Kratom Alkaloids as Probes for Opioid Receptor Function: Pharmacological Characterization of Minor Indole and Oxindole Alkaloids from Kratom

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Research Article

Kratom Alkaloids as Probes for Opioid Receptor Function:

Pharmacological Characterization of Minor Indole and Oxindole

Alkaloids from Kratom

Soumen Chakraborty, Rajendra Uprety, Amal E. Daibani, Valerie L. Rouzic, Amanda Hunkele,

Kevin Appourchaux, Shainnel O. Eans, Nitin Nuthikattu, Rahul Jilakara, Lisa Thammavong,

Gavril W. Pasternak, Ying-Xian Pan, Jay P. McLaughlin, Tao Che, and Susruta Majumdar *

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ABSTRACT: Dry leaves of kratom (mitragyna speciosa) are

anecdotally consumed as pain relievers and antidotes against

opioid withdrawal and alcohol use disorders. There are at least 54

alkaloids in kratom; however, investigations to date have focused

around mitragynine, 7-hydroxy mitragynine (7OH), and mitragy-

nine pseudoindoxyl (MP). Herein, we probe a few minor indole

and oxindole based alkaloids, reporting the receptor aﬃnity, G-

protein activity, and βarrestin-2 signaling of corynantheidine,

corynoxine, corynoxine B, mitraciliatine, and isopaynantheine at

mouse and human opioid receptors. We identify corynantheidine

as a mu opioid receptor (MOR) partial agonist, whereas its

oxindole derivative corynoxine was an MOR full agonist. Similarly, another alkaloid mitraciliatine was found to be an MOR partial

agonist, while isopaynantheine was a KOR agonist which showed reduced βarrestin-2 recruitment. Corynantheidine, corynoxine, and

mitraciliatine showed MOR dependent antinociception in mice, but mitraciliatine and corynoxine displayed attenuated respiratory

depression and hyperlocomotion compared to the prototypic MOR agonist morphine in vivo when administered supraspinally.

Isopaynantheine on the other hand was identiﬁed as the ﬁrst kratom derived KOR agonist in vivo. While these minor alkaloids are

unlikely to play the majority role in the biological actions of kratom, they represent excellent starting points for further diversiﬁcation

as well as distinct eﬃcacy and signaling proﬁles with which to probe opioid actions in vivo.

KEYWORDS: Respiration, kratom, oxindoles, corynoxine, mitraciliatine, partial agonism

The kappa²⁶and delta opioid receptor (DOR)^27,28have

been proposed as alternate targets to develop safer analgesics

INTRODUCTION

■

Opioid receptor agonists like morphine and fentanyl are

as well. Peripherally restricted kappa agonists²⁶and kappa

clinically used analgesics. However, activation of a mu opioid

antagonists²⁹have been reported to preclude or ameliorate the

receptor (MOR) is associated with adverse eﬀects like

negative aﬀect associated with pain. Similar to MOR based

respiratory depression, dependence, and abuse potential.¹⁻³

approaches, biased agonism^7,24,30and allosterism³¹have been

Mismanagement of prolonged use of opioids for treating pain,

proposed as avenues to develop functionally selective opioids

coupled with the spread of illicit synthetic opioids such as

at these two receptors. A recent paper from our group³²shows

fentanyl, has led to an opioid epidemic estimated to take an

that dual partial agonism at MOR/KOR subtypes may oﬀer

average of 130 American lives daily.⁴⁻⁶Synthesis of a

some additional advantages over targeting partial agonism at a

nonaddictive and unexploitable analgesic is a pressing scientiﬁc

single subtype.

priority in addressing the causes of the current opioid

Natural products oﬀer another approach, with unique

epidemic.

opportunities to discover probes with druglike properties.³³⁻³⁵

Various approaches to design functionally selective opioids

have been proposed in the opioid ﬁeld. These include ligands

displaying biased agonism,⁷⁻⁹targeting heteromers,^10,11

Received: March 15, 2021

Accepted: June 28, 2021

Published: July 2, 2021

allosteric modulators,¹²⁻¹⁴targeting MOR splice variants,¹⁵⁻¹⁷

and compounds with polypharmacological actions at multiple

opioid targets.¹⁸⁻²²A recent approach aims at revisiting low

eﬃcacy partial agonism in newer assays with limited signal

ampliﬁcation.²³⁻²⁵

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Figure 1. Chemical structures and conﬁgurations at C3 and C20 of select indole and oxindole moiety based kratom alkaloids.

Scheme 1. Chemical Synthesis of Corynantheidine

Nearly 30% of all FDA approved drugs are either natural

products or their derivatives.³⁶In search of templates distinct

from morphine and fentanyl, our group has been interested in

the chemical and pharmacological characterization of kratom

based natural products. Mitragyna speciosa is a psychoactive

plant from Southeast Asia, which is also known as

“kratom”.^37,38In Malaysia it is well-known as “biak biak”.

Kratom products are commercially accessible in the U.S. and

sold as dried leaves, capsules, and liquid concentrates.³⁸⁻⁴⁰

Individuals use it to relieve pain and alcoholism, and it is also

used in withdrawal medications.

Kratom leaves mostly consist of alkaloids, which contain

indole or oxindole moiety (Figure 1). Roughly 54 alkaloids

have already been extracted from this plant, with mitragynine

being the major (∼66%) alkaloid component.⁴¹⁻⁴⁵About 1−

2% of the total dry leaf material in kratom is mitragynine. The

relative concentrations of each alkaloid present in this plant are

largely dependent on its origin and geographical factors.

Variation in climates, soil types, and environment have been

proposed to play a signiﬁcant role in the overall distribution of

alkaloids in kratom.^46,47While we have studied kratom from

various sources, we have largely focused our recent studies on

the “Red Indonesian Micro Powder” from Moon Kratom

(Austin, TX). In our hands, we ﬁnd approximately 48.6% of

mitragynine (1, Figure 1), 5.8% of paynantheine (2), 3.5% of

speciogynine (3), and 7.7% of speciociliatine (4) along with a

much smaller amount (<0.02% of mitraciliatine, <0.03% of

isopaynantheine) from the crude kratom alkaloid extracts

(Figure 1). Although we did not ﬁnd other minor alkaloids like

corynantheidine (5), corynoxine (9), corynoxine B (10), and

7-hydroxymitragynine (8) from “Red Indonesian Micro

Powder”, other sources of kratom have been reported to

have these minor alkaloids.^44,48,49

Pharmacological characterization of kratom,^39,43,50mitragy-

nine,^51,527-hydroxy mitragynine (7OH),^51,53mitragynine

pseudoindoxyl (MP)^51,54,55(Figure 1), and other major

alkaloids paynantheine,⁴³speciogynine,^43,45,56and speciocillia-

tine are known in the literature. However, studies are limited

on contributions of the minor alkaloids to biological actions of

kratom, and the signaling proﬁle of these individual alkaloids

remains unknown to date. This report describes the character-

ization of low abundance indole alkaloids: corynantheidine⁵⁷

(5), isopaynantheine (6), mitraciliatine (7), and the oxindole

alkaloids⁴⁸corynoxine (9) and corynoxine B (10). Alkaloids

were characterized pharmacologically in cell lines expressing

opioid receptors using binding assays, [³⁵S]GTPγS assays, and

BRET based Gi-1 and βarrestin-2 signaling, with selected

alkaloids tested in vivo for antinociception and adverse eﬀects

like respiratory depression and locomotor activity in mice. Oﬀ-

target evaluation on three alkaloids was also carried out using

PDSP-NIMH screening.⁵⁸

Presently, we report identiﬁcation of three novel anti-

nociceptive natural products and compounds with unique

opioid receptor mediated signaling proﬁles including partial

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Table 1. Aﬃnity Determination in Mouse Opioid Receptor Transfected Cell Lines

a

aﬃnity (K_inM)

compound

mMOR

mKOR

mDOR

corynantheidine

mitraciliatine

isopaynantheine

corynoxine

57.1 8.3

135.1 7.7

91.8 5.8

140.6 73.9

5010 388

230 47

385.4 47

101.2 2.3

325.9 31.9

3081.4 1727.5

>10 μM

172 24.5

827.1 85.7

822.9 126.9

1273.9 225.7

1682 260.92

1011 49

corynoxine B

b

mitragynine

231 21

3081.4 1727.5

b

7OH mitragynine

37

4

1273.9 225.7

b

DAMGO

3.3 0.43

4.6 1.8

-

b

morphine

-

b

U50,488H

0.73 0.32

b

DPDPE

-

1.39 0.67

a

Competition studies were performed with the indicated natural prodcuts or compounds against ¹²⁵I-IBNtxA (0.1 nM) in membranes from CHO

cells stably expressing cloned mouse opioid receptors. Results are presented as nM SEM from three independent experiments performed in

b

triplicate. The dash (-) means “not determined”. Literature values from Varadi et al., 2016, 59(18), 8381−97.

Table 2. Functional Activity Characterization Using [³⁵S]GTPγS Assays in Opioid Transfected Cell Lines

a

[³⁵S]GTPγS functional assay

mMOR

mKOR

mDOR

compound

EC₅₀(nM)

E_max(%)

EC₅₀(nM)

E_max(%)

EC₅₀(nM)

no activity

E_max(%)

corynanthedine

mitraciliatine

isopaynantheine

corynoxine

104.24 4.59

228 9.6

191.8 5.8

202.8 4.4

74.13 1.24

47.5 0.05

no activity

218 6.4

282.2 18

no activity

-

98.7

93.5

5

50

5

136.4 3.3

no activity

100

no activity

corynoxine B

b

DAMGO

3.4 0.2

-

b

U50,488H

DPDPE

-

9.5 1.8

-

100

-

b

-

16.2 5.1

100

a

Eﬃcacy and potency data were obtained using agonist i [³⁵S]GTPγS binding assay. Eﬃcacy is represented as EC₅₀(nM) and percent maximal

stimulation (E_max) relative to standard agonist DAMGO (MOR), DPDPE (DOR), or U50,488H (KOR) at 1000 nM. Results are presented as nM

SEM from three independent experiments performed in triplicate. The dash (-) denotes not determined or not applicable. No activity = no

b

agonism at 1000 nM drug concentration or no antagonism of 100 nM prototypic control agonist. Literature values from Uprety et al. Elife. 2021

Feb 8;10:e56519.

agonism at MOR by corynantheidine and mitraciliatine,

antinociceptive agents with attenuated adverse eﬀects (cor-

ynoxine and mitraciliatine), and reduced oﬀ-target receptor

labeling by mitraciliatine. Another natural product, isopaynan-

theine with a C20-vinyl group instead of C20-ethyl in the case

of mitraciliatine, was a KOR agonist with reduced recruitment

of βarrestin-2 in cell lines and showed KOR dependent−MOR

independent antinociception in mice.

corresponding triﬂate 13. Synthesis of corynantheidine (5) was

achieved by palladium-catalyzed removal of the triﬂate ester

(13) in the presence of formic acid.

