Design, Synthesis, and Biological Evaluation of Novel Pyrimido[4,5-b]indole Derivatives against Gram-Negative Multidrug-Resistant Pathogens

pubs.acs.org/jmc

Article

Design, Synthesis, and Biological Evaluation of Novel

Pyrimido[4,5‑b]indole Derivatives Against Gram-Negative

Multidrug-Resistant Pathogens

Qidi Kong, Wei Pan, Heng Xu, Yaru Xue, Bin Guo, Xin Meng, Cheng Luo, Ting Wang, Shuhua Zhang,

and Yushe Yang *

Cite This: J. Med. Chem. 2021, 64, 8644 −8665

Read Online

sı

*

Metrics & More

Article Recommendations

Supporting Information

ACCESS

ABSTRACT: Due to the poor permeability across Gram-negative

bacterial membranes and the troublesome bacterial eﬄux

mechanism, only a few GyrB/ParE inhibitors with potent activity

against Gram-negative pathogens have been reported. Among

them, pyrimido[4,5-b]indole derivatives represented by GP-1

demonstrated excellent broad-spectrum antibacterial activity

against both Gram-positive and Gram-negative bacteria but were

limited by hERG inhibition and poor pharmacokinetics proﬁle. To

improve their drug-like properties, we designed a series of novel

pyrimido[4,5-b]indole derivatives based on the tricyclic scaﬀold of

GP-1 and the C-7 moiety of acoraﬂoxacin. These eﬀorts have

culminated in the discovery of a promising compound 18r with reduced hERG liability and an improved PK proﬁle. Compound 18r

exhibited superior broad-spectrum in vitro antibacterial activity compared to GP-1, including a variety of clinical multidrug G⁻

pathogens, especially Acinetobacter baumannii, and the in vivo eﬃcacy was also demonstrated in a neutropenic mouse thigh model of

infection with multidrug-resistant A. baumannii.

and are vital enzymes for DNA topology and cell viability.^10,11

GyrA/GyrB and ParC/ParE are subunits of these two

INTRODUCTION

■

Antimicrobial resistance (AMR) has become one of the biggest

enzymes, respectively. While GyrA and ParC inhibited by the

threats to global public health over the past several decades,

and we have now entered the post-antibiotic era.¹AMR causes

700,000 or more deaths globally each year, which could

well-known class of ﬂuoroquinolones are clinically validated

targets for antimicrobial agents, GyrB and ParE also have long

been studied in the ﬁeld of antibacterial drugs.^12,13Due to their

diﬀerent modes of action, GyrB/ParE inhibitors do not exhibit

cross-resistance with ﬂuoroquinolones.^14,15Varieties of novel

scaﬀolds targeting GyrB/ParE ATP-binding site have been

discovered by industry and academia during the last 50 years.¹⁴

Although several inhibitors have entered clinical trials, none of

them have been approved except novobiocin (Figure 1, 1),

which was withdrawn in 2011 for reasons of safety and eﬃcacy.

DS-2969b^16,17(2) and SPR720¹⁸(3) are the only two

compounds still in active clinical trials, while AZD-5099^14,19

(4) entered phase I but was terminated soon after due to

safety. Furthermore, few GyrB/ParE inhibitors displayed

activity against critical G⁻bacteria neither do the compounds

increase to 10 million by 2050.²Although AMR is widespread

among both Gram-positive (G⁺) and Gram-negative (G⁻)

bacteria, the latter poses a more severe and urgent problem.

This diﬀerence is derived from the outer membrane, which is a

structure unique to G⁻pathogens, along with eﬄux pumps.^3,4

The penetration problem of G⁻bacteria has been widely

debated.⁵In order to ﬁght against AMR, it is urgent to develop

novel antibiotics with new targets or new modes of action and

active against resistant bacterial strains, especially G⁻

pathogens. Nevertheless, no new class of antibiotic against

G⁻pathogens has been approved in over 50 years.⁶⁻⁸In order

to guide and promote the discovery and development of novel

antibiotics, the World Health Organization (WHO) has

published a global priority pathogen list. Nine of the twelve

important bacteria are G⁻bacteria, including all three critical

priority pathogens: Acinetobacter baumannii, Pseudomonas

aeruginosa, and extended-spectrum β-lactamases (ESBLs)

producing Enterobacteriaceae (including Klebsiella pneumoniae

and Escherichia coli).⁹

Received: April 5, 2021

Published: June 3, 2021

DNA gyrase and topoisomerase IV are type IIA bacterial

topoisomerases that have structural and functional similarities

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

8644

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 1. Structures of representative GyrB/ParE inhibitors.

Figure 2. Design of novel pyrimido[4,5-b]indole GyrB/ParE inhibitors.

mentioned above. Pyrimido[4,5-b]indole inhibitors discovered

by Trius Therapeutics and represented by GP-1 (5) broke this

condition and showed excellent broad-spectrum activity

covering A. baumannii, P. aeruginosa, and K. pneumoniae for

the ﬁrst time.²⁰However, hERG inhibition, poor property of

pharmacokinetics (PK), and insuﬃcient eﬃcacy in vivo against

Gram-negative bacteria have limited the development of these

compounds.²⁰⁻²²Recently, a series of pyrido[2,3-b]indole

derivatives²³represented by compound 6 and pyridylurea

derivatives²⁴represented by compound 7 were also demon-

strated to have strong activity against critical G⁻pathogens in

vitro. According to the “rules of penetration”,²⁵the unique G⁻

activities of these compounds probably originate from the high

rigidity and low three-dimensionality of their structures with an

ionizable nitrogen. Nevertheless, the in vivo potency remains a

problem.

the Asp73 carboxylate group and a conserved water molecule,

which are crucial to its antibacterial activity, while the methyl

pyrimidine moiety at the C-2 position is involved in

interactions with Glu50-Arg76 and Arg136 (Figure 2). The

basic amino moiety at the C-4 position is shown to interact

with Asn46 within a structured water network, which is

considered to be important for activity against G⁻bacteria.²⁰

In an attempt to overcome the drawbacks of pyrimido[4,5-

b]indole derivatives, GP-1 was selected as the lead compound

and modiﬁcations at the C-2, C-4, C-5, and C-8 were designed

(Figure 2). Actually, the (3aR,6aR)-octahydropyrrolo[3,4-

b]pyrrole moiety of GP-1 was derived from the basic amino

moiety at the C-7 position of ﬂuoroquinolone. According to

the interactions demonstrated above and the C-7 position

modiﬁcation of acoraﬂoxacin,²⁶a unique C-4 moiety was

introduced in the new design (Figure 2). Acoraﬂoxacin had

entered phase II clinical trials and its C-7 moiety is featured

with the exocyclic double bond, with which the chemical

The X-ray crystal structure of GP-1 in complex with E. coli

GyrB²⁰shows that the pyrimido[4,5-b]indole scaﬀold binds to

the ATP-binding site of GyrB, forming hydrogen bonds with

8645

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 1. Synthesis of Compounds 18a−r

a

Reagents and conditions: (a) MeI, K₂CO₃, DMF, 0 °C, 94%; (b) mCPBA, DCM, 0 °C to rt, 84%; (c) 2-methylpyrimidine-5-ol, K₂CO₃, NMP,

100 °C, 84%; (d) MeI, K₂CO₃, DMF, 0 °C, 78%; (e) mCPBA, DMF, 0 °C to rt, 85%; (f) 2-methylpyrimidine-5-ol, K₂CO₃, NMP, 100 °C, 81%;

(g) 16a−b, 16f, 16j−k, and 16m−r, K₂CO₃, NMP, microwave at 100 °C; (h) BOP, 16c−e, 16g−i, and 16l, triethylamine, NMP, 0 to 50 °C; (i)

TFA, DCM, rt; (j) hydrazine hydrate for 18a−m and 18o−r, MeOH, reﬂux, 9−82% for three steps; methylamine for 18n, EtOH, 40 °C, 30% for

three steps.

a

Scheme 2. Synthesis of Compounds 20, 22, and 26−27

a

Reagents and conditions: (a) 16k, K₂CO₃, NMP, 100 °C, 67%; (b) NaCN, DMSO, 95 °C, 86%; (c) TFA, DCM, rt; (d) hydrazine hydrate,

MeOH, reﬂux, 12−62% for three steps.; (e) NaN₃, DMSO, 60 °C, 94%; (f) H₂, Pd/C, EtOH, rt, 66%; (g) methanesulfonyl chloride, triethylamine,

pyridine, sealed tube, 90 °C, 32%.

structural novelty of new compounds could be improved and

the antibacterial activity could be changed.

accepted method to discover novel antibacterial agents.

Herein, we demonstrated our eﬀort in discovering a series of

novel pyrimido[4,5-b]indole derivatives with superior G⁻

bacterial activity, low hERG inhibition, and improved PK

properties in rodent models. Among them, eﬃcacy of

compound 18r was also studied in vivo.

As previous observations^27,28have revealed that the

inhibitory activity against DNA gyrase had minor correlation

with the antibacterial activity, phenotypic screening (MIC test)

was employed in our research, which is the most widely

8646

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 3. Synthesis of Compounds 31a−e

a

Reagents and conditions: (a) 28a−e, K₂CO₃, NMP, 100 °C; (b) 16k, K₂CO₃, NMP, microwave at 100 °C; (c) TFA, DCM, rt; (d) hydrazine

hydrate, MeOH, reﬂux, 15−34% for four steps.

a

Scheme 4. Synthesis of Compounds 34a−d and 38

a

Reagents and conditions: (a) 32a−c, K₂CO₃, NMP, microwave at 100 °C; (b) BOP, 32d, triethylamine, NMP, 0 to 50 °C; (c) TFA, DCM, rt,

20−31% for two steps; (d) 35, K₂CO₃, NMP, microwave at 100 °C, 82%; (e) 32c, K₂CO₃, NMP, microwave at 100 °C; (f) TFA, DCM, rt, 32% for

two steps.

on these key intermediates (10, 11, and 15), a series of

pyrimidoindole compounds modiﬁed at the C-2 and C-4

positions were synthesized. With the installation of aliphatic

amines 16a−r by condensation or selective substitution

reactions at the C-4 position, compounds 17a−r were

prepared from 11 or 15. The condensation reactions were

activated by the benzotriazol-1-yloxytris(dimethylamino)-

phosphonium hexaﬂuorophosphate (BOP) reagent and the

substitution reactions took place under microwave irradiation

at 100 °C. Compounds 18a−r were prepared after tert-

butoxycarbonyl (Boc) and phthaloyl (Phth) were subsequently

deprotected. Compounds 18a−b, 18f, 18j−k, and 18m−r

were obtained via key intermediate 11, but compounds 18c−e,

18g−i, and 18l failed to be prepared by the same route and

CHEMISTRY

■

The preparation of compounds 18a−r is illustrated in Scheme

1. Methyl sulﬁde 9 was aﬀorded from methylation of 8²⁹with

iodomethane. In the presence of 3-chloroperoxybenzoic acid

(mCPBA), 9 was oxidized to methylsulfonyl intermediate 10.

Via a substitution reaction with 2-methylpyrimidine-5-ol at the

C-2 position and C-4 position of 10, key intermediate 11 was

obtained. In the other branch, thiol compound 12²⁹was

converted to methyl sulﬁde 13 when one equivalent of

iodomethane was added. 13 was oxidized to 14 with mCPBA

in an ice-water bath. Key intermediate 15 was then prepared

through a substitution reaction with 2-methylpyrimidine-5-ol

at the C-2 position of 14, similar to the synthesis of 11. Based

8647

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 5. Synthesis of Compounds 42a−b

a

Reagents and conditions: (a) 39a−d, K₂CO₃, CuI, PPh₃, (1R,2R)-N,N′-dimethyl-1,2-cyclohexanediamine, NMP, 130 °C; (b) BOP, 16k,

triethylamine, NMP, 0 to 50 °C, 20−40% for two steps; (c) TFA, DCM, rt; (d) hydrazine hydrate, MeOH, reﬂux, 57−61% for two steps.

a

Scheme 6. Synthesis of Compound 47

a

Reagents and conditions: (a) NCCOOEt, 4 N HCl in 1,4-dioxane, 82 °C; (b) 10% NaOH (aq), EtOH, reﬂux, 97% for two steps; (c) 2-methyl-5-

pyrimidinamine, HATU, triethylamine, DMF, rt, 14%; (d) H₂, 10% Pd/C, 50 °C, 58%; (e) BOP, 32c, triethylamine, NMP, 0 to 50 °C, 20%.

were instead prepared from intermediate 15. An ee value

greater than 94% was achieved during the preparation of

compound 18r. Details for the synthesis of aliphatic amines

16a−r can be found in the Supporting Information.

substitution reaction with 32c followed by deprotection, 36

was converted to compound 38.

