meri Canals & Rob Lane U. Nottingham SUMMARY-3

• We have discovered several bivalent MOR/D3R partial MOR Pharmacophore linker D3R Pharmacophore agonists with nM affinities

MOR • Computational studies are ongoing to elucidate key ligand- Agonism or Partial Agonism D3R Antagonism or receptor interactions as a source for the wide range of Partial Agonism affinities observed for structurally similar compounds

MOR • Functional studies have been conducted to evaluate efficacy DA VTA and G- vs beta-arrestin bias

• In vivo studies are being conducted on our 3 lead D3R compounds Analgesia • And, of course we are designing 2nd generation compounds, further investigating novel chiral/cyclic linkers, new primary pharmacophores and improving druggability Reward

Bonifazi et al., manuscript in revision. Acknowledgements HN

O NH

Medicinal Chemistry: HO N Alessandro Bonifazi, Francisco Battiti, Vivek O N Kumar, Anver Shaik, Adrian Guerrero, Sophie Cl Cemaj, JJ Cao – NIDA-IRP

Computational Chemistry: Lei Shi – NIDA-IRP Seva Katritich - USC

Pharmacology: Zheng-Xiong Xi, Zhi-Bing You, Chloe Jordan, Guo-Hua Bi, Ewa Galaj, Bree Humburg, Pramisha Adhikari, Hideaki Yano - NIDA-IRP Mike Nader – Wake Forest Matt Banks, VCU Olivier George UCSD

PK and Metabolism: Barbara Slusher, Rana Rais – JHU Jenny Lam, Alex Gadiano – NIDA-IRP, JHU