Mitraciliatine (7), a diastereoisomer of mitragynine, has also

been isolated as a minor alkaloid from mitragyna speciosa

leaves.⁶²⁻⁶⁵This tetracyclic indole alkaloid represents the

opposite stereochemistry at both C3 (3R) and C20 (20R) to

that of mitragynine. Mitraciliatine is the least studied among all

other minor alkaloids.⁶⁶We were able to extract a considerable

amount of this alkaloid from “Red Indonesian Micro Powder”

(see procedure in Materials and Methods section).

Isopaynantheine (6) is another minor alkaloid with an

indole core.^62,65,66Compared to that of mitragynine, this

molecule has the opposite stereochemical connectivity at both

C3 (R-isomer) and C20 (R-isomer). The other diﬀerence is

the replacement of the C20 ethyl moiety with a vinyl moiety.

This compound is also the C3 epimer of paynantheine (2).

Studies on isopaynantheine as an opioid probe are unknown in

the literature. We extracted this from “Red Indonesian Micro

Powder” (detailed extraction in the Materials and Methods

section).

RESULTS AND DISCUSSION

■

Chemistry. Corynantheidine (5), one of the minor indole

alkaloids from kratom is also known as 9-desmethoxymi-

tragynine (Figure 1).^49,59,60Corynantheidine has the same

stereochemistry at C3 (3S) and C20 (20S) as mitragynine.

About 1% of alkaloid extract is believed to be corynantheidine

in some extracts according to literature reports.⁶¹We were not

able to ﬁnd this alkaloid in “Red Indonesian Micro Powder”, so

we chemically synthesized corynantheidine from mitragynine

as shown in Scheme 1.

Extraction of mitragynine (1) from kratom powder was

performed using a literature reported protocol by our group.⁵¹

9-Hydroxymitragynine (12, Scheme 1) was synthesized from 1

using AlCl₃as the Lewis acid in the presence of ethanethiol.⁵⁷

Hydroxy compound 12 was treated with N-phenyl-bis(tri-

ﬂuoromethanesulfonimide) in the presence of a base to furnish

Two oxindole alkaloids corynoxine (9) and corynoxine B

(10) have also been isolated from M. speciosa.^44,48,67These two

molecules with an oxindole⁶⁸core diﬀer in their stereo-

chemistry at spiro C7 being (7S) for 9 and (7R) for 10.⁴⁴

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Figure 2. G-protein and βarrestin-2 signaling of Corynantheidine at opioid receptors and dose dependent antinociception time course of

Corynantheidine. Corynantheidine shows MOR selective G-protein signaling while lacking measurable βarrestin-2 recruitment. (A) Gi-1 activation

measured using BRET assays at hMOR. Corynantheidine is a hMOR partial agonist. DAMGO EC₅₀(nM) (pEC₅₀SEM) = 3.56 (8.45 0.13)

nM, E_max

%

SEM = 100 4.17. Corynantheidine EC₅₀(nM) (pEC₅₀SEM) = 67.2 (7.17 0.29) nM, E_max

%

SEM = 37.2 4.30. (B) No

measurable βarrestin-2 recruitment was observed in BRET assays of corynantheidine at hMOR. DAMGO EC₅₀(nM) (pEC₅₀SEM) = 172.89

(6.76 0.08) nM, E_max

Gi-1 activation in BRET assay of corynantheidine at hKOR. U50488H EC₅₀(nM) (pEC₅₀SEM) = 2.8 (8.55 0.21) nM, E_max

5.89. Corynantheidine EC₅₀(nM) (pEC₅₀SEM) = n.d., E_max

assays of corynantheidine at hKOR. U50488H EC₅₀(nM) (pEC₅₀

Corynantheidine EC₅₀(nM) (pEC₅₀SEM) = n.d., E_maxSEM = <20. (E) Gi-1 activation in BRET assay of corynantheidine at hDOR.

DPDPE EC₅₀(nM) (pEC₅₀SEM) = 1.23 (8.91 0.28) nM, E_maxSEM = 100 11.69. Corynantheidine EC₅₀(nM) (pEC₅₀SEM) = n.d.,

%

SEM = 100 3.48. Corynantheidine EC₅₀(nM) (pEC₅₀SEM) = not determined (n.d.), E_max

%

SEM = <20. (C)

%

SEM = 100

%

SEM = <20. (D) No measurable βarrestin-2 recruitment was observed in BRET

SEM) = 229.54 (6.64 0.11) nM, E_maxSEM = 100 4.63.

%

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Figure 2. continued

E_max%

SEM = <20%. (F) No measurable βarrestin-2 recruitment was observed in BRET assays of corynantheidine at hDOR.

DPDPE EC₅₀(nM) (pEC₅₀SEM) = 12.28 (7.91 0.12) nM, E_maxSEM = 100 4.08. Corynantheidine EC₅₀(nM) (pEC₅₀

SEM) = n.d., E_maxSEM = <20. n.d. = not determined. Data from both Gi-1 activation and βarrestin-2 assays using human

%

opioid receptors were normalized to E_maxof the corresponding controls, DAMGO, U50,488H, and DPDPE. The data were

processed in GraphPad Prism using a three-parameter logistic equation to ﬁt the dose response curves. Mean EC₅₀(pEC₅₀SEM)

represents the data for assays run in triplicate (n = 3). See Table S1 for values. (G) Time course of tail withdrawal antinociception of

corynantheidine: Groups of C57BL/6J mice (n = 8 each group) received (i.c.v) corynantheidine, and antinociceptive response was

measured at doses of 10, 30, and 100 nmol in WT mice at the indicated time points using the 55 °C tail withdrawal assay. Each point

represents mean% antinociception SEM corynantheidine displayed potent dose dependent antinociceptive response. Attenuated

eﬀect of antinociception of corynantheidine (100 nmol, i.c.v.) was observed in MOR KO mice at 10−20 min (*p = 0.05, unpaired t

test per row corrected for multiple comparisons using Holm-Sidak method).

Although the biological property of these templates have not

been studied extensively, total synthesis has been carried out

on these two scaﬀolds.⁶⁹For our studies, we purchased

corynoxine and corynoxine B from BOC sciences (NY, USA).

Pharmacology. Corynantheidine. Corynantheidine has

been characterized ex vivo in guinea pig ileum assays. It

showed no agonist actions in these assays and has been

reported to be an MOR antagonist.⁵⁷In recent years, the

binding aﬃnity at MOR and α adrenergic receptor subtype 1D

(α_1D) has been reported to be 118 and 41 nM, respectively.⁵⁶

We reevaluated this natural product in binding assays and

studied the signaling properties of this molecule at both mouse

opioid receptors as well as at human opioid receptors. In our

hands, corynantheidine had moderate aﬃnity of K_i= 57 nM

for the mouse mu opioid receptor (mMOR) (Table 1), about

∼4-fold higher than that of mitragynine (K_i= 230 nM)⁵¹in the

same assay. It also had moderate aﬃnity at the mouse kappa

opioid receptor (mKOR) and mDOR (Table 1). In

[³⁵S]GTPγS assays (Table 2), corynantheidine was a partial

agonist (E_max= 74%) with eﬃcacy comparable to that of

mitragynine at mMOR (E_maxof mitragynine = 65%), and its

potency was comparable to its binding aﬃnity. It did not signal

through mKOR or mDOR and can be classiﬁed as an MOR

selective agonist in this assay (Table 2).

The signaling of this molecule was further characterized at

human opioid receptors using BRET assays.^32,70The Gi-1

activation proﬁle at the human mu opioid receptor (hMOR)

was similar to that of [³⁵S]GTPγS assay in mMOR (Figure

2A). Corynantheidine was a selective partial agonist at MOR

(E_max= 37%) in this assay but was 20 times less potent

compared to [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin

(DAMGO). In the [³⁵S]GTPγS assay, it was about 30-fold

less potent than DAMGO. It showed no signaling through the

human kappa opioid receptor (hKOR) (Figure 2C) or hDOR

(Figure 2E) as well as no βarrestin-2 recruitment (Figure

2B,D,F) at any opioid subtypes consistent with the

pharmacology of the mitragynine template where βarrestin-2

recruitment is not seen.^43,51,71Potential oﬀ-target labeling of

this molecule was examined using radioligand assays at ∼50

CNS human receptors using the PDSP-NIMH (Table 3C).

Corynantheidine was found to bind to as many as 11 targets in

addition to hMOR (K_i= 339 nM) and hKOR, with the highest

aﬃnity demonstrated for hα_2A(K_i= 74 nM) and hNMDA (K_i

= 83 nM). The antinociceptive actions of corynantheidine, to

the best of our knowledge, are unknown. To evaluate in vivo

agonist activity of corynantheidine, we utilized the 55 °C

warm-water tail withdrawal assay^32,51with C57BL/6J mice.