The synthesis routine toward C-2 thioether analogues 42a−

b is illustrated in Scheme 5. The thiol group in 12 underwent

an Ullmann coupling reaction with the corresponding iodide

39a−b to provide thioether intermediates 40a−b. CuI, PPh₃,

and (1R,2R)-N,N′-dimethyl-1,2-cyclohexanediamine were en-

gaged in the coupling. Via a condensation reaction with the

BOP reagent, amine 16k was installed at the C-4 position of

40a−b to aﬀord 41a−b. Subsequent Boc and Phth

deprotection led to the formation of compounds 42a−b.

Compound 47 with an amide linker was prepared from

indole 43 as outlined in Scheme 6. Intermediate 43²⁹was

reacted in a solution of 4 M hydrogen chloride in 1,4-dioxane

with ethyl cyanoformate to produce acid 44. In the presence of

2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hex-

aﬂuorophosphate (HATU) and triethylamine, a condensation

reaction between acid 44 and 2-methyl-5-pyrimidinamine

aﬀorded amide 45. Catalyzed by 10% palladium on carbon,

the benzyl group of intermediate 45 was deprotected to obtain

amide 46 under a hydrogen atmosphere. After condensing 32c

with BOP, amide 46 was converted to compound 47.

The synthesis of three key intermediates 53a−c is displayed

in Scheme 7. Triﬂuoride 48²⁹was substituted by malononitrile

at the C-3 position by an S_NAr reaction in the presence of

NaOH and then reduced by sodium dithionite to aﬀord

cyanide 49. Catalyzed by DMSO, intermediate 49 reacted with

carbon disulﬁde (CS₂) in a sealed tube to form a

pyrimidoindole and provide dithiol 50. Diﬂuoride 52 was

obtained via subsequent methylation by MeI and oxidation by

mCPBA. 2-Methylpyrimidine-5-ol was installed at the C-2 and

C-4 positions of the pyrimidoindole to aﬀord 53a. The ﬁrst ﬁve

steps of the synthesis of deuterated intermediate 53b were

diﬀerent, with 3,5-diﬂuoroaniline as the starting material.

Acetic anhydride was used to acetylate 3,5-diﬂuoroaniline and

then deuterated iodomethane was used to obtain diﬂuoride 55.

In concentrated sulfuric acid, diﬂuoride 56 was produced after

The divergent synthesis of analogues with various functional

groups at the C-2 position is illustrated in Scheme 2.

Intermediate 10 was selectively reacted with 16k at the C-4

position to aﬀord key intermediate 19, which was then

deprotected by TFA and hydrazine hydrate to produce

compound 20. Intermediate 19 was exposed to sodium

cyanide in DMSO at 95 °C to obtain intermediate 21,

followed by deprotection of the Phth and Boc groups to give

compound 22. Intermediate 19 was also substituted with NaN₃

in DMSO at 60 °C and reduced by H₂with palladium on

carbon to obtain amine derivative 24, which was subsequently

reacted with methanesulfonyl chloride in a sealed tube to

provide sulfamide 25. Compounds 26 and 27 were produced

after deprotection of amine 24 and sulfamide 25, respectively.

Analogues 31a−e were prepared from intermediate 10

(Scheme 3). This key intermediate reacted with 28a−e to

aﬀord 29a−e and then underwent substitution with amine 16k

at the C-4 position to obtain 30a−e. Subsequent removal of

the Boc- and Phth-protecting groups with TFA and hydrazine

hydrate provided compounds 31a−e. Compounds 28a and

28e were purchased, while intermediates 28b−d were

synthesized, the details of which can be found in the

Supporting Information.

The procedures for the synthesis of compounds 34a−d and

38 are depicted in Scheme 4 and are similar to those for 18a−

r. Compounds 33a−d were also prepared from the key

intermediates 11 or 15 by condensation or substitution with

aliphatic alcohols 32a−d. Analogues 34a−d were obtained

after ﬁnal deprotection with TFA for 2 min. As the C-4 moiety

containing a hydroxy group would be cleaved by TFA, the

reaction time should be controlled. Key intermediate 10 was

reacted with 35 to aﬀord intermediate 36. After subjection to a

8648

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 7. Synthesis of Key Intermediates 53a−c

a

Reagents and conditions: (a) malononitrile, NaOH, NaHCO₃, sodium dithionite, DMF, 0 to 40 °C, 71%; (b) CS₂, NaOH, DMSO, EtOH, 80 °C;

(c) MeI, K₂CO₃, DMF, 0 °C, 42% for two steps; (d) mCPBA, DCM, 0 °C to rt, 51%; (e) 2-methylpyrimidine-5-ol, K₂CO₃, NMP, 100 °C, 55%; (f)

acetic anhydride, 0 °C, 83%; (g) deuterated iodomethane, NaH, THF, 0 °C, 60%; (h) KNO₃, conc. H₂SO₄, 0 °C, 62%; (i) 4 M HCl (aq), 1,4-

dioxane, reﬂux, 94%; (j) Boc₂O, NaH, THF, 0 °C to rt, 82%; (k) ethylamine, THF, 0 °C, 70%; (l) Boc₂O, DMAP, THF, 0 °C to rt, 58%.

nitration of 55 in the presence of potassium nitrate. Acetyl-56

was deprotected by 4 M HCl (aq), and the naked amino was

subsequently Boc-protected to aﬀord diﬂuoride 58. In this

reaction, 4-dimethylaminopyridine (DMAP) did not work

well, and NaH was chosen to facilitate the eﬀects of Boc₂O.

Key intermediate 56b was then accessed through a ﬁve-step

synthetic sequence similar to that for 56a. Diﬂuoride 59 was

prepared from 1,3,5-triﬂuoro-2-nitrobenzene by an S_NAr

reaction with ethylamine. Diﬂuoride 60 was aﬀorded via Boc

protection catalyzed by DMAP. Key intermediate 56c was then

obtained through a ﬁve-step process similar to that for 56a

outlined in Scheme 7a.

The synthesis of analogues 61, 63a−f, and 65 is shown in

Scheme 8. Intermediate 53a was substituted by 32c and then

deprotected to aﬀord compound 61. With the participation of

amines 16k, 16p, 16r, and 62, compounds 63a−f were

accessed by carrying 53a−c through the three-step synthetic

sequence (g−i) outlined in Scheme 1. The ee values of

compounds 63d−e were both greater than 92%. Intermediate

64 was prepared from diﬂuoride 58 through a ﬁve-step

synthetic process similar to that of 56a, as shown in Scheme

7a. Compound 65 was then produced after installation of 16k

at the C-4 position of 64 along with Boc and Phth

deprotection.

RESULTS AND DISCUSSION

■

SARs and hERG Inhibition of the Newly Synthesized

Pyrimidoindoles. The in vitro activities of all new

pyrimidoindoles were evaluated. To better correlate the in

vitro activities with future clinical applications, most of the

tested bacterial strains were clinical isolates, including

ﬂuoroquinolone-resistant and methicillin-resistant Staphylococ-

cus aureus (MRSA), ESBL-producing E. coli, oxacillinase

(OXA-23)-producing multidrug-resistant (MDR) A. bauman-

8649

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 8. Synthesis of Compounds 61, 63a−f, and 65

a

Reagents and conditions: (a) 32c, K₂CO₃, NMP, microwave at 100 °C; (b) TFA, DCM, rt, 21% for two steps; (c) 16k, 16p, 16r and 62, K₂CO₃,

NMP, microwave at 100 °C; (d) TFA, DCM, rt; (e) hydrazine hydrate, MeOH, reﬂux, 10−38% for three steps; (f) 16k, K₂CO₃, NMP, microwave

at 100 °C; (g) TFA, DCM, rt; (h) hydrazine hydrate, MeOH, reﬂux, 24% for three steps.

nii, imipenemase (IMP-4)-producing MDR P. aeruginosa, and

K. pneumoniae carbapenemase (KPC-2)-producing MDR K.

pneumoniae. We also tested compounds with standard strains,

such as E. coli ATCC35218. The activities are presented as the

minimum inhibitory concentrations (MICs), and the results

are displayed as ranges of MIC values. The racemate of GP-1

was used here as a positive control. More details about the

antibacterial activity and strains can be found in the Supporting

Information.

inhibition problem was surprisingly solved by the azetidine

ring at the beginning of our exploration. Combined with its

advantages in conﬁguration and metabolic stability, the

azetidine ring provided more attractive potential for further

investigation. Additionally, CH₃and F substituents at the R1

position contributed signiﬁcant improvement in antibacterial

activity and indicated that the R1 position deserved more

modiﬁcations.

Based on the azetidine ring, a comprehensive SAR

investigation was implemented around the C-4 moiety on R1

and R2, as displayed in Table 3. Since the G⁺bacterial activity

demonstrated by 18g was potent enough, we cast more

attention to improve the activity against G⁻pathogens. To our

delight, analogues 18i and 18k−o exhibited superior broad-

spectrum antibacterial activity over rac-GP-1, especially against

ECO⁺. Compared to 18g, Cl and Br substitutions, as in 18h

and 18i, improved the S. aureus and ECO⁺activity, suggesting

that weak electron-withdrawing groups are preferred. However,

the corresponding C-4 moieties 16h−i were not used in later

investigations because the physicochemical properties of 18h−

i were poor. Compound 18j, with a methyl substitution on the

double bond, showed better antibacterial activity than 18f but

was not as potent as rac-GP-1 in G⁻activity. Surprisingly, the

ethyl group of 18k led to a large improvement in activity

against G⁻bacteria, which was 2−64-fold more potent than

rac-GP-1. After that, the eﬀects of isopropyl, n-propyl, and allyl

groups at R1 were studied. Compounds 18l−n all demon-

strated excellent G⁻activity, although they were less potent

than 18k, indicating that hydrophobic R1 groups were

tolerated. Due to the relatively high hERG inhibition of

compounds 18m−n, substitutions larger than n-propyl were

not tried.

The C-4 moiety on pyrimidoindole is essential for

antibacterial activity, especially the G⁻bacterial activity, and

can modulate metabolic property, safety, antibacterial spec-

trum, and structure novelty by diversiﬁed substituents. Trius

Therapeutics have explored varieties of saturated cyclo-

heteroaliphatic amine rings in this position, including single

rings, fused rings, bridge rings, and spiro rings, but failed to

balance the activity against G⁻pathogens and hERG

inhibition.²⁹We wanted to not only improve the activity but

also evade the hERG K⁺channel inhibition problem that exists

in pyrimido[4,5-b]indole series. Inspired by the patent of

acoraﬂoxacin,³⁰we modiﬁed the C-4 moiety with an exocyclic

double bond linked to the ring system for the initial

investigation, as shown in Table 1. By adjusting the ring size

with the change in m and n, we ﬁrst explored the eﬀect of

azetidine, pyrrolidine, and piperidine rings. The diﬀerent

broad-spectrum performances of compounds 18a−g demon-

strated that the pyrrolidine ring was most beneﬁcial for activity,

and azetidine was the second. However, the hERG inhibition

results of 18c, 18e, and 18g in Table 2 showed that the

azetidine had less hERG liability than the pyrrolidine or

piperidine scaﬀold, especially the compound 18g whose

maximum inhibition rate was as low as 3.2%. The hERG

8650

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 1. In Vitro Antibacterial Activity of Compounds 18a−g

a

MRSA, methicillin-resistant S. aureus, 4 strains; MSSA, methicillin-sensitive S. aureus, 4 strains; MRSE, methicillin-resistant S. epidermidis, 4

strains; MSSE, methicillin-sensitive S. epidermidis, 4 strains; ECO⁺, ESBL-producing E. coli, 3 strains; ECO⁻, non-ESBL-producing E. coli, 3 strains;

and PAE, IMP-4 producing MDR P. aeruginosa, 3 strains.

Table 2. hERG K⁺Channel Inhibition of Selected

Compounds

compound 18o signiﬁcantly improved G⁻activity with a 4−16-

fold potency shift in the MIC. Compound 18p, which is a

racemate, was designed and synthesized based on compound

18o and maintained potency under the eﬀect of a methyl

group at R1. Nevertheless, the introduction of an ethyl group

at R2 (18q) led to a great decrease in activity, especially for G⁻

pathogens, indicating that a large group was not tolerated at

the R2 position. Compounds 18h and 18j−p were also tested

for their inhibition of the hERG K⁺channel, and their IC₅₀

values were all greater than the highest tested concentration

(40 μM), suggesting good safety to a certain degree.

Given the solubilities of pyrimido[4,5-b]indole derivatives

were generally very poor and compounds with hydroxy groups

could be readily transformed into prodrugs whose water

solubility would be signiﬁcantly improved, compounds 34a−d

with terminal hydroxy groups at the Y position in the C-4

moiety were prepared. These compounds suﬀered a signiﬁcant

loss in activity against G⁻bacteria compared with their amine

analogues, especially for P. aeruginosa, further proving that

terminal amines in the C-4 moiety are of importance to G⁻

bacterial activity. However, hydroxyl groups would show an

advantage when another ﬂuorine atom was installed at the C-5

position of the pyrimidoindole, since the activity of hydroxy

compound 61 was much better than that of amine 63a.

Compared to compound 18k, amine 63a lost most activity,

indicating that the hydrogen atom at the C-5 position was

more beneﬁcial than the ﬂuorine atom in this series.

a

compd

18c

18e

maximum inhibition rate (%)

IC₅₀(μM)

70.4

41.6

3.2

18.4

>40

51.5

>40

23.4

>40

12.3

0.04

18g

18h

18j

34.3

4.8

18k

18l

18m

18n

18o

18p

18r

31a

31b

31c

63c

rac-GP-1

Cisapride

41.1

26.5

50.6

49.1

32.3

37.0

18.3

62.0

30.9

36.0

14.2

70.5

97.7

a

The maximum tested concentration of cisapride was 3 μM, and the

maximum tested concentrations of the other compounds were 40 μM.

With regard to another position, R2 of the C-4 moiety,

subtle modiﬁcations could greatly inﬂuence the potency in

vitro. Compared to 18j, a methyl group at the R2 position of

8651

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Table 3. In Vitro Antibacterial Activity of Compounds 18h−q, 34a−d, 61, and 63a

a

compd

R1

R2

R4

MIC (μg/mL)

MRSE

<0.008

MSSA

<0.008

MRSA

<0.008

MSSE

<0.008

ECO⁺

ECO⁻

0.25−2

0.015−0.5

0.06−1

<0.008−0.06

0.03−0.5

0.06−0.5

0.015−0.25

0.25−1

PAE

18h

Cl

H

F

1

8−16

2−4

4−>16

0.5−1

1−2

2

18i

Br

<0.008

<0.008−0.015

<0.008

<0.008−0.015

0.5

<0.008

0.03−0.125

<0.008

<0.03

<0.008

0.03−0.25

<0.008

<0.03

0.06−0.5

2−4

18j

Me

Et

0.015−0.06

<0.008

18k

<0.008−0.015

0.125−0.25

0.5−1

18l

i-Pr

n-Pr

allyl

H

<0.008

18m

18n

<0.008

1

18o

Me

H

<0.008

0.5−1

1−2

>16

18p

Me

Et

<0.008

18q

0.25−0.5

<0.008

≥16

8−>16

34a

H

<0.008

<0.03

0.5−1

>64

0.125−1

0.25−>64

0.125−0.5

0.25−1

0.125−1

32−64

>16

34b

Me

Et

<0.03

>64

34c

H

<0.008

<0.03

<0.008

<0.03

<0.008

<0.03

0.25−0.5

0.5−1

0.25−0.5

64

4−16

≥64

8−>64

>64

34d

i-Pr

Et

H

<0.03

61

H

<0.03

63a

Et

H

F

0.25−2

<0.008

4−>64

0.25−0.5

<0.008−0.015

0.125−4

<0.03

<0.03−2

0.25−1

≤0.03−>64

rac-GP-1

levoﬂoxacin

0.015−0.06

0.25−8

<0.008−0.015

0.5−16

0.06−0.5

0.5−4

0.5−2

16−64

a

MRSA, methicillin-resistant S. aureus, 4 strains; MSSA, methicillin-sensitive S. aureus, 4 strains; MRSE, methicillin-resistant S. epidermidis, 4

strains; MSSE, methicillin-sensitive S. epidermidis, 4 strains; ECO⁺, ESBL-producing E. coli, 3 strains; ECO⁻, non-ESBL-producing E. coli, 3 strains;

and PAE, IMP-4 producing MDR P. aeruginosa, 3 strains.

According to explorations of Trius Therapeutics²⁹and Redx

Pharma,³¹modiﬁcation in the C-2 moiety could greatly

inﬂuence the antibacterial activity. Given the encouraging

outcome of 18k, its C-4 moiety was ﬁxed in our following

investigation of the C-2 moiety. Table 4 shows the in vitro

activity of compounds with modiﬁcations to L and R₃.

Methylsulfonyl, cyano, amino, and methanesulfonamide

groups were explored at the C-2 position, but these

compounds (20, 22, and 26−27) lost almost all activity,

indicating that aromatic rings in the C-2 moiety were required

for potency. With a cyclopropyl group introduced at the

pyrimidine of the C-2 moiety, compound 31a still exhibited

better potency than rac-GP-1 but had moderate inhibition of

the hERG K⁺channel with an IC₅₀of 23.4 μM. Hence, we ﬁne-

tuned the substitution at the 2′-position on the pyrimidine of

R3 where the vector is pointing toward the solvent-exposed

area. Hydroxymethyl groups and 1-hydroxyethyl groups at this

position (31b and 31c) could maintain potency against G⁺

bacteria but partly lost potency against ECO⁻and P. aeruginosa

compared to rac-GP-1. The antibacterial activity of 31d with a

2-hydroxypropan-2-yl group suﬀered a larger decrease in

activity against G⁻pathogens, which was in accordance with

the research by Hu et al.²³Compounds 31b−c performed well

in the hERG inhibition assay and further suggested that the

azetidine moiety resolved the hERG inhibition problem.

Compound 31e with an aliphatic ring fused to a benzene

ring and compound 42b with 5-bromopyridin-2-yl lost most

activity, while compound 38, with pyrazolo[1,5-a]pyrimidinyl,

retained activity compared to 34c with a methyl pyrimidine

moiety. Ether, thioether, and amide linkers (such as

compounds 18k, 42a, and 47) were investigated at the L

position, and the ether moiety proved to be the best linker

among them. While a thioether led to a slight reduction in

potency, the amide linker resulted in a total loss of activity

against G⁻bacteria. The SARs at C-2 of the pyrimidoindoles

indicated that the (2-methylpyrimidin-5-yl)oxy moiety of 18k

seemed to be the optimal selection.

According to the excellent data shown above, compounds

18k−p and 31b−c were selected to test against more strains

and species of G⁻bacteria (Table 5, more results are detailed

in the Supporting Information; the MDR E. coli and P.

aeruginosa tested in this table are diﬀerent from the strains

tested in Tables 1, 3, and 4). These compounds all exhibited

broad-spectrum antibacterial activity covering all three critical

priority pathogens emphasized by the WHO, especially MDR

A. baumannii. In this test, the potency of compound 18k was

2−8-fold stronger than that of rac-GP-1, while the activities of

18n, 18p, and 31c were comparable to this lead compound.

We also compared these compounds with meropenem and

ceﬁderocol which is a novel antibiotic potent against critical

MDR G⁻pathogens, and our compounds showed signiﬁcant

superior activity in vitro over these controls. The great

diﬀerence in activity between levoﬂoxacin and these com-

pounds further proved their quite diﬀerent mechanisms of

action.

8652

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Table 4. In Vitro Antibacterial Activity of Compounds 20, 22, 26−27, 31a−e, 38, 42a−b, and 47

a

MRSA, methicillin-resistant S. aureus, 4 strains; MSSA, methicillin-sensitive S. aureus, 4 strains; MRSE, methicillin-resistant S. epidermidis, 4

strains; MSSE, methicillin-sensitive S. epidermidis, 4 strains; ECO⁺, ESBL-producing E. coli, 3 strains; ECO⁻, non-ESBL-producing E. coli, 3 strains;

and PAE, IMP-4 producing MDR P. aeruginosa, 3 strains.

Microsomal Stability and PK Proﬁle of Selected

Pyrimido[4,5-b]indoles. Based on their in vitro antibacterial

activities, compounds 18k−m, 18o−p, 31b−c, and 34c were

selected to examine their metabolic stability in a liver

microsome assay. The results are summarized in Table 6.

Although the liver microsomal stability of compounds 18k−m,

18o−p, and 31b−c in humans was acceptable, with the

metabolic bioavailability (MF) of 18p reaching 82.2%, the data

in rats were poor, and their MFs were all below 40%. Hence,

the metabolite identiﬁcation study of compound 18k was

conducted in vitro in rat liver microsomes. It was demonstrated

that metabolite M5 with demethylation at R5 was the major

metabolite, accounting for 72.85% of the metabolites observed

(more details can be found in the Supporting Information),

and the methyl group at R5 was considered to be a soft spot.

To improve the metabolic properties of these pyrimidoin-

doles, CD₃was exploited in the R5 position of compounds

63b−e, which is a classic strategy to solve PK problems caused

by methyl groups.³²The G⁻activities of these compounds

were also evaluated. They maintained superior activity against

G⁻pathogens in accordance with CH₃analogues, as shown in

Table 5. Since the activity of compound 63d was better than

that of 63e, the (R)-conﬁguration was preferred for this C-4

moiety. Compounds 63b−d was then tested in liver micro-

8653

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 5. Broad-Spectrum Antibacterial Activity of Compounds 18k−p, 18r, 31b−c, 63b−f, and 65 against G⁻Pathogens

a

b

ER, ESBL-producing MDR E. coli clinical isolates, 4 strains (diﬀerent from the strains tested in Tables 1, 3, and 4). Representative G⁻wild-type

c

d

bacterial strains. KR, KPC-2-producing MDR K. pneumoniae clinical isolates, 4 strains. AR, OXA-23-producing MDR A. baumannii clinical

isolates, 5 strains. PR, IMP-4-producing MDR P. aeruginosa clinical isolates, 3 strains (diﬀerent from the strains tested in Tables 1, 3 and 4).

e

somes. Compared to 18k and 18p, the MFs of compounds

63b−c were slightly improved in rat liver microsomes but

decreased in human liver microsomes. To further evaluate their

PK properties in vivo, 18k, 18m, 18o−p, 31c, 61, and 63c−d

were evaluated in Sprague−Dawley (SD) rats after intravenous

administration. As summarized in Table 7, compound 18k

with the best antibacterial potency in vitro demonstrated low

plasma exposure (AUC_0−∞= 776 ng h/mL), rapid clearance

(Cl = 86.7 mL/min/kg), and a high volume of distribution (V_ss

= 14.7 L/kg). The parameters of 18m, 18o, and 31c were

similar to those of 18k, although compound 31c performed

was only half that of 18p, and its clearance was three times

greater, indicating the poor consistency of PK data in vitro and

in vivo. We also traced the formation of M5 during the

metabolic process of 63c and found that the concentration of

M5 was very low throughout the process. After a deuterated

methyl group was installed at R5, the soft spot was likely

transferred to another position.

On the basis of compound 18p, compound 18r was

designed and synthesized with the same (R)-conﬁguration

moiety as 63d at the C-4 position. As displayed in Tables 5 and

7, compound 18r demonstrated excellent in vitro activity

against G⁻bacteria better than rac-GP-1 and showed an

improved PK proﬁle and a favorable dose−exposure relation-

ship in rats (5 mg/kg vs 20 mg/kg), although its microsomal

stability in vitro was still poor (Table 6). Low hERG inhibition

was also shown with compound 18r. Thus, this compound was

used in an animal eﬃcacy study. Additionally, compound 63f

slightly better. Surprisingly, good plasma exposure (AUC_0−∞

=

1513 ng h/mL) and moderate clearance (Cl = 44.1 mL/min/

kg) were observed for compound 18p. This does not correlate

well with the in vitro rat microsomal stability data. Never-

theless, the in vivo PK proﬁles of those deuterated compounds

were quite diﬀerent from our assumption. The AUC of 63c

8654

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 6. Microsomal Stability of Selected Compounds

a

b

c

compd

species

T_1/2(min) Cl_int(mL/min/g protein) MF (%)

18k

Rat

Human

Rat

Human

Rat

Human

Rat

Human

Rat

Human

Rat

Human

Rat

Human

Rat

Human

Rat

Human

Rat

Human

Rat

Human

Rat

28.3

147

74.3

14.3

118.3

55.2

84.4

44.3

180.9

19.6

126

37.4

57.8

27.3

26.2

34.5

30.7

19.7

50.0

26.1

82.2

15.0

40.8

20.1

35.0

24.3

33.8

14.8

27.6

41.0

49.6

30.0

66.0

17.2

62.6

18l

17.8

38.0

24.9

47.5

12.0

107

16.7

494

8.3

73.9

11.9

57.8

15.2

54.7

45.6

76.0

32.9

105

20.2

208

9.8

179

18m

18o

18p

18r

31b

31c

34c

63b

63c

63d

4.25

252

28.4

Figure 3. Eﬃcacy study of compound 18r in a neutropenic mouse

thigh model. The ﬁgure shows the log unit cfu levels in the OXA-23-

producing MDR A. baumannii clinical isolate after 2 h of infection

with treatment with either vehicle control, levoﬂoxacin (LEV), or

compound 18r. The line for each animal group represents the mean.