Supraspinal administration (intracerebroventricular, i.c.v.) of

Table 3. Oﬀ-Target Proﬁling of Three Alkaloids at ∼50

CNS Human Receptors through PDSP

(a) mitraciliatine PDSP screening

a

target

aﬃnity (K_i(pEC₅₀SEM) nM

hNET

hMOR

hKOR

hα2A

6604 (5.2 0.1)

304 (6.52 0.06)

73 (7.14 0.07)

2153 (5.67 0.09)

(b) Isopaynantheine PDSP Screening

hSERT

5111 (5.3 0.2)

5066 (5.3 0.2)

122 (6.91 0.07)

27 (7.57 0.07)

206 (6.7 0.1)

2152 (5.7 0.1)

hDAT

hMOR

hKOR

hH1

hα2A

(c) Corynantheidine PDSP Screening

h5HT1A

hSERT

hDAT

hMOR

hKOR

hH2

hα1A

hα1B

hα2A

1156 (5.96 0.06)

2615 (5.6 0.2)

3915 (5.4 0.2)

339 (6.47 0.07)

2109 (5.68 0.07)

5126 (5.29 0.07)

397 (6.4 0.06)

3304 (5.48 0.06)

74 (7.1 0.1)

hα2B

hα2C

hSigma 2

hNMDA

1539 (5.81 0.08)

269 (6.57 0.09)

1073 (6 0.1)

83 (7.08 0.09)

a

National Institute of Mental Health Psychoactive Drug Screening

Program (NIMH-PDSP) conducted the binding aﬃnities reported in

Table 3. The methods and radioligands details used for the binding

assays are available on the NIMH PDSP website and Besnard et al.,

2012, 492(7428), 215−20.

the compound was utilized to avoid potential blood−brain

barrier (BBB) permeability issues and to minimize the eﬀects

of metabolism. The compound showed a ceiling eﬀect in

antinociception assays (with a maximal 50% maximum possible

eﬀect (MPE)) consistent with the observed partial agonism

seen in cell lines at mMOR in the [³⁵S]GTPγS assays (Table

2). To probe for MOR dependent actions, we also evaluated

corynantheidine in MOR KO mice, ﬁnding antinociception to

be MOR dependent (Figure 2G). It is possible that the

antinociception is mediated by multiple targets in addition to

MOR based on the oﬀ-target labeling displayed by this

molecule, notably the higher aﬃnity for α adrenergic receptor

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Figure 3. G-protein and βarrestin-2 signaling of corynoxine at opioid receptors. Corynoxine shows MOR selective G-protein signaling while not

recruiting βarrestin-2. (A) Gi-1 activation measured using BRET assays at hMOR. Corynoxine is a full agonist at hMOR compared to DAMGO.

DAMGO EC₅₀(nM) (pEC₅₀SEM) = 28.12 (7.55 0.08) nM, E_max

(5.79 0.09) nM, E_maxSEM = 96.54 4.69. (B) No measurable βarrestin-2 recruitment was observed in BRET assays of corynoxine at

hMOR. DAMGO EC₅₀(nM) (pEC₅₀SEM) = 90.6 (7.04 0.07) nM, E_maxSEM = 100 2.29. Corynoxine EC₅₀(nM) (pEC₅₀SEM) =

n.d. E_maxSEM < 20%. (C) Gi-1 activation measured using BRET assays at hKOR. U50488H EC₅₀(nM) (pEC₅₀SEM) = 6.93 (8.16 0.06)

nM, E_maxSEM = 100 1.61. Corynoxine EC₅₀(nM) (pEC₅₀SEM) = n.d. E_maxSEM < 20%. (D) No measurable βarrestin-2 recruitment

was observed in BRET assays of corynoxine at hKOR. U50488H EC₅₀(nM) (pEC₅₀SEM) = 48.95 (7.31 0.04) nM, E_maxSEM = 100

1.47. Corynoxine EC₅₀(nM) (pEC₅₀SEM) = n.d., E_maxSEM < 20. (E) Gi-1 activation measured using BRET assays at hDOR. DPDPE EC₅₀

(nM) (pEC₅₀SEM) = 3.65 (8.43 0.37) nM, E_maxSEM = 100 14.51. Corynoxine EC₅₀(nM) (pEC₅₀SEM) = n.d. E_maxSEM <

20%. (F) No measurable βarrestin-2 recruitment was observed in BRET assays of corynoxine at hDOR. DPDPE EC₅₀(nM) (pEC₅₀SEM) =

17.69 (7.75 0.17) nM, E_maxSEM = 100 5.8. Corynoxine EC₅₀(nM) (pEC₅₀SEM) = n.d., E_maxSEM < 20. n.d. = not determined.

%

SEM = 100 2.78. Corynoxine EC₅₀(nM) (pEC₅₀SEM) = 1630

%

Data from both Gi-1 activation and βarrestin-2 assays using human opioid receptors were normalized to E_maxof the corresponding controls,

DAMGO, U50,488H, and DPDPE. The data were processed in GraphPad Prism using a three-parameter logistic equation to ﬁt the dose response

curves. Mean EC₅₀(pEC₅₀SEM) represents the data for assays run in triplicate (n = 3). See Table S1 for values.

subtype 2A (α_2A) aﬃnity over MOR. The adverse eﬀects of

corynantheidine were not investigated further because of its

structural similarity to mitragynine and other congeners.

Corynoxine and Corynoxine B. We next looked at the

oxindoles of corynantheidine, i.e., corynoxine and corynoxine

B. In addition to Mitragyna speciosa, these alkaloids are also

found in Cat’s claw.⁷²⁻⁷⁴The binding aﬃnities at hMOR and

antinociception in rats have been recently reported, although

functional characterization was not reported for both

molecules.⁴⁸Corynoxine exhibited moderate aﬃnity for

mMOR (K_i= 140 nM, Table 1) and poor aﬃnity for

mKOR and mDOR (K_i> 1000 nM). In [³⁵S]GTPγS functional

assays (Table 2), corynoxine was similar to mitragynine with

respect to its potency, but it had a higher eﬃcacy than both

mitragynine as well as DAMGO (E_max= 136%). It did not

signal through mKOR or mDOR, consistent with its binding

aﬃnity at these targets.

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Figure 4. Dose dependent antinociception time course, respiratory depression, and locomotor eﬀects of corynoxine. Corynoxine shows MOR

dependent antinociception. Attenuated respiratory depression and locomotor eﬀects were observed for corynoxine at equianalgesic morphine

doses. (A) Time course of tail withdrawal antinociception of corynoxine: Groups of C57BL/6J mice (n = 8 each group) received (i.c.v.)

corynoxine, and antinociceptive response was evaluated at doses of 3, 10, and 30 nmol in WT mice at the indicated time points using the 55 °C tail

withdrawal assay. Each point represents mean% antinociception SEM. Corynoxine exhibited potent potent dose dependent antinociceptive

response. Reduced eﬀect of antinociception of corynoxine (30 nmol, i.c.v.) was observed in MOR KO mice at 10−30 min (****p < 0.0001,

unpaired t test per row corrected for multiple comparisons using Holm-Sidak method). (B) Antinociception time course of Morphine: Groups of

C57BL/6J mice (n = 8 each group) received (i.c.v.) morphine, and antinociceptive response was evaluated at doses of 0.3, 1, 3, and 10 nmol in WT

mice at the indicated time points using the 55 °C tail withdrawal assay. Each point represents mean% antinociception SEM. (C) Respiratory rate

corynoxine: Groups of mice received either vehicle (n = 12), saline (n = 12), morphine (100 nmol, i.c.v.; n = 12), or corynoxine (100 nmol, i.c.v.; n

= 12), and the measurement of breath rates was done every 20 min for 180 min. Corynoxine showed an increase in breath rates at 60 min (***p =

0.0002), 80−160 min (****p < 0.0001), and 180 min (***p = 0.001) compared to those of the vehicle, while morphine decreased breadth rates at

20−40 min (*p = 0.0387). The p values were calculated by 2-way ANOVA followed by Dunnett’s multiple-comparison test. (D) Locomotor eﬀect

of corynoxine: Groups of mice received either vehicle (n = 24), saline (n = 12), morphine (100 nmol, i.c.v.; n = 12), or corynoxine (100 nmol, i.c.v.;

n = 12), and the distance traveled by charcoal for each group of mice was measured. Corynoxine showed no signiﬁcant hyperlocomotion compared

to that of the vehicle as determined by 2-way ANOVA followed by Dunnett’s multiple-comparison test. However, signiﬁcant locomotor eﬀect was

observed for morphine at 80−100 min (****p < 0.0001), 120 min (**p = 0.0038), 140−160 min (****p < 0.0001), and 180 min (**p = 0.0093)

compared to that of the vehicle. The p values were calculated by 2-way ANOVA followed by Dunnett’s multiple-comparison test.

At human opioid receptors, similar to [³⁵S]GTPγS assays,

corynoxine was a selective MOR full agonist (Figure 3A) with

a potency 58-fold less than that of DAMGO. Corynoxine did

not signal through other opioid receptor subtypes (Figure

3C,E) nor did it recruit βarrestin-2 at any opioid receptor

subtypes (Figure 3B,D,F), similar to mitragynine and

corynantheidine as we described before. In mice, corynoxine

showed potent antinociception in the tail-withdrawal assay

with an ED₅₀(and 95% C.I.) value of 3.43 (0.53−6.41) nmol

using i.c.v. administration and comparable to the ED₅₀(and

95% C.I.) value of morphine (4.77 (1.49−28.8) nmol) (Figure

4A,B). Receptor selectivity in vivo was assessed in MOR KO

mice, and corynoxine displayed MOR dependent antinocicep-

tion, consistent with its in vitro activity (Figure 4A). Because

most classical MOR agonists are known to lead to respiratory

depression and hyperlocomotion, we evaluated this structurally

unique natural product in the CLAMS (Comprehensive

Laboratory Animal Monitoring System) assay.^32,39Whereas a

100 nmol, i.c.v. dose of morphine showed a signiﬁcant decrease

in breath rate, this dose of corynoxine showed no depression of

breath rate; instead, it showed statistically signiﬁcant

respiratory stimulation (Figure 4C). Moreover, in contrast to

morphine, this dose of corynoxine did not produce signiﬁcant

hyperlocomotion (Figure 4D).

Screening of corynoxine B showed poor binding aﬃnity at

all opioid subtypes (K_i> 1000 nM) (Table 1) and no activity

in [³⁵S]GTPγS assays (Table 2). Accordingly, this molecule

was not screened in BRET assays for either G-protein or

βarrestin-2 recruitment nor is it tested in mice.