Statistical signiﬁcance (two-tailed t-test) vs vehicle; one asterisk

represents adjusted p values 0.01−0.05, and three asterisks represent p

< 0.0001. In addition, ns represents no signiﬁcant diﬀerence.

176.4

36.3

138.4

38.4

256.1

51.4

63.9

20.0

104

bactericidal eﬃcacy comparable to that of levoﬂoxacin at the

same dose.

Cytotoxicity of Compounds 18k, 18n, and 18r. Other

than the hERG inhibition test, we also identiﬁed the safety

proﬁle of compounds by cytotoxicity analysis with two

mammalian cells (human L02 and HEK-293). Compounds

18k, 18n, and 18r were evaluated (Table 8) and all CC₅₀

10.1

215

11.7

Human

a

b

c

T

_1/2: elimination half-life. Cl_int: intrinsic body clearance. Calculated

metabolic bioavailability.

Table 8. CC₅₀of Selected Compounds in L02 Cells and

HEK 293 Cells after Incubation for 48 h

with an ethyl group at the R5 position was designed in order to

further improve the PK properties of 18r. It was slightly better

than 18r in the PK proﬁle, but the activity was totally lost

suggesting that only small groups were tolerated at the R5

position.

In Vivo Eﬃcacy of Compound 18r. Compound 18r was

evaluated for its in vivo eﬃcacy after intravenous injection in a

neutropenic mouse thigh infection model. The animals were

infected by a clinically isolated strain of MDR A. baumannii

that produces OXA-23. Levoﬂoxacin was tested as a positive

control as it was reported that the in vivo potency of GP-1

against G⁻bacteria was worse than levoﬂoxacin.²⁰As shown in

Figure 3, the corresponding log units of cfu reduction under

diﬀerent dosing regimens were used to demonstrate the

eﬃcacy. A dose−response trend was observed for compound

18r (10, 20, and 30 mg/kg), and at 30 mg/kg, 18r showed

a

CC₅₀(μM; μg/mL )

compd

18k

18n

18r

rac-GP-1

L02

HEK 293

9.74; 5.07

6.99; 3.72

16.04; 8.34

24.03; 10.43

16.96; 8.82

29.00; 15.53

19.02; 9.89

21.80; 9.47

a

For better reading, μg/mL values for cytotoxicity are also included.

values were calculated after 48 h incubation. Cytotoxicity of

18k and 18n was relatively high against the L02 cell line (CC₅₀

< 10 μM) while that of compounds 18r and rac-GP-1 was

modest. As for HEK 293, these compounds displayed a similar

eﬀect and all exerted minor cytotoxicity. The cytotoxicity of

a

Table 7. In Vivo PK Properties of Selected Compounds in Rats after Intravenous Administration

compd

dose (mg/kg)

AUC_0−∞(ng·h/mL)

T_1/2(h)

MRT (h)

Cl (mL/min/kg)

V_ss(L/kg)

18k

18m

18o

18p

31c

61

63c

63d

63f

4

5

776 44.0

778 103.9

836 153

1513 48

983 294.5

517 169

696 93

3.7 0.8

4.7 1.1

9.0 1.7

5.7 0.5

2.7 0.4

6.5 0.4

7.2 0.9

6.5 0.6

6.7 0.7

8.5 1.3

5.9 0.2

2.8 1.0

4.4 1.0

6.8 0.5

4.7 0.6

2.1 0.7

8.3 0.9

5.9 0.6

5.6 0.1

4.3 0.3

7.8 1.0

3.1 0.4

86.7 4.8

86.8 12.4

81.5 14.4

44.1 1.4

71.5 18.4

141 54

14.7 5.9

22.5 2.3

33.4 6.9

12.3 1.1

9.3 4.5

68.4 19.5

43.2 9.1

44.5 3.7

16.7 0.5

35.7 5.7

10.8 0.9

121 16

634 47

132

9

1292 84

1099 134

5787 258

64.7 4.4

76.6 8.9

57.7 2.5

18r

5

20

a

Sprague−Dawley rats (male), iv, n = 3. All values are represented as the mean

standard deviation. Abbreviations: AUC, area under the

concentration−time curve; T_1/2, elimination half-life; MRT, mean residence time; CL, clearance; V_ss, volume of distribution at the steady state.

8655

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 4. Docking results for compounds 18k and 18r. (A) Binding mode of GP-1 in a crystal structure of E. coli gyrase (PDB id: 4KFG) was

analyzed. (B,C) Binding modes of 18k (B) and 18r (C) were predicted using molecular docking. (D) Binding conformation superposition of GP-1,

18k, and 18r.

Figure 5. In Vitro SAR of Pyrimido[4,5-b]indole Derivatives.

compound 18r (9.89 μg/mL), which was 20−40-fold lower

than its antibacterial activity against A. baumannii, was

comparable with that of rac-GP-1. Novel pyrimido[4,5-

b]indole derivatives did not show superiority over rac-GP-1

in this assay.

interaction was observed for GP-1 with Asn46, which may

account for the better antibacterial activity of 18k. Compound

18r, however, failed to form the same interaction with Asp49,

which may explain its slightly reduced activity compared to

that of 18k. It seems that contact with Asp106 is important for

the activity of pyrimido[4,5-b]indoles, as all three compounds

interacted with this residue. More attention should be paid to

the interaction with Asp106 in further studies.

Binding Mode of Compounds 18k and 18r with E. coli

Gyrase. In order to gain insights into the binding modes of

compounds 18k and 18r, we then carried out molecular

modeling and docking studies based on the X-ray crystal

structure of the DNA gyrase B ATP-binding domain of E. coli

in complex with GP-1 (PDB code: 4KFG, 1.60 Å resolution).²⁰

The docking results are displayed in Figure 4, which show that

compounds 18k and 18r bind to GyrB in a similar mode to

that of GP-1. Hydrogen bonds between the pyrimido[4,5-

b]indole tricycle and Asp73 and a water molecule were shared

among GP-1, compounds 18k and 18r. However, the terminal

amine in the C-4 moiety forms a unique hydrogen bond with

the carboxylic group of Asp49, while a hydrogen-bond

CONCLUSIONS

■

Development of the pyrimido[4,5-b]indole derivatives was

previously limited due to the hERG liability, poor PK property,

and insuﬃcient eﬃcacy in vivo against G⁻bacteria. In this

study, based on the pyrimidoindole scaﬀold of GP-1 and the

C-7 moiety of acoraﬂoxacin, novel analogues with varied

substitutions at multiple positions were designed and

synthesized, and their SARs, hERG inhibition, PK proﬁles, in

vivo eﬃcacy as well as cytotoxicity, and binding mode were

8656

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Afterward, the reaction mixture was cooled to room temperature. The

reaction mixture was poured into 15 mL of water and a white

precipitate appeared. After ﬁltration and drying, the white solid was

redissolved in 2 mL of dichloromethane (DCM), and triﬂuoroacetic

acid (TFA) (2.0 mL, 26.9 mmol) was added. The reaction mixture

was allowed to stir at room temperature for 30 min and then

evaporated to dryness in vacuo. The residue was redissolved in 20 mL

of methanol, and 85% hydrazine hydrate (1.0 mL, 17.4 mmol) was

added. After being reheated to reﬂux for 1 h, the reaction mixture was

then concentrated in vacuo and diluted with water (20 mL). The

crude solid product was formed during concentration and was ﬁltered

and dried. The crude product was puriﬁed by chromatography on

silica gel with DCM/methanol (10:1) to aﬀord 18a as a pale-yellow

studied. A summary of the in vitro SAR of these pyrimido[4,5-

b]indole compounds is presented in Figure 5. Compounds

with a unique C-4 moiety consisting of an azetidine ring, an

exocyclic double bond, and a terminal amine group

demonstrated excellent broad-spectrum antibacterial activity,

including a variety of clinical MDR G⁻pathogens, especially A.

baumannii. Simultaneously, these compounds successfully

evaded hERG K⁺channel inhibition. A structure−metabolism

relationship study led to the discovery of compound 18r, with

an improved in vivo PK proﬁle and therapeutic eﬃcacy in a

neutropenic mouse thigh infection model. Nevertheless,

compound 18r still displayed minor cytotoxicity as other

pyrimido[4,5-b]indole derivatives, and the in vivo eﬃcacy

against MDR A. baumannii of this compound is moderate

which is probably due to the modest AUC and high clearance.

A broad-spectrum antibacterial agent is of importance to the

treatment of MDR bacterial infection in clinical application,

but the development of these agents with a novel mechanism

or novel target is slow.³³Compound 18r as a novel

pyrimido[4,5-b]indole derivative demonstrated outstanding

broad-spectrum activity against both G⁺and G⁻pathogens.

It provides a substantial foundation for the discovery of a new

class of broad-spectrum antibiotic. Based on the excellent lead

18r, we would carry out more following research on this series

to further improve the PK proﬁle, safety, and in vivo eﬃcacy

against MDR G⁻bacteria.

1

solid (41 mg, 32% yield). H NMR (600 MHz, DMSO-d₆): δ 11.81

(s, 1H), 8.73 (s, 2H), 7.82 (s, 3H), 6.66 (d, J = 9.5 Hz, 1H), 6.36 (d, J

= 11.6 Hz, 1H), 5.35 (t, J = 6.9 Hz, 1H), 3.65 (d, J = 21.1 Hz, 6H),

2.86 (s, 3H), 2.67 (s, 3H), 2.41 (s, 2H), 2.33 (s, 2H). ¹³C NMR (151

MHz, DMSO-d₆): δ 163.23, 161.07, 160.52, 159.84, 158.31, 157.98,

157.64, 150.62 (2C), 146.04, 141.89, 136.56, 120.67, 118.52, 115.80,

95.84, 94.19, 92.18, 48.52, 47.92, 35.55, 34.80, 29.67, 27.91, 24.86.

MS (ESI) m/z: 449.2 (M + H)⁺. High-resolution mass spectrometry

(HRMS) (ESI): Anal. Calcd for C₂₃H₂₆FN₈O [(M + H)⁺]: 449.2208.

Found: 449.2211.

4-(4-(1-Aminopropan-2-ylidene)piperidin-1-yl)-6-ﬂuoro-N-meth-

yl-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-8-

amine (18b). Compound 18b (40 mg, 58% yield) was prepared from

11 (80 mg, 150 μmol), 16b (81 mg, 300 μmol), K₂CO₃(62 mg, 451

μmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine hydrate (1.0

mL, 17.4 mmol) in the same manner as described for 18a but puriﬁed

by prep-HPLC (solvent A, 0.1% TFA aqueous solution; solvent B,

1

acetonitrile; gradient, 20−80% B). H NMR (500 MHz, DMSO-d₆):

EXPERIMENTAL SECTION

■

δ 11.70 (s, 1H), 8.67 (s, 2H), 7.89 (s, 3H), 6.63 (d, J = 9.8 Hz, 1H),

6.30 (d, J = 12.0 Hz, 1H), 3.65−3.59 (m, 4H), 3.48 (d, J = 5.3 Hz,

2H), 2.81 (s, 3H), 2.63 (s, 3H), 2.42 (s, 2H), 2.37 (s, 2H), 1.74 (s,

3H). ¹³C NMR (126 MHz, DMSO-d₆): δ 163.79, 161.55, 160.98,

160.57, 159.27, 158.73, 158.18, 151.11 (2C), 146.64, 137.12, 136.25,

121.25, 120.92, 119.27, 96.23, 94.90, 92.74, 48.59, 48.01, 41.04, 30.22,

29.66, 29.41, 25.28, 16.86. MS (ESI) m/z: 463.2 (M + H)⁺. HRMS

(ESI): Anal. Calcd for C₂₄H₂₈FN₈O [(M + H)⁺]: 463.2375. Found:

463.2365.