Mitraciliatine. We next screened mitraciliatine. This natural

product has been described in the literature to be present in

kratom,⁴⁴but to the best of our knowledge, this natural

product has not been evaluated pharmacologically. Similar to

other kratom natural products, this molecule had moderate

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Figure 5. G-protein and βarrestin-2 signaling, antinociception time course, respiratory depression, and locomotor eﬀects of mitraciliatine.

Mitraciliatine shows agonism at hMOR and hKOR while showing diﬀerential βarrestin-2 signaling at the same subtypes. Mitraciliatine shows MOR

dependent antinociception. Attenuated respiratory depression and locomotor eﬀects were observed for mitraciliatine at equianalgesic morphine

doses. (A) Gi-1 activation measured using BRET assays at hMOR. Mitracilliatine shows G-protein signaling at hMOR while showing no βarrestin-2

signaling, compared to DAMGO. DAMGO EC₅₀(nM) (pEC₅₀SEM) = 3.56 (8.45 0.13) nM, E_max

(nM) (pEC₅₀SEM) = 23.69 (7.62 0.16) nM, E_maxSEM = 50.7 3.22. (B) No measurable βarrestin-2 recruitment was observed in BRET

assays of mitracilatine at hMOR. DAMGO EC₅₀(nM) (pEC₅₀SEM) = 90.6 (7.04 0.07) nM, E_maxSEM = 100 2.29. Mitraciliatine EC₅₀

(nM) (pEC₅₀SEM) = n.d., E_maxSEM < 20. (C) Gi-1 activation measured using BRET assays at hKOR. Mitracilliatine shows G-protein

signaling at hKOR as well as robust βarrestin-2 signaling at hKOR. U50488H EC₅₀(nM) (pEC₅₀SEM) = 6.93 (8.16 0.06) nM, E_maxSEM

= 100 1.61. Mitraciliatine EC₅₀(nM) (pEC₅₀SEM) = 269.19 (6.57 0.06) nM, E_maxSEM = 114.32 2.42. (D) Signiﬁcant βarrestin-2

recruitment was observed in BRET assays of mitracilatine at hKOR. U50488H EC₅₀(nM) (pEC₅₀SEM) = 48.95 (7.31 0.04) nM, E_max

SEM = 100 1.47. Mitraciliatine EC₅₀(nM) (pEC₅₀SEM) = 383.12 (6.42 0.06) nM, E_maxSEM = 104.13 2.57. (E) Gi-1 activation

measured using BRET assays at hDOR. Mitraciliatine did not show G-protein or βarrestin-2 signaling at hDOR. DPDPE EC₅₀(nM) (pEC₅₀

SEM) = 3.65 (8.43 0.37) nM, E_maxSEM = 100 14.54. Mitraciliatine EC₅₀(nM) (pEC₅₀SEM) = n.d., E_maxSEM = n.d. (F) No

measurable βarrestin-2 recruitment was observed in BRET assays of mitracilatine at hDOR. DPDPE EC₅₀(nM) (pEC₅₀SEM) = 17.69 (7.75

0.17) nM, E_maxSEM = 100 5.8. Mitraciliatine EC₅₀(nM) (pEC₅₀SEM) = n.d., E_maxSEM < 20. n.d. = not determined. Data from both

%

SEM = 100 4.17. Mitraciliatine EC₅₀

%

Gi-1 activation and βarrestin-2 assays using human opioid receptors were normalized to E_maxof the corresponding controls, DAMGO, U50,488H,

and DPDPE. The data were processed in GraphPad Prism using a three-parameter logistic equation to ﬁt the dose response curves. Mean EC₅₀

(pEC₅₀SEM) represents the data for assays run in triplicate (n = 3). See Table S1 for values. (G) Antinociception time course of mitraciliatine:

Groups of C57BL/6J mice (n = 8 each group) received (i.c.v.) mitraciliatine, and antinociceptive response was evaluated at doses of 10, 30, and

100 nmol in WT mice at the indicated time points using the 55 °C tail withdrawal assay. Each point represents mean% antinociception SEM. We

observed potent dose dependent antinociceptive response of mitraciliatine. Antinociception eﬀect of mitraciliatine (100 nmol, i.c.v.) remained

intact in KOR KO mice, although a statistically not signiﬁcant increase in %MPE was seen. Antinociception was found attenuated in MOR KO

mice at 10−30 min (***p = 0.0001, 0.001, and 0.0001 at 10, 20, and 30 min, respectively, unpaired t test per row corrected for multiple

comparisons using Holm-Sidak method). (H) Respiratory rate mitraciliatine: Groups of mice received either vehicle (n = 12), saline (n = 12),

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Figure 5. continued

morphine (100 nmol, i.c.v.; n = 12), or mitraciliatine (100 nmol, i.c.v.; n = 12), and the measurement of breath rates was done every 20 min for 180

min. Mitraciliatine showed increase in breath rates at 100 min (*p = 0.0233), 120 min (**p = 0.0017), and 140−180 min (****p < 0.0001)

compared to vehicle, while morphine decreased breadth rates at 20−40 min (*p = 0.0387). The p values were calculated by 2-way ANOVA

followed by Dunnett’s multiple-comparison test. (I) Locomotor eﬀect mitraciliatine: Groups of mice received either vehicle (n = 24), saline (n =

12), morphine (100 nmol, i.c.v.; n = 12), or mitraciliatine (100 nmol, i.c.v.; n = 12), and the distance traveled by charcoal for each group of mice

was measured. Mitraciliatine showed no signiﬁcant hyperlocomotion compared to that of the vehicle as determined by 2-way ANOVA followed by

Dunnett’s multiple-comparison test. However, signiﬁcant locomotor eﬀect was observed for morphine at 80−100 min (****p < 0.0001), 120 min

(**p = 0.0038), 140−160 min (****p < 0.0001), and 180 min (**p = 0.0093) compared to that of the vehicle. The p values were calculated by 2-

way ANOVA followed by Dunnett’s multiple-comparison test.

mMOR (Table 1) aﬃnity (K_i= 135 nM). To our surprise, it

had similar mKOR aﬃnity (K_i= 101 nM), in contrast to the

majority of mitragynine template based compounds which

tend to have higher aﬃnity and activity at MOR.³⁴In

[³⁵S]GTPγS assays, it was a mixed mMOR−mKOR dual

agonist (Table 2). Although its potency was similar to that of

mitragynine (EC₅₀∼ 200 nM), at both subtypes, it was found

to be a partial agonist (E_max= 47%) at mMOR and displayed

full agonism at mKOR (E_max= 98%). At mDOR, mitraciliatine

had no activity (Table 2). In BRET based assays with hMOR

and hKOR (Figure 5B−D), mitraciliatine diﬀerentially

recruited βarrestin-2. Consistent with mitragynine template

based compounds, no βarrestin-2 recruitment was seen

through hMOR (Figure 5B). In contrast, mitraciliatine

robustly recruited βarrestin-2 (E_max= 104%) through hKOR

(Figure 5D). At hDOR, it showed no signaling at both Gi as

well as βarrestin-2 (Figure 5E,F). Consistent with results

utilizing rodent receptors, mitraciliatine was a partial agonist

(E_max= 51%) at hMOR while displaying full agonism at hKOR.

Unlike other mitragynine congeners, this compound had a

cleaner oﬀ-target proﬁle (Table 3a). It had a moderate hKOR

aﬃnity (K_i= 73 nM)/hMOR aﬃnity (K_i= 304 nM) and

labeled two other nonopioid targets: the norepinephrine

transporter (hNET) (K_i= 6604 nM) and hα_2Areceptor (K_i

= 2153 nM). When administered supraspinally (i.c.v.) to mice,

mitraciliatine produced a ceiling eﬀect in antinociception

(Figure 5G), with maximal antinociception of 67% at a dose of

100 nmol, i.c.v. Testing in opioid receptor knockout mice

suggested that the antinociception was MOR dependent and,

surprisingly, independent of KOR (Figure 5G). When

evaluated in the CLAMS assay at a 100 nmol, i.c.v. dose,

mitraciliatine showed no respiratory depression or hyper-

locomotion. (Figure 5H,I).

Isopaynantheine. Isopaynantheine is the C20-vinyl ana-

logue of mitraciliatine. Similar to mitraciliatine, it has been

described in the literature to be present in kratom but has

never been characterized pharmacologically. In binding assays,

it was similar to mitraciliatine with mixed aﬃnity (Table 1) at

both mMOR (K_i= 92 nM) and mKOR (K_i= 325 nM). In

[³⁵S]GTPγS assays, too, it was found to act as a partial agonist

at mMOR (E_max= 50%) and showed full agonism at mKOR

(Table 2). The potencies were comparable to mitraciliatine. In

BRET assays at human opioid receptors, isopaynantheine

showed no G-protein activity at hMOR (Figure 6A), in sharp

contrast to the structurally similar natural product mitracilia-

tine, which was a partial agonist at hMOR. No measurable

activity was seen in the βarrestin-2 signaling pathway as well

(Figure 6B). Because it was found to have modest aﬃnity as

well as partial agonism at mMOR, we next examined this

natural product to see if it was an antagonist at hMOR. We

found that isopaynanthine was a modestly potent MOR

antagonist (IC₅₀= 1.26 μM) in comparison to diprenorphine

(IC₅₀= 4.2 nM) in the Gi-1 pathway (Figure 6C). Similarly, it

was an antagonist in the βarrestin-2 pathway as well (Figure

6D). At hKOR, it was found to be an agonist with a E_max= 80%

and modest potency (EC₅₀= 560 nM) compared to that of

U50,488h (EC₅₀= 7 nM) in the Gi-1 pathway (Figure 6E). In

sharp contrast to mitraciliatine, it did not recruit βarrestin-2 at

hKOR (Figure 6F). Similar to other alkaloids, no signaling was

seen through hDOR (Figure 6G,H). Oﬀ-target screening

(Table 3b) revealed it was less cleaner than mitraciliatine but

far superior than mitragynine.^45,56It had a high hKOR aﬃnity

(K_i= 27 nM)/lower hMOR aﬃnity (K_i= 122 nM) but also

had a labeled H1 receptor with a 206 nM aﬃnity (Table 3b).

While hSERT (serotonin transporter), hDAT (dopamine

transporters), and hα_2Awere also labeled by this molecule,

the aﬃnities were poorer for these targets.

When assessed supraspinally, isopaynanthine showed

antinociceptive activity with an ED₅₀(and 95% CI) value of

20.6 (6.97, 36.98 nmol) compared to that of U50,488H (ED₅₀

= 8.8 (5.7, 13.5 nmol) (Figure 6I,J). Receptor selectivity was

also assessed in vivo using MOR and KOR KO mice (Figure

6I). Antinociception was MOR independent and KOR

dependent.

DISCUSSION

■

Kratom, a plant which grows in Southeast Asia has been under

intense investigation in recent years. Earlier work prior to the

1990s and 2000s^38,41,52on kratom and its alkaloids focused on

characterization with in vivo⁷⁵⁻⁷⁸and ex vivo^57,79functional

assays. In the past ﬁve years, the signaling properties of the

component alkaloids have been assessed in cell lines expressing

opioid receptors, as well as more detailed re-evaluation of

kratom itself in animal models.^39,50Chemical characterization

of individual alkaloids⁴⁴present in kratom are also being

evaluated.

Kratom tea (a concoction of numerous alkaloids) and

kratom alkaloid extracts⁵⁰have been described as agents with

much-reduced symptoms of opioid withdrawal compared to

those of traditional MOR agonists like morphine. Moreover,

antiopioid withdrawal eﬀects in subjects physically dependent

on morphine with minimal adverse eﬀects has also been

observed in animal models.³⁹The major component in kratom,

mitragynine, has been characterized pharmacologically in in

vitro assays as well as in vivo assays. In cellular studies,

mitragynine was found to exhibit partial agonism at MOR in

ampliﬁed G-protein assays while showing no recruitment of

βarrestin-2 by us and others.^{34,43,50,51,53,80}Mitragynine also has

been proposed to be a low eﬃcacy agonist⁸¹in vivo and has

been found to have no abuse liability on its own while also

blunting the self-administration of opioid agonists like heroin

in rats.⁸²In mice, it has also been reported to have less physical

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Figure 6. continued

(pEC₅₀SEM) = 1276.4 (5.89 0.14), E_max

%

SEM = 106 8.67.

n.d. = not determined. (E) Gi-1 activation measured using BRET

assays at KOR. Isopaynantheine shows G-protein signaling at KOR

while showing no βarrestin-2 signaling. U50488H EC₅₀(nM) (pEC₅₀

SEM) = 7.03 (8.15

Isopaynantheine EC₅₀(nM) (pEC₅₀SEM) = 560.4 (6.25 0.066)

nM, E_maxSEM = 80.5 2.9. (F) No βarrestin-2 recruitment was

observed in BRET assays of isopaynantheine at KOR. U50488H EC₅₀

nM (pEC₅₀SEM) = 128.31 (6.89 0.10) nM, E_maxSEM = 100

4.12. Isopaynantheine EC₅₀(nM) (pEC₅₀SEM) = 599.29 (6.22

0.43) nM, E_maxSEM < 20. (G) Gi-1 activation measured using

0.04) nM, E_max

%

SEM = 100

1.58.

%

BRET assays at DOR. Isopaynantheine did not show G-protein or

βarrestin-2 signaling at DOR. SNC80 EC₅₀(nM) (pEC₅₀SEM) =

13.37 (7.87

0.05) nM, E_max

%

SEM = 100

1.89.

SEM

Isopaynantheine EC₅₀(nM) (pEC₅₀SEM) = n.d., E_max

%

< 20. (H) No measurable βarrestin-2 recruitment was observed in

BRET assays of isopaynantheine at DOR. DPDPE EC₅₀(nM) (pEC₅₀

SEM) = 7.76 (8.11

Isopaynantheine EC₅₀(nM) (pEC₅₀SEM) = n.d., E_max

0.05) nM, E_max

%

SEM = 100

1.81.

SEM <

%

20. n.d. = not determined. Data from both Gi-1 activation and

βarrestin-2 assays using human opioid receptors were normalized to

E_maxof the corresponding controls, DAMGO, U50,488H, DPDPE,

and diprenorphine (for anatogonism). The data were processed in

GraphPad Prism using a three-parameter logistic equation to ﬁt the

dose response curves. Mean EC₅₀(pEC₅₀SEM) represents the data

for assays run in triplicate (n = 3). See Table S1 for values. (I)

Antinociception time course of isopaynantheine: Groups of C57BL/

6J mice (n = 8 each group) received (i.c.v.) isopaynantheine, and

antinociceptive response was evaluated at doses of 10, 30, and 100

nmol in WT mice at the indicated time points using the 55 °C tail

withdrawal assay. Each point represents mean% antinociception

SEM. Isopaynantheine exhibited strong dose dependent antinocicep-

tive response. Isopaynantheine (100 nmol, i.c.v.) retained the

antinociceptive response in MOR KO mice. Antinociception was

found reduced in KOR KO mice at 10−20 min (***p = 0.0001,

*0.013 at 10 and 20 min, respectively, unpaired t test per row

corrected for multiple comparisons using Holm-Sidak method). (J)

Antinociception time course of U50488H: Groups of C57BL/6J mice

(n = 8 each group) received (i.c.v.) U50488H, and antinociceptive

response was evaluated at doses of 3, 10, and 30 nmol in WT mice at

the indicated time points using the 55 °C tail withdrawal assay. Each

point represents mean% antinociception SEM.

dependence than morphine alone and to block morphine

withdrawal. The involvement of adrenergic receptors^56,83and

serotonergic receptors^45,78,84as well as MOR itself in the

biological actions of mitragynine has been recognized and

remains a topic of emerging study.⁵⁶

Another alkaloid, 7-hydroxy mitragynine (7OH, present in

far less amounts <1%, not seen in our extracts),⁸⁵is a partial

agonist at MOR, with enhanced eﬃcacy and potency

compared to those of mitragynine while also showing reduced

βarrestin-2 recruitment.^43,51It is currently believed that the

antinociceptive actions of mitragynine are mediated by its

bioconversion to 7OH through a CYP450 dependent path-

way.⁵³Unlike mitragynine, 7OH shows adverse eﬀects similar

to those of classical MOR agonists and is a potent MOR

dependent antinociceptive agent with tolerance, dependence,

and abuse potential similar to those of typical opioid agonists

in mice.^43,51,53,76

Figure 6. G-protein, βarrestin-2 signaling, and dose dependent

antinociception time course of isopaynantheine. Isopaynantheine was

an MOR antagonist−KOR biased agonist. Antinociception of

Isopaynantheine is KOR dependent. (A) Gi-1 activation measured

using BRET assays at MOR. Isopaynantheine did not show G-protein

signaling at MOR compared to DAMGO. DAMGO EC₅₀(nM)

(pEC₅₀SEM) = 31.27 (7.50 0.03) nM, E_max

1.51. Isopaynantheine EC₅₀(nM) (pEC₅₀SEM) = n.d., E_max

%

SEM = 100

%

SEM < 20. (B) No measurable βarrestin-2 recruitment was observed

in BRET assays of isopaynantheine at MOR. DAMGO EC₅₀(nM)

(pEC₅₀SEM) = 81.62 (7.09 0.06) nM, E_max

2.55. Isopaynantheine EC₅₀(nM) (pEC₅₀SEM) = n.d., E_max

%

SEM = 100

%

SEM < 20. (C) Gi-1 antagonism measured using BRET assays at

MOR. Diprenorphine EC₅₀(nM) (pEC₅₀SEM) = 4.28 (8.37

0.05) nM, E_max

(pEC₅₀SEM) = 1259 (5.9 0.12) nM, E_max

%

SEM = 100 2.90. Isopaynantheine EC₅₀(nM)

SEM = 156.91

The semisynthetic analog mitragynine pseudoindoxyl (MP)

was initially reported in the literature as a fungal metabolite of

mitragynine.⁵⁵The spiro-pseudoindoxyl core of MP (Figure 1)

resulted from the Lewis acidic rearrangement of 7OH. MP is

%

13.49. (D) βarrestin-2 antagonism measured using BRET assays at

MOR. Diprenorphine EC₅₀(nM) (pEC₅₀SEM) = 0.43 (9.37

0.10) nM, E_max

%

SEM = 100 2.47. Isopaynantheine EC₅₀(nM)

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also an antinociceptive agent through opioid receptors and

shows G-protein partial agonism at MOR with minimal

βarrestin-2 recruitment.⁵¹Recent reports suggest that this

alkaloid is a metabolite formed in vitro from 7OH.⁸⁶

Recent eﬀorts from the Filizola group have probed the

binding of these three alkaloids and suggested subtle but

important diﬀerences in the binding poses of these three

alkaloids using computational simulations coupled with

mutational validation.⁵⁴

Through this work, we now report the binding and

functional characterization of minor kratom alkaloids and

develop a structure−activity relationship (SAR) for subtype

selectivity, eﬃcacy, and βarrestin-2 recruitment which may

help us and others in the ﬁeld toward additional derivatization

eﬀorts in the future as well as help us map out the individual

contribution of kratom alkaloids toward its bioactivity in

humans.

Similar to the related parent natural product, corynanthei-

dine or 9-desmethoxy mitragynine shows MOR partial

agonism, MOR subtype selectivity, and no recruitment of

βarrestin-2, and it labels multiple nonopioid receptors while

displaying MOR dependent antinociception in mice. The

present aﬃnity of corynantheidine at MOR, and higher aﬃnity

for adrenergic receptors over MOR, was comparable to recent

reports by Obeng et al.,⁵⁶although in our case, we ﬁnd a

higher aﬃnity at α_2Ainstead of α_1D.