Chemistry. All solvents and chemicals were used as purchased

without further puriﬁcation. Inert atmosphere operations were

conducted under argon in ﬂame-dried glassware. Room temperature

refers to 20−25 °C. The microwave-assisted reactions were carried

out in a CEM Discover microwave synthesizer. Intermediates not

described below were purchased from commercial vendors and were

used as supplied unless stated otherwise. All reaction mixtures were

monitored using thin-layer chromatography (TLC) on silica gel F-254

TLC plates. Column chromatography was carried out using silica gel

(200−300 mesh). ¹H and ¹³C NMR spectra were recorded on a

Bruker 400, a Bruker 500, or a Bruker 600 NMR spectrometer using

the solvent residue as the internal standards. Chemical shifts (δ) are

reported in parts per million (ppm), and coupling constants (J) are

reported in hertz (Hz). Electron ionization (EI)-mass spectrometry

(MS) images were obtained on a Finnigan MAT95 spectrometer, and

electrospray ionization (ESI)-MS images were obtained on a Krats

MS 80 mass spectrometer. Puriﬁcation of all intermediates or ﬁnal

compounds was determined by either column chromatography or

preparative reverse-phase high-performance liquid chromatography

(HPLC) (SunFire C¹⁸OBD 100 mm × 30 mm; particle size, 5 μm)

with acetonitrile/buﬀer (0.1 CF₃COOH in water) as the mobile

phase. Purity of all ﬁnal compounds was determined by analytical

HPLC (PLATISIL ODS 250 mm × 4.6 mm, particle size 5 μm) with

acetonitrile/buﬀer (0.1 CF₃COOH in water) as the mobile phase.

The ee values were determined using chiral HPLC (CHIRALCEL

OD-H column 250 mm × 4.6 mm, particle size 5 μm or

CHIRALPAK IA column 250 mm × 4.6 mm, particle size 5 μm)

with ethanol/n-hexane or methanol/ethanol/0.1% diethylamine

(DEA) as the mobile phase. A purity of >95% was achieved for all

tested compounds. Detailed synthetic procedure and spectral

characterization for all intermediates are provided in the Supporting

Information.

4-(4-(2-Amino-1-ﬂuoroethylidene)piperidin-1-yl)-6-ﬂuoro-N-

methyl-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-

8-amine (18c). Compound 15 (120 mg, 272 μmol) and triethylamine

(83 mg, 817 μmol) was dissolved in 5 mL of NMP. The BOP reagent

(169 mg, 381 μmol) was added into the solution at 0 °C under an

argon atmosphere, and the reaction mixture was stirred for 30 min.

Then, intermediate 16c (155 mg, 564 μmol) dissolved in 5 mL of

NMP was added dropwise to the mixture. The reaction mixture was

heated to 50 °C and stirred for 1 h. The mixture was diluted with 20

mL of water and a white solid was precipitated out. The precipitate

was obtained after ﬁltration and drying. It was redissolved in 2 mL of

DCM, and TFA (2.0 mL, 26.9 mmol) was added. The reaction

mixture was allowed to stir at room temperature for 30 min and then

evaporated to dryness in vacuo. The residue was redissolved in 20 mL

of methanol, and 85% hydrazine hydrate (1.0 mL, 17.4 mmol) was

added. After being reheated to reﬂux for 1 h, the reaction mixture was

then concentrated in vacuo and diluted with water (20 mL). The

crude solid was product formed during concentration and was ﬁltered

and dried. The crude product was puriﬁed by chromatography on

silica gel with DCM/methanol (10:1) to aﬀord 18c as a pale-yellow

solid (72 mg, 57% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 8.74 (s,

2H), 6.66 (dd, J = 10.0, 2.0 Hz, 1H), 6.36 (dd, J = 12.1, 1.9 Hz, 1H),

5.64 (d, J = 4.5 Hz, 1H), 3.71−3.61 (m, 4H), 3.35 (d, J = 23.5 Hz,

2H), 2.86 (d, J = 4.8 Hz, 3H), 2.67 (d, J = 9.8 Hz, 3H), 2.40−2.36

(m, 2H), 2.36−2.31 (m, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ

163.39, 161.25, 160.79, 160.00, 158.47, 157.77, 150.80 (2C), 146.22,

136.70, 120.84, 118.70, 111.44, 95.98, 94.42, 92.30, 48.35, 47.55,

38.32, 29.83, 26.64, 25.02, 24.67. MS (ESI) m/z: 467.2 (M + H)⁺.

HRMS (ESI): Anal. Calcd for C₂₃H₂₅F₂N₈O [(M + H)⁺]: 467.2114.

Found: 467.2128.

4-(4-(2-Aminoethylidene)piperidin-1-yl)-6-ﬂuoro-N-methyl-2-

((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-8-amine

(18a). Intermediate 16a (144 mg, 563 μmol) was dissolved in 1-

methyl-2-pyrrolidinone (NMP) (5 mL) within a microwave vial.

Compound 11 (150 mg, 281 μmol) and potassium carbonate

(K₂CO₃) (117 mg, 845 μmol) were added into the solution. The vial

was capped and heated in a microwave reactor at 100 °C for 3 h.

8657

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(Z)-4-(3-(2-Amino-1-ﬂuoroethylidene)pyrrolidin-1-yl)-6-ﬂuoro-N-

methyl-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-

8-amine (18d). Compound 18d (33 mg, 21% yield) was prepared

from 15 (150 mg, 341 μmol), 16d (133 mg, 511 μmol), BOP reagent

(211 mg, 477 μmol), triethylamine (103 mg, 1.02 mmol), TFA (2.0

mL, 26.9 mmol), and 85% hydrazine hydrate (1.0 mL, 17.4 mmol) in

the same manner as described for 18c. ¹H NMR (400 MHz, DMSO-

d₆): δ 8.70 (s, 2H), 7.02 (d, J = 10.9 Hz, 1H), 6.28 (d, J = 11.9 Hz,

1H), 5.54 (s, 1H), 4.49 (s, 2H), 4.00 (t, J = 6.9 Hz, 2H), 3.27 (s, 2H),

2.82 (d, J = 4.4 Hz, 3H), 2.69 (s, 2H), 2.65 (s, 3H). ¹³C NMR (126

MHz, DMSO-d₆): δ 163.73, 161.40, 160.55, 158.72, 158.34, 157.81,

153.61, 151.21 (2C), 146.65, 136.98, 121.27, 119.32, 113.84, 95.15,

95.00, 92.33, 49.71, 49.20, 30.28, 26.43, 25.45. MS (ESI) m/z: 453.3

(M + H)⁺. HRMS (ESI): Anal. Calcd for C₂₂H₂₃F₂N₈O [(M + H)⁺]:

453.1957. Found: 453.1966.

(s, 2H), 2.85 (s, 3H), 2.68 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆):

δ 163.41, 161.34, 160.20, 159.31, 158.82−158.14, 156.82, 150.71

(2C), 146.14, 136.64, 134.31, 120.88, 118.17, 117.62, 115.48, 94.62,

93.95, 92.32, 59.12, 58.92, 41.12, 29.80, 25.01. MS (ESI) m/z: 455.3

(M + H)⁺. HRMS (ESI): Anal. Calcd for C₂₁H₂₁ClFN₈O [(M +

H)⁺]: 455.1505. Found: 455.1517.

4-(3-(2-Amino-1-bromoethylidene)azetidin-1-yl)-6-ﬂuoro-N-

methyl-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-

8-amine (18i). Compound 18i (63 mg, 46% yield) was prepared from

15 (120 mg, 272 μmol), 16i (100 mg, 409 μmol), BOP reagent (169

mg, 381 μmol), triethylamine (83 mg, 817 μmol), TFA (2.0 mL, 26.9

mmol), and 85% hydrazine hydrate (1.0 mL, 17.4 mmol) in the same

manner as described for 18c. ¹H NMR (400 MHz, DMSO-d₆): δ 8.73

(s, 2H), 6.88 (d, J = 9.4 Hz, 1H), 6.31 (d, J = 10.6 Hz, 1H), 5.74 (s,

1H), 5.17 (s, 2H), 4.77 (s, 2H), 3.51 (s, 2H), 2.84 (d, J = 4.6 Hz,

3H), 2.67 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆): δ 163.31,

161.31, 160.26, 159.19, 158.42, 156.71, 150.66 (2C), 146.13, 136.58,

129.83, 120.80, 118.23, 94.48, 93.80, 92.12, 61.88−61.10, 60.22,

46.63, 29.73, 24.98. MS (ESI) m/z: 499.2 (M + H)⁺. HRMS (ESI):

Anal. Calcd for C₂₁H₂₁BrFN₈O [(M + H)⁺]: 499.1000. Found:

499.1011.

(E)-4-(3-(2-Amino-1-ﬂuoroethylidene)pyrrolidin-1-yl)-6-ﬂuoro-N-

methyl-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-

8-amine (18e). Compound 18e (45 mg, 50% yield) was prepared

from 15 (70 mg, 159 μmol), 16e (83 mg, 303 μmol), BOP reagent

(98 mg, 223 μmol), triethylamine (48 mg, 477 μmol), TFA (2.0 mL,

26.9 mmol), and 85% hydrazine hydrate (1.0 mL, 17.4 mmol) in the

same manner as described for 18c but puriﬁed by prep-HPLC

(solvent A, 0.1% TFA aqueous solution; solvent B, acetonitrile;

4-(4-(1-Aminopropan-2-ylidene)piperidin-1-yl)-6-ﬂuoro-N-meth-

yl-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-8-

amine (18j). Compound 18j (25 mg, 26% yield) was prepared from

11 (120 mg, 225 μmol), 16j (109 mg, 451 μmol), K₂CO₃(93 mg,

676 μmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine hydrate

(1.0 mL, 17.4 mmol) in the same manner as described for 18a but

puriﬁed by prep-HPLC (solvent A, 0.1% TFA aqueous solution;

1

gradient, 20−80% B). H NMR (500 MHz, DMSO-d₆): δ 11.73 (s,

1H), 8.69 (s, 2H), 8.32 (s, 3H), 6.96 (dd, J = 10.9, 1.4 Hz, 1H), 6.30

(dd, J = 11.9, 1.6 Hz, 1H), 4.48 (s, 2H), 4.04 (t, J = 7.0 Hz, 2H), 3.80

(d, J = 20.7 Hz, 2H), 2.83 (s, 3H), 2.77 (t, J = 6.3 Hz, 2H), 2.65 (s,

3H). ¹³C NMR (126 MHz, DMSO-d₆): δ 163.79, 161.40, 160.60,

158.68, 158.53−158.51, 158.37, 157.88, 151.22 (2C), 146.65, 144.68,

137.06, 121.36, 120.54, 119.25, 95.17, 95.00, 92.43, 49.60, 49.22,

37.48, 30.28, 26.84, 25.45. MS (ESI) m/z: 453.3 (M + H)⁺. HRMS

(ESI): Anal. Calcd for C₂₂H₂₃F₂N₈O [(M + H)⁺]: 453.1957. Found:

453.1966.

1

solvent B, acetonitrile; gradient, 20−80% B). H NMR (500 MHz,

DMSO-d₆): δ 11.71 (s, 1H), 8.68 (s, 2H), 7.98 (s, 3H), 6.90 (d, J =

10.1 Hz, 1H), 6.29 (d, J = 12.1 Hz, 1H), 5.06 (s, 2H), 4.90 (s, 2H),

3.38 (d, J = 4.9 Hz, 2H), 2.81 (s, 3H), 2.64 (s, 3H), 1.68 (s, 3H). ¹³C

NMR (126 MHz, DMSO-d₆): δ 163.80, 161.86, 160.74, 159.74,

158.91, 157.20, 151.12 (2C), 146.62, 136.97, 130.28, 121.64, 121.21,

118.89, 118.79, 94.84, 94.48, 92.63, 59.72, 59.48, 40.68, 30.25, 25.41,

15.69. MS (ESI) m/z: 435.3 (M + H)⁺. HRMS (ESI): Anal. Calcd for

C₂₂H₂₄FN₈O [(M + H)⁺]: 435.2052. Found: 435.2056.

4-(3-(2-Aminoethylidene)azetidin-1-yl)-6-ﬂuoro-N-methyl-2-((2-

methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-8-amine (18f).

Compound 18f (97 mg, 82% yield) was prepared from 11 (150 mg,

282 μmol), 16f (78 mg, 338 μmol), K₂CO₃(117 mg, 845 μmol), TFA

(2.0 mL, 26.9 mmol), and 85% hydrazine hydrate (1.0 mL, 17.4

mmol) in the same manner as described for 18a. ¹H NMR (500 MHz,

DMSO-d₆): δ 8.70 (s, 2H), 6.87 (d, J = 9.1 Hz, 1H), 6.28 (d, J = 12.0

Hz, 1H), 5.67 (d, J = 4.3 Hz, 1H), 5.51 (s, 1H), 5.06 (s, 2H), 4.90 (s,

2H), 3.22 (d, J = 6.3 Hz, 2H), 2.83 (d, J = 4.5 Hz, 3H), 2.65 (s, 3H).

¹³C NMR (126 MHz, DMSO-d₆): δ 163.72, 161.85, 159.67, 158.86,

157.18, 151.14 (2C), 146.65, 137.03, 130.96, 121.60, 121.21, 118.88,

94.75, 94.37, 92.48, 60.38, 29.86. MS (ESI) m/z: 421.2 (M + H)⁺.

HRMS (ESI): Anal. Calcd for C₂₁H₂₂FN₈O [(M + H)⁺]: 421.1895.

Found: 421.1886.

4-(3-(2-Amino-1-ﬂuoroethylidene)azetidin-1-yl)-6-ﬂuoro-N-

methyl-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-

8-amine (18g). Compound 18g (58 mg, 49% yield) was prepared

from 15 (120 mg, 272 μmol), 16g (101 mg, 409 μmol), BOP reagent

(169 mg, 381 μmol), triethylamine (83 mg, 817 μmol), TFA (2.0 mL,

26.9 mmol), and 85% hydrazine hydrate (1.0 mL, 17.4 mmol) in the

same manner as described for 18c. ¹H NMR (600 MHz, DMSO-d₆):

δ 8.68 (s, 2H), 6.84 (dd, J = 10.2, 2.0 Hz, 1H), 6.27 (dd, J = 12.1, 2.0

Hz, 1H), 5.50 (d, J = 4.3 Hz, 1H), 5.09 (s, 2H), 4.89 (s, 2H), 3.27 (d,

J = 13.6 Hz, 2H), 2.81 (d, J = 4.9 Hz, 3H), 2.63 (s, 3H). ¹³C NMR

(151 MHz, DMSO-d₆): δ 163.35, 161.41, 159.17, 158.58, 156.74,

150.76 (2C), 146.16, 136.53, 120.74 s, 118.32, 118.10, 105.93,

105.77, 94.27, 93.96, 92.16, 56.84, 56.43, 29.81, 25.02, 1.17. MS (ESI)

m/z: 439.3 (M + H)⁺. HRMS (ESI): Anal. Calcd for C₂₁H₂₁F₂N₈O

[(M + H)⁺]: 439.1801. Found: 439.1810.

4-(3-(2-Amino-1-chloroethylidene)azetidin-1-yl)-6-ﬂuoro-N-

methyl-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-

8-amine (18h). Compound 18h (73 mg, 49% yield) was prepared

from 15 (120 mg, 272 μmol), 16h (100 mg, 381 μmol), BOP reagent

(169 mg, 381 μmol), triethylamine (83 mg, 817 μmol), TFA (2.0 mL,

26.9 mmol), and 85% hydrazine hydrate (1.0 mL, 17.4 mmol) in the

same manner as described for 18e. ¹H NMR (600 MHz, DMSO-d₆):

δ 11.82 (s, 1H), 8.73 (s, 2H), 8.36 (s, 3H), 6.89 (dd, J = 10.0, 1.8 Hz,

1H), 6.33 (dd, J = 12.1, 1.9 Hz, 1H), 5.18 (s, 2H), 4.93 (s, 2H), 3.82

4-(3-(1-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-N-methyl-

2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-8-amine

(18k). Compound 18k (31 mg, 24% yield) was prepared from 11

(120 mg, 225 μmol), 16k (81 mg, 316 μmol), K₂CO₃(93 mg, 676

μmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine hydrate (1.0

mL, 17.4 mmol) in the same manner as described for 18a but puriﬁed

by prep-HPLC (solvent A, 0.1% TFA aqueous solution; solvent B,

1

acetonitrile; gradient, 20−80% B). H NMR (500 MHz, DMSO-d₆):

δ 11.76 (s, 1H), 8.72 (s, 2H), 7.93 (s, 3H), 6.94 (dd, J = 10.1, 1.8 Hz,

1H), 6.32 (dd, J = 12.1, 1.9 Hz, 1H), 5.13 (s, 2H), 5.01 (s, 2H), 3.43

(d, J = 5.3 Hz, 2H), 2.85 (s, 3H), 2.67 (s, 3H), 2.12 (d, J = 7.5 Hz,

2H), 1.03 (t, J = 7.5 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆): δ

163.81, 161.91, 160.75, 159.82, 158.92, 157.20, 151.15 (2C), 146.64,

137.02, 130.55, 127.13, 121.23, 118.83, 118.29, 94.83, 94.53, 92.64,

59.92, 59.42, 38.26, 30.28, 25.46, 22.69, 12.41. MS (ESI) m/z: 449.2

(M + H)⁺. HRMS (ESI): Anal. Calcd for C₂₃H₂₆FN₈O [(M + H)⁺]:

449.2208. Found: 449.2207.

4-(3-(1-Amino-3-methylbutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-

N-methyl-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]-

indol-8-amine (18l). Compound 18l (39 mg, 25% yield) was

prepared from 15 (120 mg, 272 μmol), 16l (130 mg, 490 μmol), BOP

reagent (169 mg, 381 μmol), triethylamine (83 mg, 817 μmol), TFA

(2.0 mL, 26.9 mmol), and 85% hydrazine hydrate (1.0 mL, 17.4

mmol) in the same manner as described for 18e. ¹H NMR (500 MHz,

DMSO-d₆): δ 11.75 (s, 1H), 8.69 (s, 2H), 7.93 (s, 3H), 6.91 (dd, J =

10.2, 1.9 Hz, 1H), 6.30 (dd, J = 12.2, 2.0 Hz, 1H), 5.10 (d, J = 45.9

Hz, 4H), 4.20 (d, J = 5.8 Hz, 1H), 3.39 (d, J = 5.3 Hz, 2H), 2.83 (s,

3H), 2.65 (s, 3H), 1.06 (d, J = 6.9 Hz, 6H). ¹³C NMR (126 MHz,

DMSO-d₆): δ 163.33, 161.43, 160.25, 159.25, 158.42, 158.16, 156.69,

150.67 (2C), 146.14, 136.53, 130.58, 129.92, 120.74, 118.33, 115.40,

94.32, 94.02, 92.13, 59.97, 59.29, 37.50, 29.90, 29.77, 24.95, 20.53

(2C). MS (ESI) m/z: 463.2 (M + H)⁺. HRMS (ESI): Anal. Calcd for

C₂₄H₂₈FN₈O [(M + H)⁺]: 463.2365. Found: 463.2365.

8658

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

4-(3-(1-Aminopentan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-N-meth-

yl-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-8-

amine (18m). Compound 18m (61 mg, 35% yield) was prepared

from 11 (200 mg, 376 μmol), 16m (183 mg, 676 μmol), K₂CO₃(156

mg, 1.13 mmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine

hydrate (1.0 mL, 17.4 mmol) in the same manner as described for

MS (ESI) m/z: 448.4 (M + H)⁺. HRMS (ESI): Anal. Calcd for

C₂₃H₂₆FN₈O [(M + H)⁺]: 449.2208. Found: 449.2199.

4-(3-(2-Aminopentan-3-ylidene)azetidin-1-yl)-6-ﬂuoro-N-meth-

yl-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-8-

amine (18q). Compound 18q (12 mg, 9% yield) was prepared from

11 (150 mg, 282 μmol), 16q (107 mg, 394 μmol), K₂CO₃(117 mg,

845 μmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine hydrate

1

18a. H NMR (600 MHz, DMSO-d₆): δ 8.72 (s, 2H), 6.92 (d, J =

1

(1.0 mL, 17.4 mmol) in the same manner as described for 18a. H

10.3 Hz, 1H), 6.30 (d, J = 12.0 Hz, 1H), 5.60 (s, 1H), 5.12 (s, 2H),

4.93 (s, 2H), 3.15 (d, J = 9.6 Hz, 2H), 2.84 (s, 3H), 2.68 (s, 3H), 2.00

(s, 2H), 1.43 (d, J = 7.3 Hz, 2H), 0.90 (t, J = 7.1 Hz, 3H). ¹³C NMR

(151 MHz, DMSO-d₆): δ 163.31, 161.52, 159.28, 158.54, 156.69,

150.76 (2C), 146.22, 136.49, 133.26, 122.38, 120.68, 118.52, 94.25,

94.05, 92.05, 59.85, 59.33, 41.98, 31.32, 31.17, 29.83, 25.02, 20.32,

13.95. MS (ESI) m/z: 463.2 (M + H)⁺. HRMS (ESI): Anal. Calcd for

C₂₄H₂₈FN₈O [(M + H)⁺]: 463.2365. Found: 463.2353.

NMR (500 MHz, DMSO-d₆): δ 8.70 (s, 2H), 6.90 (d, J = 8.8 Hz,

1H), 6.28 (d, J = 10.9 Hz, 1H), 5.52 (s, 1H), 5.09 (s, 2H), 4.90 (s,

2H), 3.56 (d, J = 22.3 Hz, 1H), 2.83 (d, J = 3.8 Hz, 3H), 2.66 (s, 3H),

2.09−1.87 (m, 2H), 1.36 (ddd, J = 63.2, 27.3, 14.0 Hz, 3H), 0.88 (t, J

= 7.2 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆): δ 165.15, 163.36,

161.12, 160.25, 158.57, 152.59 (2C), 148.09, 138.36, 134.94, 124.45,

122.56, 120.38, 96.14, 95.90d, (J = 26.1 Hz), 93.90, 61.74, 61.17,

43.75, 33.04, 31.69, 26.87, 22.17, 15.80. MS (ESI) m/z: 463.5 (M +

H)⁺. HRMS (ESI): Anal. Calcd for C₂₄H₂₈FN₈O [(M + H)⁺]:

463.2365. Found: 463.2365.

(R)-4-(3-(3-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-N-

methyl-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-

8-amine (18r). Compound 18r (97 mg, 35% yield) was prepared

from 11 (320 mg, 601 μmol), 16r (231 mg, 901 μmol), K₂CO₃(249

mg, 1.80 mmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine

hydrate (1.0 mL, 17.4 mmol) in the same manner as described for

18a. ¹H NMR (500 MHz, DMSO-d₆): δ 8.72 (s, 2H), 6.92 (d, J = 9.7

Hz, 1H), 6.30 (d, J = 11.6 Hz, 1H), 5.52 (s, 1H), 5.06 (s, 2H), 4.87

(s, 2H), 3.50 (d, J = 6.3 Hz, 1H), 2.85 (d, J = 4.4 Hz, 3H), 2.67 (s,

3H), 1.55 (s, 3H), 1.07 (d, J = 6.4 Hz, 3H). ¹³C NMR (126 MHz,

DMSO-d₆): δ 163.74, 161.95, 160.67, 159.67, 158.84, 157.17, 151.19

(2C), 146.67, 136.89, 133.32, 121.09, 118.99, 94.61, 94.41, 92.49,

60.63−60.21, 59.95, 48.88, 30.28, 25.47 s, 21.90, 12.41. MS (ESI) m/

z: 449.2 (M + H)⁺. HRMS (ESI): Anal. Calcd for C₂₃H₂₆FN₈O [(M

+ H)⁺]: 449.2208. Found: 449.2210. The ee value was 94.2%. Chiral

HPLC retention time 32.95 min; column: CHIRALPAK IA column

(250 mm × 4.6 mm, 5 μm); column temperature 30 °C; ﬂow rate 0.5

mL/min; detection UV 254 nm; mobile phase: solvent A (80%) =

methanol + 0.1% DEA, solvent B (20%) = ethanol + 0.1% DEA; total

run time 40 min.