The oxindole of corynantheidine, corynoxine retains the

parent template’s MOR selectivity but interestingly shows

higher eﬃcacy at both mouse receptors measured using

[³⁵S]GTPγS assays as well as Gi-1 BRET assays at human

receptors compared to those of mitragynine and corynanthei-

dine. It is possible that the change of central core indole to

oxindole leads to this increase in eﬃcacy, although additional

analogues need to be synthesized before this SAR can be

validated. The binding aﬃnity at MOR for corynoxine has

been reported to be 16.4 nM at hMOR⁴⁸compared to 140 nM

for mMOR in our hands. It is possible that the diﬀerences may

be attributed to the use of diﬀerent assays or to the screening

with receptors from diﬀerent species. It is worth noting that

the functional activity at hMOR for corynoxine was also

moderate and in line with the binding aﬃnity in mMOR. Our

ﬁndings of corynoxine antinociception in mice are in line with

antinociception reported in rats following intravenous (IV)

administration.⁴⁸Of interest, despite displaying full MOR

agonism, corynoxine showed no respiratory depression or

hyperlocomotion characteristic of typical MOR agonists. Exact

mechanisms for this separation of adverse eﬀects is unknown at

this point and will be investigated in detail later on.

Two alkaloids mitraciliatine and isopaynantheine have never

been characterized in the literature. Both natural products

show unique mixed actions at MOR as well as KOR in vitro at

mouse and human receptors. Mitragynine is reported to have

weak KOR antagonism,^51,87to label adrenergic receptors,^45,56

and to have serotonergic actions with in vivo testing.⁷⁸In

contrast, mitraciliatine demonstrated KOR full agonism and

MOR partial agonism at both mouse and human receptors.

Mitraciliatine does not recruit βarrestin-2 at MOR but shows

robust βarrestin-2 recruitment at KOR and, based on

preliminary in vitro screening, appears to have higher receptor

selectivity for opioid over adrenergic receptors with fewer oﬀ-

target interactions. It is possible that the stereochemistry at C3

(3S in mitragynine but 3R for mitraciliatine) and C20 (20S in

mitragynine but 20R for mitraciliatine) plays a key role in the

SAR as well as a cleaner oﬀ-target proﬁle for these compounds.

The signaling proﬁle as well as receptor activity of

isopaynantheine was unique as well. At mouse receptors

similar to mitraciliatine, it showed MOR partial agonism and

KOR full agonism. At human opioid receptors in sharp

contrast to mitraciliatine, it was an MOR antagonist. At KOR,

it was a high eﬃcacy agonist. Diﬀerences were also seen in

βarrestin-2 recruitment; at MOR, it was an antagonist, and at

KOR, it showed reduced βarrestin-2 recruitment.

Mitraciliatine also showed no respiratory depression or

hyperlocomotion, potentially due to its partial agonism in both

cell lines and mice, where a ceiling eﬀect was observed.

Recently, partial agonism has been revisited as a means to

develop MOR-mediated antinociception with attenuated

respiratory depression.^88,89

A discrepancy between the in vitro/in vivo receptor activity

was observed in the present activity of mitraciliatine. In spite of

full agonism at mKOR in [³⁵S]GTPγS assays, antinociceptive

actions were independent of KOR in mice and only dependent

on MOR. While further study is needed, there is precedence

for discrepancies of this type in the opioid ﬁeld. For example, a

lack of correlation between in vitro/in vivo actions exist with

MP1104, which possesses pM aﬃnity⁹⁰and agonist activity⁷⁰

in functional assays at MOR but was found to lack MOR

activity in vivo.²⁰

An unexpected increase in breathing rate was observed with

both corynoxine as well as mitraciliatine in the CLAMS assay.

In the literature, enhanced respiration has been attributed to

KOR agonism,⁹¹serotonin,⁹²and adrenergic receptors;⁹³the

exact mechanism of this respiratory stimulation remains

unknown and requires more detailed examination in due

course.

C20 substituent (vinyl vs ethyl) was identiﬁed as a

functional switch for in vivo MOR vs KOR receptor selectivity.

Mitraciliatine with C20 ethyl substituent was an MOR agonist,

while isopaynantheine with a vinyl group was a KOR agonist in

mice.

CONCLUSION

■

In conclusion, we report a thorough and complete in vitro

pharmacological characterization of ﬁve kratom based minor

alkaloids. Given their low abundance, it seems unlikely that

these alkaloids play a major mediating role in the biological

actions of kratom consumed by humans. However, these

alkaloids represent novel starting points for optimizing probes

to better understand opioid receptor function.

There are three major ﬁndings from this present work. First,

we identify three new templates present in kratom with

antinociceptive activity in mice, with corynoxine being

equipotent to morphine. Second, we identify ligands with an

array of pharmacological proﬁles, ranging from the partial

opioid agonism displayed by corynantheidine and mitracilia-

tine and full agonism of corynoxine and KOR agonism with

isopaynantheine. Finally, we identify corynoxine and mitraci-

liatine to be structurally unique natural products with safer,

MOR dependent antinociception, and we identify isopaynan-

theine as the ﬁrst kratom alkaloid with KOR mediated

antinociceptive actions.

Our studies add to the growing chorus of reports

highlighting kratom as a valuable source of opioid-active

templates. Nearly ∼41 individual alkaloids in kratom still

remain uncharacterized. It is hoped that kratom and its

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4.46 (s, 1H), 3.91 (d, J = 1.0 Hz, 3H), 3.76 (s, 3H), 3.68 (d, J = 1.1

Hz, 3H), 3.29−3.23 (m, 2H), 3.19 (qd, J = 7.9, 6.6, 3.7 Hz, 1H),

3.01−2.92 (m, 1H), 2.85 (dd, J = 15.5, 2.8 Hz, 1H), 2.64 (q, J = 4.4,

3.4 Hz, 2H), 2.46 (dq, J = 13.4, 8.0, 4.9 Hz, 1H), 2.37 (ddd, J = 13.0,

10.8, 2.8 Hz, 1H), 1.99 (d, J = 13.4 Hz, 1H). Purity = 98.6%

Mitraciliatine (7). ¹H NMR of 7 matched well with the literature

reported value.⁴⁴¹H NMR (500 MHz, CDCl₃): δ 7.92 (s, 1H), 7.31

(s, 1H), 7.04 (t, J = 7.8 Hz, 1H), 6.99 (dd, J = 8.1, 0.8 Hz, 1H), 6.49

(dd, J = 7.7, 0.9 Hz, 1H), 4.45 (dd, J = 5.2, 2.5 Hz, 1H), 3.90 (s, 3H),

3.76 (s, 3H), 3.69 (s, 3H), 3.26 (ddt, J = 8.1, 6.2, 2.6 Hz, 2H), 3.22−

3.17 (m, 1H), 2.89−2.82 (m, 1H), 2.78 (dd, J = 11.2, 3.4 Hz, 1H),

2.48−2.38 (m, 2H), 2.25−2.13 (m, 2H), 1.99−1.93 (m, 1H), 1.34−

1.27 (m, 2H), 0.78−0.75 (m, 3H). Purity = 95.1%

alkaloids (including indole and oxindole templates reported in

this manuscript) may eventually lead to a new generation of

analgesics.

MATERIALS AND METHODS

■

Drugs and Chemicals. The Research Technology Branch of the

National Institute on Drug Abuse (Rockville, MD) provided the

opiates. Buﬀers and miscellaneous chemicals were purchased from

Sigma-Aldrich. We purchased kratom “Red Indonesian Micro

Powder” from Moon Kratom (Austin, TX). Corynoxine and

corynoxine B were purchased from BOC Sciences (NY, USA).

DMSO was used to dissolve all nonradioactive compounds and

diluted with water for conducting assays. The assays were conducted

with 1−2.5% of ﬁnal concentration of DMSO.

Chemical Charecterization of Synthesized Compounds. Methyl-

(E)-2-((2S,3S,12bS)-3-ethyl-8-(((triﬂuoromethyl)sulfonyl)oxy)-

1,2,3,4,6,7,12,12b-octahydroindolo[2,3-a]quinolizin-2-yl)-3-me-

thoxyacrylate (13). To a solution of 12 (9-hydroxymitragynine, 130

mg, 0.32 mmol) in dry dichloromethane (6 mL), N-phenyl-

bis(triﬂuoromethanesulfonimide) (133 mg, 0.36 mmol) was added

under argon at RT. Et₃N (0.14 mL, 1 mmol) was added to the

reaction mixture, and stirring was continued for 12 h. Next, the

reaction mixture was concentrated and worked up with EtOAc (40

mL) and brine (2 × 30 mL) and dried over anhydrous Na₂SO₄. The

solvent was evaporated under reduced pressure, and the residue was

puriﬁed in ﬂash chromatography using 15−70% EtOAc in hexane.

Desired triﬂate 13 (102 mg) was obtained as a white solid (yield,

Chemistry. We purchased all the chemicals from Sigma-Aldrich,

and they were used directly with no further puriﬁcation. Flame-dried

reaction ﬂasks were used to carry out the reactions. All reactions were

performed under inert atmosphere using argon. Puriﬁcation of the

reaction mixtures were achieved by ﬂash column chromatography on

E. Merck 230−400 mesh silica gel 60 using a Teledyne ISCO

CombiFlash R_finstrument with UV detection at 280 and 254 nm. We

used RediSep R_fsilica gel normal phase columns. Isolated yields are

reported in all the cases. A Varian 400/500 MHz NMR spectrometer

was used to record the NMR spectra. All the NMR data were

processed with MestReNova software. The chemical shifts are

reported in parts per million (ppm) downﬁeld of tetramethylsilane

and referenced to the residual solvent peak unless otherwise noted

(CDCl₃¹H = 7.26, ¹³C = 77.3). Peak multiplicity is reported using the

following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m,

multiplet; br, broad. Coupling constants (J) are expressed in hertz

(Hz). A Bruker Daltonics 10 T Apex Qe Fourier Transform Ion

Cyclotron Resonance-Mass Spectrometer (ESI-MS) was used to

record the high resolution mass spectra. The accurate masses of the

molecular ion [M + H]⁺are presented and matched well with the

calculated value. High pressure liquid chromatography (HPLC) was

carried out to determine the purity of the synthesized and isolated

compounds. Instrumentation details of HPLC are provided in the

Supporting Information (SI).