4-(3-(1-Aminopent-4-en-2-ylidene)azetidin-1-yl)-6-ﬂuoro-N-

methyl-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-

8-amine (18n). Intermediate 16n (154 mg, 575 μmol) was dissolved

in NMP (5 mL) within a microwave vial. Compound 11 (170 mg,

319 μmol) and K₂CO₃(132 mg, 958 μmol) were added into the

solution. The vial was capped and heated in a microwave reactor at

100 °C for 3 h. Afterward, the reaction mixture was cooled to room

temperature. The reaction mixture was poured into 15 mL of water

and a white precipitate appeared. After ﬁltration and drying, the white

solid was redissolved in 2 mL of DCM, and TFA (2.0 mL, 26.9

mmol) was added. The reaction mixture was allowed to stir at room

temperature for 30 min and then evaporated to dryness in vacuo. The

residue was redissolved in 10 mL of ethanol and 2 mL of 27−32%

methylamine ethanol solution was added. After being reheated to 40

°C and stirred for 1 h, the reaction mixture was then concentrated in

vacuo. The residue was puriﬁed by chromatography on silica gel with

DCM/methanol (10:1) to aﬀord 18a as a pale-yellow solid (44 mg,

1

30% yield). H NMR (500 MHz, DMSO-d₆): δ 8.72 (s, 2H), 6.91

(dd, J = 10.2, 1.6 Hz, 1H), 6.36−6.19 (m, 1H), 5.91−5.77 (m, 1H),

5.58 (d, J = 4.2 Hz, 1H), 5.12 (d, J = 15.4 Hz, 3H), 5.03 (d, J = 10.0

Hz, 1H), 4.92 (s, 2H), 3.14 (s, 2H), 2.85 (d, J = 4.7 Hz, 3H), 2.79 (d,

J = 6.5 Hz, 2H), 2.67 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆): δ

163.74, 161.94, 159.65, 158.85, 157.15, 151.18 (2C), 146.66, 136.97,

136.06, 131.22, 124.42, 121.15, 118.94, 116.51, 94.70, 94.45, 92.48,

60.41, 59.80, 42.65, 34.38, 30.26, 25.46. MS (ESI) m/z: 461.4 (M +

H)⁺. HRMS (ESI): Anal. Calcd for C₂₄H₂₆FN₈O [(M + H)⁺]:

461.2208. Found: 461.2219.

4-(3-(1-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-N-methyl-

2-(methylsulfonyl)-9H-pyrimido[4,5-b]indol-8-amine (20). Com-

pound 19 (100 mg, 154 μmol) was dissolved in 2 mL of DCM,

and TFA (2.0 mL, 26.9 mmol) was added. The reaction mixture was

allowed to stir at room temperature for 30 min and then evaporated

to dryness in vacuo. The residue was redissolved in 20 mL of

methanol, and 85% hydrazine hydrate (1.0 mL, 17.4 mmol) was

added. After being reheated to reﬂux for 1 h, the reaction mixture was

then concentrated in vacuo and diluted with water (20 mL). The

crude solid product was formed during concentration and was ﬁltered

and dried. The crude product was puriﬁed by chromatography on

silica gel with DCM/methanol (10:1) to aﬀord 20 as a pale-yellow

4-(3-(2-Aminopropylidene)azetidin-1-yl)-6-ﬂuoro-N-methyl-2-

((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-8-amine

(18o). Compound 18o (190 mg, 38% yield) was prepared from 11

(500 mg, 939 μmol), 16o (341 mg, 1.41 mmol), K₂CO₃(389 mg,

2.82 mmol), TFA (4.0 mL, 53.9 mmol), and 85% hydrazine hydrate

1

(1.0 mL, 17.4 mmol) in the same manner as described for 18k. H

NMR (400 MHz, DMSO-d₆): δ 11.79 (s, 1H), 8.72 (s, 2H), 7.98 (s,

3H), 6.92 (dd, J = 10.2, 1.8 Hz, 1H), 6.32 (dd, J = 12.2, 2.0 Hz, 1H),

5.50 (d, J = 9.0 Hz, 1H), 5.15 (s, 2H), 4.96 (s, 2H), 3.91 (s, 1H), 2.85

(s, 3H), 2.67 (s, 3H), 1.30 (d, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz,

DMSO-d₆): δ 163.25, 161.31, 160.18, 159.20, 158.14, 156.66, 150.61

(2C), 146.05, 136.46, 134.71, 120.66, 119.70, 118.22, 115.24, 94.28,

93.89, 92.09, 59.83, 59.49, 45.17, 29.71, 24.91, 18.85. MS (ESI) m/z:

435.3 (M + H)⁺. HRMS (ESI): Anal. Calcd for C₂₂H₂₄FN₈O [(M +

H)⁺]: 435.2052. Found: 435.2061.

1

solid (25 mg, 39% yield). H NMR (500 MHz, DMSO-d₆): δ 12.26

(s, 1H), 7.02 (d, J = 8.5 Hz, 1H), 6.44−6.41 (m, 1H), 5.21 (s, 2H),

5.12 (s, 2H), 3.43 (d, J = 4.8 Hz, 2H), 3.35 (d, J = 4.6 Hz, 3H), 2.88

(d, J = 2.0 Hz, 3H), 2.12 (q, J = 7.2 Hz, 2H), 1.01 (dd, J = 13.1, 7.4

Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆): δ 161.09, 159.13, 158.40,

158.37−157.93, 154.46, 137.60, 134.98, 130.23, 127.33, 122.71,

118.18, 98.72, 94.85, 93.85, 53.93, 53.64, 38.23, 36.18, 30.24 (s),

22.71, 12.44. MS (ESI) m/z: 419.1 (M + H)⁺. HRMS (ESI): Anal.

Calcd for C₁₉H₂₄FN₆O₂S [(M + H)⁺]: 419.1660. Found: 419.1666.

4-(3-(1-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-8-(methyl-

amino)-9H-pyrimido[4,5-b]indole-2-carbonitrile (22). Compound

21 (500 mg, 839 μmol) was dissolved in 20 mL of methanol, and

85% hydrazine hydrate (1.0 mL, 17.4 mmol) was added. After being

heated to reﬂux for 1 h, the reaction mixture was then concentrated in

vacuo and diluted with water (20 mL). A white solid was formed

during concentration and was ﬁltered and dried. The white solid was

redissolved in 2 mL of DCM, and TFA (2.0 mL, 26.9 mmol) was

added. The reaction mixture was allowed to stir at room temperature

for 30 min and then evaporated to dryness in vacuo. The residue was

puriﬁed by chromatography on silica gel with DCM/methanol (10:1)

4-(3-(3-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-N-methyl-

2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-8-amine

(18p). Compound 18p (105 mg, 34% yield) was prepared from 11

(290 mg, 545 μmol), 16p (195 mg, 762 μmol), K₂CO₃(225.78 mg,

1.63 mmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine hydrate

1

(1.0 mL, 17.4 mmol) in the same manner as described for 18k. H

NMR (400 MHz, DMSO-d₆): δ 11.78 (s, 1H), 8.72 (s, 2H), 8.03 (s,

3H), 6.95 (dd, J = 10.2, 2.0 Hz, 1H), 6.32 (dd, J = 12.2, 2.1 Hz, 1H),

5.13 (s, 2H), 4.93 (s, 2H), 4.00−3.90 (m, 1H), 2.85 (s, 3H), 2.67 (s,

3H), 1.65 (s, 3H), 1.29 (d, J = 6.8 Hz, 3H). ¹³C NMR (126 MHz,

DMSO-d₆): δ 163.81, 161.91, 160.75, 159.79, 159.09−158.38, 157.21,

151.15 (2C), 146.64, 137.01, 128.77, 125.29, 121.22, 118.84, 115.72,

94.82, 94.54, 92.64, 59.73, 59.56, 47.76, 30.28, 25.46, 17.45, 11.88.

8659

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

to aﬀord 22 as a pale-yellow solid (36 mg, 12% yield). ¹H NMR (400

MHz, CD₃OD_SPE): δ 7.30 (d, J = 9.3 Hz, 1H), 6.45 (d, J = 11.6 Hz,

1H), 4.40 (s, 2H), 3.90 (s, 2H), 3.86 (s, 2H), 2.98 (s, 3H), 2.49 (q, J

= 7.4 Hz, 2H), 1.14 (t, J = 7.6 Hz, 3H). ¹³C NMR (126 MHz,

DMSO-d₆): δ 160.89, 159.05, 157.90, 153.96, 138.51, 137.57, 134.91,

122.67, 122.14, 117.60, 99.17, 94.76, 93.86, 60.69, 60.37, 42.10, 30.19,

22.90, 12.74. MS (ESI) m/z: 366.1 (M + H)⁺. HRMS (ESI): Anal.

Calcd for C₁₉H₂₁FN₇[(M + H)⁺]: 366.1873. Found: 366.1872.

4-(3-(1-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-N⁸-meth-

yl-9H-pyrimido[4,5-b]indole-2,8-diamine (26). Compound 26 (75

mg, 62% yield) was prepared from 24 (150 mg, 256 μmol), TFA (2.0

mL, 26.9 mmol), and 85% hydrazine hydrate (1.0 mL, 17.4 mmol) in

the same manner as described for 19 but puriﬁed by prep-HPLC

(solvent A, 0.1% TFA aqueous solution; solvent B, acetonitrile;

mixture was then concentrated in vacuo and diluted with water (20

mL). The crude solid product was formed during concentration and

was ﬁltered and dried. The crude product was puriﬁed by

chromatography on silica gel with DCM/methanol (10:1) to aﬀord

1

31b as a pale-yellow solid (47 mg, 24% yield). H NMR (500 MHz,

DMSO-d₆): δ 8.80 (s, 2H), 6.91 (d, J = 9.7 Hz, 1H), 6.29 (d, J = 11.8

Hz, 1H), 5.54 (s, 1H), 5.09 (s, 2H), 4.95 (s, 2H), 4.65 (s, 2H), 3.24

(s, 2H), 2.83 (d, J = 3.9 Hz, 3H), 2.05 (d, J = 7.2 Hz, 2H), 0.99 (t, J =

7.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆): δ 165.23, 161.41,

159.21, 158.37, 156.63, 150.74 (2C), 146.75, 136.48, 131.65, 124.78,

120.69, 118.41, 94.26, 94.01, 92.04, 64.53, 59.74, 59.12, 29.77, 29.00,

22.30, 12.14. MS (ESI) m/z: 465.3 (M + H)⁺. HRMS (ESI): Anal.

Calcd for C₂₃H₂₆FN₈O₂[(M + H)⁺]: 465.2157. Found: 465.2157.

1-(5-((4-(3-(1-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-8-

(methylamino)-9H-pyrimido[4,5-b]indol-2-yl)oxy)pyrimidin-2-yl)-

ethan-1-ol (31c). Compound 31b (39 mg, 19% yield) was prepared

from 10 (200 mg, 423 μmol), 28b (131 mg, 931 μmol), 16k (152 mg,

593 μmol), K₂CO₃(176 mg, 1.27 mmol), TFA (2.0 mL, 26.9 mmol),

and 85% hydrazine hydrate (1.0 mL, 17.4 mmol) in the same manner

1

gradient, 20−80% B). H NMR (500 MHz, DMSO-d₆): δ 11.64 (s,

1H), 8.00 (s, 3H), 6.83 (dd, J = 10.2, 1.6 Hz, 1H), 6.25 (dd, J = 12.2,

1.8 Hz, 1H), 5.20 (s, 2H), 5.06 (s, 2H), 3.40 (s, 3H), 2.82 (s, 3H),

2.10 (q, J = 7.3 Hz, 2H), 1.00 (t, J = 7.5 Hz, 3H). ¹³C NMR (126

MHz, DMSO-d₆): δ 160.72, 158.89, 158.58, 158.32, 158.04, 136.87,

127.05, 119.76, 119.61, 118.40, 116.02, 93.68, 91.96, 90.65, 60.34,

59.82, 37.94, 29.78, 22.46, 12.02. MS (ESI) m/z: 356.1 (M + H)⁺.

HRMS (ESI): Anal. Calcd for C₁₈H₂₃FN₇[(M + H)⁺]: 356.1993.

Found: 356.2001.

1

as described for 31b. H NMR (500 MHz, DMSO-d₆): δ 8.80 (s,

2H), 6.89 (dd, J = 10.2, 1.8 Hz, 1H), 6.27 (dd, J = 12.1, 1.7 Hz, 1H),

5.48 (d, J = 4.1 Hz, 1H), 5.06 (s, 2H), 4.91 (s, 2H), 4.84 (q, J = 6.5

Hz, 1H), 3.11 (s, 2H), 2.82 (d, J = 4.5 Hz, 3H), 2.00 (q, J = 7.3 Hz,

2H), 1.45 (d, J = 6.6 Hz, 3H), 0.97 (t, J = 7.6 Hz, 3H). ¹³C NMR

(126 MHz, DMSO-d₆): δ 168.47, 161.94, 159.62, 158.83, 157.08,

151.24 (2C), 147.21, 136.92, 135.50, 121.75, 121.14, 118.96, 94.71,

94.50, 94.40, 92.48, 70.08, 60.33, 59.68, 42.39, 30.27, 23.26, 22.86,

12.77. MS (ESI) m/z: 479.2 (M + H)⁺. HRMS (ESI): Anal. Calcd for

C₂₄H₂₈FN₈O₂[(M + H)⁺]: 479.2314. Found: 479.2319.