1

61%). Only HNMR was recorded, because this is an intermediate

compound. ¹H NMR (400 MHz, CDCl₃): δ 8.01 (s, 1H), 7.44 (d, J =

0.8 Hz, 1H), 7.28 (dt, J = 8.0, 0.8 Hz, 1H), 7.07 (dd, J = 8.5, 7.6 Hz,

1H), 6.98 (d, J = 7.9 Hz, 1H), 3.74 (s, 3H), 3.71 (s, 3H), 3.23−3.10

(m, 2H), 3.02 (tt, J = 17.0, 4.9 Hz, 3H), 2.91 (dd, J = 15.4, 3.6 Hz,

1H), 2.62−2.51 (m, 2H), 2.47 (dd, J = 11.7, 3.1 Hz, 1H), 1.85−1.69

(m, 2H), 1.69−1.60 (m, 1H), 1.25−1.16 (m, 1H), 0.87 (t, J = 7.3 Hz,

3H). HRMS (ESI-TOF) m/z: [M + H]⁺calcd for C₂₃H₂₈F₃N₂O₆S

517.1620; found, 517.1611.

Corynantheidine (5). To a solution of triﬂate 13 (77.5 mg, 0.15

mmol) in dry DMF (3 mL) in a sealed tube, Pd(OAc)₂(11 mg, 0.05

mmol), dppp (31 mg, 0.07 mmol), Et₃N (0.4 mL, 3 mmol), and

HCOOH (8 uL, 0.27 mmol) were added. The stirring was continued

at 80 °C for 8 h. Next, the reaction mixture was allowed to cool to RT

and diluted with EtOAc (50 mL). Regular work up was done using

EtOAc and brine (15 × 4 mL). The EtOAc part was dried over

anhydrous Na₂SO₄and evaporated under reduced pressure. The

residue was puriﬁed by ﬂash column chromatography using 20−70%

EtOAc in hexanes. Compound 5 (40 mg, 65%) was obtained as a

white amorphous solid after puriﬁcation. ¹H NMR of 5 matched well

Isolation of Mitragynine from Mitragyna speciosa (Kratom).

Extraction of mitragynine was carried out from the dry powdered

kratom leaves adopting a modiﬁcation of previously reported

protocol.⁵¹Kratom powder (500 g) was reﬂuxed in MeOH, 500

mL, for 30 min. Next, the suspension was ﬁltered, and the alcoholic

extraction process was repeated two more times (2 × 500 mL). The

solvent from 3 combined extract was evaporated under reduced

pressure, and the alkaloid content was dried in high vacuum. The

residue was suspended in 10% aqueous AcOH (1 L) and washed

several times with hexane (4 × 500 mL). Then, the aqueous layer was

cooled to 0 °C in an ice bath and basiﬁed (pH ∼ 10) slowly with

aqueous NaOH solution (2.5M. ∼1L). EtOAc (4 × 400 mL) was

used to extract the alkaloids from the aqueous layer. The combined

EtOAc layer was washed with brine (250 mL) and dried over

anhydrous Na₂SO₄. The solvent was evaporated under reduced

pressure, and the residue was dried in high vacuum to obtain the

crude alkaloid extract (9.8 g). This crude kratom extract was subjected

to silica gel column chromatography, using 0−15% MeOH in

dichloromethane to isolate mitragynine (4.7 g), paynantheine (568

mg), speciogynine (343 mg), and speciociliatine (754 mg). After

removing the major alkaloid (mitragynine, paynantheine, speciogy-

nine, and speciociliatine) fractions out, the rest of the crude material

was again injected to silica gel column chromatography, using 5−20%

MeOH in dichlomethane to isolate mitraciliatine (2 mg; 0.02% of

total alkaloid content) and isopaynantheine (3 mg; 0.03% of total

alkaloid content) as the minor alkaloids.

1

with the literature reported value. H NMR (500 MHz, CDCl₃): δ

7.76 (s, 1H), 7.47 (dd, J = 7.6, 1.2 Hz, 1H), 7.44 (s, 1H), 7.29 (dt, J =

7.9, 1.0 Hz, 1H), 7.13−7.07 (m, 2H), 3.72 (s, 3H), 3.71 (s, 3H), 3.24

(dq, J = 11.4, 2.2 Hz, 1H), 3.09−2.99 (m, 4H), 2.76−2.69 (m, 1H),

2.63−2.58 (m, 1H), 2.56−2.50 (m, 2H), 1.87−1.81 (m, 1H), 1.81−

1.75 (m, 1H), 1.66 (dt, J = 11.1, 3.1 Hz, 1H), 1.27−1.20 (m, 1H),

0.89 (t, J = 7.4 Hz, 3H). Purity = 98.9%

Biological Assays. Aﬃnity Determination Using Binding

Assays. Radioligand binding assays using [¹²⁵I]IBNtxA as the

radioactive ligand were used to determine the aﬃnity (K_i) of natural

products by using previosuly published protocols.^90,94,95Membranes

from Chinese hamster ovary cells (CHOs) which stably express

mMOR, mDOR, and mKOR were used in the assay carried out at 25

^οC for 90 min. For mMOR, 50 mM potassium phosphate buﬀer with

5 mM MgSO4 was used, and 20 μg/μL membranes was used. For

mKOR and mDOR binding, only 50 mM potassium phosphate buﬀer

was used, and 40 μg/μL membranes was used. Following incubation,

assay contents were ﬁltered through glass ﬁber ﬁlters (obtained from

Whatman Schleicher and Schuell, Keene, NH), and assay tubes were

washed with 3 mL of ice-cold 50 mM Tris−HCl buﬀer at pH 7.4

thrice on a semiautomatic cell harvester. Levallorphan (8 μM) was

used to determine nonspeciﬁc binding and speciﬁc binding

determined by subtracting total binding from nonspeciﬁc binding.

Protein concentrations were determined using the Lowry assay using

Isopaynantheine (6). ¹H NMR of 6 matched well with the

literature reported value.⁴⁴¹H NMR (500 MHz, CDCl₃): δ 7.88 (s,

1H), 7.27 (d, J = 1.1 Hz, 1H), 7.05 (t, J = 7.8 Hz, 1H), 6.99 (d, J = 8.2

Hz, 1H), 6.50 (d, J = 7.6 Hz, 1H), 5.37 (ddd, J = 16.8, 10.3, 8.4 Hz,

1H), 4.92 (dd, J = 17.1, 2.2 Hz, 1H), 4.85 (dd, J = 10.3, 2.1 Hz, 1H),

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BSA as control as described before.⁹⁶Aﬃnities (K_i) were determined

using the Cheng Prusoﬀ equation: K_i= (IC₅₀)/(1 + L), where L =

(concentration of radioligand [¹²⁵I[IBNtxA used in asasy)/(K_dof

radioligand [¹²⁵I]IBNtxA) by nonlinear regression analysis in

GraphPad Prism.^97,98

Assessing Antinociception: The 55 °C Warm-Water Tail-With-

drawal Assay. The 55 °C warm-water tail-withdrawal assay was

conducted in C57BL/6J mice as a measure of acute thermal

antinociception as described previously.⁵¹Brieﬂy, warm (55 °C)

water in a 1.5 L heated water bath was used as the thermal nociceptive

stimulus, with the latency of each mouse to withdraw its tail taken as

the end point. After ﬁrst determining baseline latencies (1.44 0.02 s

across tested subjects), mice were administered a single graded dose

of the tested compound supraspinally, through the intracerebroven-

tricular (i.c.v.) route. Following drug administration, the tail-

withdrawal latency for each mouse was repeatedly determined every

10 min for 1 h or until latencies returned to the baseline values. A

cutoﬀ time of 15 s was utilized to prevent tissue damage; if the mouse

failed to display a tail-withdrawal response within this maximum

response, the tail was removed from the water, and the animal was

assigned a maximal antinociceptive score of 100%. At each time point,

antinociception was calculated by the formula: % antinociception =

100[(test latency − baseline latency)/(15 − baseline latency)].

Presentation of antinociception was utilized to account for innate

variability of initial latency values between mice. ED₅₀antinociceptive

values for compounds was calculated from peak eﬀect observed.

Assessment of Respiratory Depression and Locomotor Eﬀects.

Animal breathing rates (measured as breaths per minute) and

spontaneous locomotive activity (measured as ambulations per

minute) were measured using the automated, computer-controlled

Comprehensive Lab Animal Monitoring System (CLAMS) (Colum-

bus Instruments, Columbus, OH) as described previously.³⁹Mice

were allowed to move freely through the closed, sealed individual

cages (23.5 cm × 2.2 cm × 13 cm) during all testing, which began

immediately after a 60 min habituation period. After habituation, mice

were administered i.c.v. compound or vehicle and, 5 min later,

conﬁned to the CLAMS testing cages for 180 min. Pressure

transducers built into each sealed CLAMS cage measured the

frequency of respiration (at breaths/min) of each occupant mouse.

Infrared beams located in the ﬂoor measured locomotion by counting

sequential breaks of adjacent beams (as ambulations). Data were

averaged over 20 min periods for the 3 h test and are expressed as %

vehicle response SEM.

Functional Assays [³⁵S]GTPγS. To determine if the natural

products behaved as full agonists or partial agonists, the

[³⁵S]GTPγS assay in the same membranes (mMOR, mKOR, and

mDOR stably expressed in CHO cell lines) as those used in the

binding assays were used. Assay incubation time was 60 min at 30 °C

in buﬀer (50 mM Tris−HCl, pH 7.4, 3 mM MgCl₂, 0.2 mM EGTA,

and 10 mM NaCl) containing 0.05 nM [³⁵S]GTPγS; 2 μg/mL each of

protease inhibitors, namely, leupeptin, pepstatin, aprotinin, and

bestatin; and 30 μM GDP.⁹⁸Following incubation, the assay tubes

were ﬁltered through glass ﬁber ﬁlters (obtained from Whatman

Schleicher and Schuell, Keene, NH), and assay tubes were washed

with 3 mL of ice-cold 50 mM Tris−HCl buﬀer at pH 7.4 thrice on a

semiautomatic cell harvester. Following ﬁltration, ﬁlters were placed

in vials containg 3 mL of Liquiscint (obtained National Diagnostics,

Atlanta, GA), and counts were measured using scintillation spectros-

copy using a Tri-Carb 2900TR counter (obtained from PerkinElmer

Life and Analytical Sciences). Basal activity was determined in the

presence of GDP and in the absence of the tested drug. Data obtained

was then normalized against appropriate controls, e.g., 1000 nM

DAMGO, DPDPE, and U50,488h for mMOR, mDOR, and mKOR,

respectively. Agonistic potency (EC₅₀), antagonistic potency (IC₅₀),

and maximal stimulation (%E_max) values were calculated by nonlinear

regression analysis in GraphPad Prism.

Bioluminescence Resonance Energy Transfer (BRET) Assay.^70,99

To measure the Gαi1 protein mediated activation, human embryonic

kidney (HEK293T) cells were cotransfected using a 1:1:1:1 DNA

ratio of receptor:Gαi-Rluc:Gβ1:Gγ2-GFP2, while a 1:5 DNA ratio of

receptor-Rluc:mVenus-arrestin2 was used to measure the β-arrestin2

recruitment. Transfection was performed in OptiMEM using Transit

2020 at a 2:1 ratio of Transit:micrograms (μg) of DNA. After at least

18 h, 0.05% trypsin-EDTA was added to gently detach the cells, and

subsequently, the cells were plated in a plating media (DMEM

supplemented with 1% dialyzed FBS) at a density of 30 000−50 000

cells per well in poly-^D-lysine-coated white and clear-bottom 96-well

assay plates. The next day, a white adhesive bottom seal was applied,

and the culture medium was carefully decanted and replaced by 60 μL

of a drug buﬀer (1× Hank’s balanced salt solution (HBSS) and 20

mM HEPES, pH 7.4). Following assay buﬀer aspiration, cells were

treated with 60 μL of freshly prepared coelenterazine 400a (5 μM)

ﬁnal concentration for Gαi1 activation or coelenterazine h (5 μM)

concentration for β-arrestin2 recruitment. After 5 min of incubation,

30 μL of indole and oxindole based alkaloids (mitraciliatine,

corynoxine, corynantheidine, and isopaynantheine) was individually

added and incubated for an additional 5 min. Subsequently, the plates

were placed in an LB940 Mithras plate reader (Berthold

Technologies) to measure BRET ratios by reading each well for 1

s. With respect to Gαi1 protein mediated activation, the BRET2 ratio

was expressed as the ratio of the GFP2 emission to RLuc8 emission at

510 and 395 nm, respectively. In regard to β-arrestin2 recruitment,

the BRET1 ratio was deﬁned as the ratio of mVenus/RLuc with 485

and 530 nm. The ratio of mVenus/RLuc was calculated per well, and

the net BRET ratio was calculated by subtracting the mVenus/RLuc

per well from the mVenus/RLuc ratio in wells without the presence of

mVenus-arrestin2. The Graphpad Prism 8 software was used to plot

the net BRET ratio versus the corresponding drug concentration.

Mice. Male C57BL/6J mice (24−34 g) were purchased from

Jackson Laboratories (Bar Harbor, ME) at 7 weeks of age. MOR KO

and KOR KO mice were bred in the McLaughlin laboratory at

University of Florida. Progenitors of the colonies for MOR KO and

KOR KO were obtained from Jackson Laboratories. All mice used

Data Analysis. Data analysis was carried out using Graphpad Prism

8.0 software. Results for both radioligand binding assays and

[³⁵S]GTPγS are presented as nM

SEM from n = 3 experiments

performed in triplicate. Data from both Gi-1 activation and βarrestin-

2 assays using human opioid receptors were normalized to E_maxof the

corresponding agonist controls, DAMGO, U50,488H, and DPDPE.

The three-parameter logistic equation using Graph Pad Prism was

used to ﬁt the dose response curves obtained. All data is presented as

mean EC₅₀(pEC₅₀SEM) for assays run in triplicate.

Statistical analysis used either one-way or two-way ANOVA or

unpaired t test wherever appropriate with p < 0.05 being considered

to be signiﬁcant.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acschemneuro.1c00149.

■

sı

HPLC experimental method and HPLC traces of

compounds 5−7 and functional binding data of the

compounds at opioid receptors (PDF)

AUTHOR INFORMATION

Corresponding Author

■

Susruta Majumdar − Center for Clinical Pharmacology,

University of Health Sciences & Pharmacy at St. Louis and

Washington University School of Medicine, St. Louis,

Missouri 63110, United States; Department of

̈

throughout the manuscript were opioid naive. All mice were

maintained on a 12 h light/dark cycle with Purina rodent chow and

water available ad libitum and housed in groups of ﬁve until testing.

For all behavior experiments, 50% DMSO:saline was used as a vehicle

to dissolve drugs.

Anesthesiology, Washington University School of Medicine, St.

Louis, Missouri 63110, United States; orcid.org/0000-

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0002-2931-3823; Phone: 314-446-8162;

Email: susrutam@email.wustl.edu

Hill, North Carolina 27599, United States; orcid.org/

0000-0002-1620-3027

Complete contact information is available at:

https://pubs.acs.org/10.1021/acschemneuro.1c00149

Authors

Soumen Chakraborty − Center for Clinical Pharmacology,

University of Health Sciences & Pharmacy at St. Louis and

Washington University School of Medicine, St. Louis,

Missouri 63110, United States; Department of

Anesthesiology, Washington University School of Medicine, St.

Louis, Missouri 63110, United States

Rajendra Uprety − Department of Neurology, Memorial Sloan

Kettering Cancer Center, New York, New York 10065,

United States

Amal E. Daibani − Center for Clinical Pharmacology,

University of Health Sciences & Pharmacy at St. Louis and

Washington University School of Medicine, St. Louis,

Missouri 63110, United States; Department of

Anesthesiology, Washington University School of Medicine, St.

Louis, Missouri 63110, United States

Author Contributions

S.C. and R.U. equally contributed in this work. S.M. and T.C.

conceived the experiments. S.C., R.U., A.E.D., V.L.R., A.H.,

N.N., K.A., L.T., and R.J. carried out the experiments and

analyzed the data. J.P.M. supervised S.O.E. and analyzed data

for the behavioral characterization of analogues. S.C. and S.M.

wrote the manuscript with edits from all authors.

Notes

The authors declare the following competing ﬁnancial

interest(s): S.M. is a co-founder of Sparian Inc. S.M. is an

inventor on patent applications related to mitragynine

analogues, which may lead to royalties or other licensing

revenues from future commercial products.

^∇G.W.P. deceased (22nd February, 2019).

Valerie L. Rouzic − Department of Neurology, Memorial

Sloan Kettering Cancer Center, New York, New York 10065,

United States

Amanda Hunkele − Department of Neurology, Memorial

Sloan Kettering Cancer Center, New York, New York 10065,

United States

Kevin Appourchaux − Center for Clinical Pharmacology,

University of Health Sciences & Pharmacy at St. Louis and

Washington University School of Medicine, St. Louis,

Missouri 63110, United States

Shainnel O. Eans − Department of Pharmacodynamics,

University of Florida, Gainesville, Florida 032610, United

States

Nitin Nuthikattu − Center for Clinical Pharmacology,

University of Health Sciences & Pharmacy at St. Louis and

Washington University School of Medicine, St. Louis,

Missouri 63110, United States

Rahul Jilakara − Center for Clinical Pharmacology, University

of Health Sciences & Pharmacy at St. Louis and Washington

University School of Medicine, St. Louis, Missouri 63110,

United States

ACKNOWLEDGMENTS

■

S.M. is supported by funds from NIH grants DA045884 and

DA048379 and start-up funds from Center for Clinical

Pharmacology, University of Health Sciences & Pharmacy

and Washington University. Y.-X.P. is supported by NIH

grants DA042888 and DA046714. This research was funded in

part through the NIH/NCI Cancer Center Support Grant P30

CA008748 to MSKCC. The authors would like to thank

Professor Bryan L. Roth and the National Institute of Mental

Health’s Psychoactive Drug Screening Program for their

assistance in determining binding aﬃnities at nonopioid

CNS targets. We are thankful to Dr. Mariko Kitajima

(Graduate School of Pharmaceutical Sciences, Chiba Uni-

versity, Japan) for providing isopaynantheine, used as standard.

ABBREVIATIONS

■

mMOR, mouse mu opioid receptor; hMOR, human mu opioid

receptor; mKOR, mouse kappa opioid receptor; hKOR, human

kappa opioid receptor; NET, norepinephrine transporter;

DAT, dopamine transporter; SERT, serotonin transporter;

α2A, α adrenergic receptor subtype 2A; α1D, α adrenergic

receptor subtype 1D; 7OH, 7-hydroxy mitragynine; MP,

mitragynine pseudoindoxyl; DAMGO, [D-Ala2,N-MePh-

e4,Gly-ol5]-enkephalin; DCM, dichloromethane; BBB,

blood−brain barrier; MPE, maximum possible eﬀect; IV,

intravenous; i.c.v., intracerebroventricular; CLAMS, Compre-

hensive Laboratory Animal Monitoring System

Lisa Thammavong − Center for Clinical Pharmacology,

University of Health Sciences & Pharmacy at St. Louis and

Washington University School of Medicine, St. Louis,

Missouri 63110, United States

^∇Gavril W. Pasternak − Department of Neurology, Memorial

Sloan Kettering Cancer Center, New York, New York 10065,

United States

Ying-Xian Pan − Department of Neurology, Memorial Sloan

Kettering Cancer Center, New York, New York 10065,

United States; Department of Anesthesiology, Rutgers New

Jersey Medical School, Newark, New Jersey 07103, United

States

Jay P. McLaughlin − Department of Pharmacodynamics,

University of Florida, Gainesville, Florida 032610, United

States; orcid.org/0000-0001-9851-9342

Tao Che − Center for Clinical Pharmacology, University of

Health Sciences & Pharmacy at St. Louis and Washington

University School of Medicine, St. Louis, Missouri 63110,

United States; Department of Anesthesiology, Washington

University School of Medicine, St. Louis, Missouri 63110,

United States; Department of Pharmacology, University of

North Carolina at Chapel Hill School of Medicine, Chapel

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Article Doi

Kratom Alkaloids as Probes for Opioid Receptor Function: Pharmacological Characterization of Minor Indole and Oxindole Alkaloids from Kratom

DOI: 10.1021/acschemneuro.1c00149

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