N-(4-(3-(1-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-8-

(methylamino)-9H-pyrimido[4,5-b]indol-2-yl)methanesulfonamide

(27). Compound 27 (35 mg, 33% yield) was prepared from 25 (130

mg, 196 μmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine

hydrate (1.0 mL, 17.4 mmol) in the same manner as described for 26.

¹H NMR (500 MHz, DMSO-d₆): δ 11.44 (s, 1H), 9.21 (s, 2H),

2-(5-((4-(3-(1-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-8-

(methylamino)-9H-pyrimido[4,5-b]indol-2-yl)oxy)pyrimidin-2-yl)-

propan-2-ol (31d). Compound 31d (42 mg, 20% yield) was prepared

from 10 (200 mg, 423 μmol), 28d (143 mg, 931 μmol), 16k (152 mg,

593 μmol), K₂CO₃(176 mg, 1.27 mmol), TFA (2.0 mL, 26.9 mmol),

and 85% hydrazine hydrate (1.0 mL, 17.4 mmol) in the same manner

7.45−7.30 (m, 2H), 6.26 (d, J = 11.9 Hz, 1H), 4.29 (s, 2H), 3.97 (s,

2H), 3.92 (s, 2H), 3.36 (s, 3H), 2.83 (s, 3H), 2.31 (d, J = 7.2 Hz,

2H), 0.99 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆): δ

160.33, 158.77−157.93, 156.74, 155.36, 154.62, 136.29, 133.48,

127.85, 119.84, 118.93, 115.49, 93.22, 92.60, 91.75, 53.31, 53.12,

41.38, 35.32, 29.68, 18.56, 12.32. MS (ESI) m/z: 434.2 (M + H)⁺.

HRMS (ESI): Anal. Calcd for C₁₉H₂₅FN₇O₂S [(M + H)⁺]: 434.1769.

Found: 434.1768.

1

as described for 31b. H NMR (500 MHz, DMSO-d₆): δ 8.83 (s,

2H), 6.94 (d, J = 9.6 Hz, 1H), 6.32 (d, J = 11.7 Hz, 1H), 5.58 (s, 1H),

5.15 (s, 2H), 4.99 (s, 2H), 3.34 (s, 2H), 2.85 (d, J = 3.8 Hz, 3H), 2.10

(d, J = 6.9 Hz, 2H), 1.56 (s, 6H), 1.02 (t, J = 7.3 Hz, 3H). ¹³C NMR

(126 MHz, DMSO-d₆): δ 170.03, 161.33, 159.22, 158.32, 156.51,

150.35 (2C), 146.27, 136.49, 129.52, 129.03, 120.62, 118.32, 94.29,

93.90, 92.00, 72.53, 59.52, 58.91 (s), 31.03, 29.66, 28.91, 22.20, 11.92.

MS (ESI) m/z: 493.3 (M + H)⁺. HRMS (ESI): Anal. Calcd for

C₂₅H₃₀FN₈O₂[(M + H)⁺]: 493.2470. Found: 493.2482.

4-(3-(1-Aminobutan-2-ylidene)azetidin-1-yl)-2-((5-cyclopropyl-

pyrimidin-2-yl)oxy)-6-ﬂuoro-N-methyl-9H-pyrimido[4,5-b]indol-8-

amine (31a). Compound 31a (105 mg, 34% yield) was prepared

from 29a (250 mg, 428 μmol), 16k (164 mg, 641 μmol), K₂CO₃(177

mg, 1.28 mmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine

hydrate (1.0 mL, 17.4 mmol) in the same manner as described for

1

18k. H NMR (500 MHz, DMSO-d₆): δ 8.66 (s, 2H), 6.92 (dd, J =

6-((4-(3-(1-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-8-

(methylamino)-9H-pyrimido[4,5-b]indol-2-yl)oxy)isoindolin-1-one

(31e). Compound 31e (37 mg, 15% yield) was prepared from 10

(200 mg, 423 μmol), 28e (139 mg, 931 μmol), 16k (152 mg, 593

μmol), K₂CO₃(176 mg, 1.27 mmol), TFA (2.0 mL, 26.9 mmol), and

85% hydrazine hydrate (1.0 mL, 17.4 mmol) in the same manner as

described for 31b but puriﬁed by prep-HPLC (solvent A, 0.1% TFA

10.3, 2.1 Hz, 1H), 6.30 (dd, J = 12.2, 2.1 Hz, 1H), 5.59 (d, J = 4.3 Hz,

1H), 5.09 (s, 2H), 4.95 (s, 2H), 3.18 (s, 2H), 2.85 (d, J = 4.8 Hz,

3H), 2.31−2.22 (m, 1H), 2.04 (dd, J = 15.1, 7.5 Hz, 2H), 1.07 (dt, J =

8.0, 2.9 Hz, 2H), 1.04−0.97 (m, 5H). ¹³C NMR (126 MHz, DMSO-

d₆): δ 167.41, 162.03, 159.69, 158.84, 157.15, 151.20 (2C), 146.49,

136.97, 134.27, 123.07, 121.14, 118.91, 94.66, 94.47, 92.46, 60.31,

59.63, 41.79, 30.26, 22.86, 17.95, 12.72, 10.83 (2C). MS (ESI) m/z:

475.3 (M + H)⁺. HRMS (ESI): Anal. Calcd for C₂₅H₂₈FN₈O [(M +

H)⁺]: 475.2365. Found: 475.2374.

(5-((4-(3-(1-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-8-

(methylamino)-9H-pyrimido[4,5-b]indol-2-yl)oxy)pyrimidin-2-yl)-

methanol (31b). Intermediate 10 (200 mg, 423.27 μmol) was

dissolved in NMP (5 mL) within a microwave vial. Compound 28b

(117 mg, 931.20 μmol) and K₂CO₃(176 mg, 1.27 mmol) were added

into the solution. The vial was capped and heated in a microwave

reactor at 100 °C for 3 h. After that, compound 16k (152 mg, 592.58

μmol) was added into the solution. The vial was capped and reheated

again in the microwave reactor at 100 °C for 3 h. Afterward, the

reaction mixture was cooled to room temperature. The reaction

mixture was poured into 15 mL of water and a white precipitate

appeared. After ﬁltration and drying, the white solid was redissolved in

2 mL of DCM, and TFA (2.0 mL, 26.9 mmol) was added. The

reaction mixture was allowed to stir at room temperature for 30 min

and then evaporated to dryness in vacuo. The residue was redissolved

in 20 mL of methanol, and 85% hydrazine hydrate (1.0 mL, 17.4

mmol) was added. After being reheated to reﬂux for 1 h, the reaction

1

aqueous solution; solvent B, acetonitrile; gradient, 20−80% B). H

NMR (500 MHz, DMSO-d₆): δ 11.74 (s, 1H), 8.66 (s, 1H), 7.97 (s,

3H), 7.63 (d, J = 8.0 Hz, 1H), 7.49−7.40 (m, 2H), 6.94 (d, J = 9.8

Hz, 1H), 6.31 (d, J = 12.0 Hz, 1H), 5.15 (s, 2H), 5.01 (s, 2H), 4.42

(s, 2H), 3.43 (d, J = 4.4 Hz, 2H), 2.84 (s, 3H), 2.12 (d, J = 7.2 Hz,

2H), 1.03 (t, J = 7.5 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆): δ

169.86, 162.67, 160.70, 160.13, 158.86, 158.79−158.31, 157.44,

153.54, 140.65, 136.94, 134.35, 130.80, 127.03, 125.75, 125.15,

122.19, 118.87, 116.27, 94.64, 94.44, 92.52, 59.97, 59.41, 45.17, 38.29,

30.29, 22.71, 12.41. MS (ESI) m/z: 488.3 (M + H)⁺. HRMS (ESI):

Anal. Calcd for C₂₆H₂₇FN₇O₂[(M + H)⁺]: 488.2205. Found:

488.2202.

1-(1-(6-Fluoro-8-(methylamino)-2-((2-methylpyrimidin-5-yl)-

oxy)-9H-pyrimido[4,5-b]indol-4-yl)azetidin-3-ylidene)propan-2-ol

(34a). Intermediate 32a (80 mg, 703 μmol) was dissolved in NMP (5

mL) within a microwave vial. Compound 11 (130 mg, 244 μmol) and

K₂CO₃(156 mg, 1.13 mmol) were added into the solution. The vial

was capped and heated in a microwave reactor at 100 °C for 2 h.

Afterward, the reaction mixture was cooled to room temperature. The

reaction mixture was poured into 15 mL of water and a white

8660

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

precipitate appeared. After ﬁltration and drying, the white solid was

redissolved in TFA (2.0 mL, 26.9 mmol). The reaction mixture was

allowed to stir at room temperature for 2 min and then evaporated to

dryness in vacuo. The residue was adjusted to pH ∼8 by saturated

solution of sodium bicarbonate. The mixture was extracted with ethyl

acetate and the organic phase was washed with water and brine, dried

over anhydrous sodium sulfate (Na₂SO₄), and ﬁltered, and the ﬁltrate

was concentrated in vacuo. The residue was puriﬁed by chromatog-

raphy on silica gel with DCM/methanol (10:1) to aﬀord 34a as a

5.5 Hz, 1H), 3.96 (d, J = 4.3 Hz, 2H), 2.82 (d, J = 4.7 Hz, 3H), 2.64

(s, 3H), 2.44−2.33 (m, 1H), 1.03 (s, 3H), 1.02 (s, 3H). ¹³C NMR

(151 MHz, DMSO-d₆): δ 163.74, 161.96, 159.61, 158.96, 157.14,

151.20 (2C), 146.66, 137.45, 136.95, 122.54, 121.11, 118.96, 94.65,

94.40, 92.47, 59.66, 56.49, 49.06, 30.27, 29.86, 25.46, 21.30 (2C). MS

(ESI) m/z: 462.3 (M − H)⁻. HRMS (ESI): Anal. Calcd for

C₂₄H₂₅FN₇O₂[(M − H)⁻]: 462.2059. Found: 462.2051.

2-(1-(6-Fluoro-8-(methylamino)-2-(pyrazolo[1,5-a]pyrimidin-6-

yloxy)-9H-pyrimido[4,5-b]indol-4-yl)azetidin-3-ylidene)butan-1-ol

(38). Compound 38 (18 mg, 32% yield) was prepared from 36 (70

mg, 120 μmol), 32c (39 mg, 308 μmol), K₂CO₃(49.82 mg, 360.48

μmol), and TFA (2.0 mL, 26.9 mmol) in the same manner as

described for 34a. ¹H NMR (500 MHz, DMSO-d₆): δ 11.69 (s, 1H),

9.37 (s, 1H), 8.70 (s, 1H), 8.25 (s, 1H), 6.91 (d, J = 9.8 Hz, 1H), 6.81

(s, 1H), 6.30 (d, J = 11.8 Hz, 1H), 5.48 (s, 1H), 5.06 (s, 2H), 4.99 (s,

2H), 4.77 (s, 1H), 3.96 (s, 2H), 2.84 (s, 3H), 1.99 (d, J = 6.7 Hz,

2H), 1.00 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆): δ

162.46, 159.71, 158.83, 157.18, 148.41, 146.41, 145.60, 138.46,

137.00, 134.10, 128.76, 122.52, 121.12, 118.97, 96.91, 94.69, 94.47,

92.52, 60.75, 60.58, 59.76, 30.28, 22.49, 12.76. MS (ESI) m/z: 475.4

(M + H)⁺. HRMS (ESI): Anal. Calcd for C₂₄H₂₄FN₈O₂[(M + H)⁺]:

475.2001. Found: 475.2008.

4-(3-(1-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-N-methyl-

2-((2-methylpyrimidin-5-yl)thio)-9H-pyrimido[4,5-b]indol-8-amine

(42a). Compound 42a (62 mg, 61% yield) was prepared from 41a

(123 mg, 177 μmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine

hydrate (1.0 mL, 17.4 mmol) in the same manner as described for 26.

¹H NMR (500 MHz, DMSO-d₆): δ 11.58 (s, 1H), 8.87 (s, 2H), 7.92

(s, 3H), 6.88 (dd, J = 10.0, 1.7 Hz, 1H), 6.30 (dd, J = 12.1, 1.8 Hz,

1H), 5.05 (s, 2H), 4.89 (s, 2H), 3.39 (d, J = 5.3 Hz, 2H), 2.82 (s,

3H), 2.69 (s, 3H), 2.09 (q, J = 7.2 Hz, 2H), 1.00 (t, J = 7.5 Hz, 3H).

¹³C NMR (126 MHz, DMSO-d₆): δ 169.10, 165.22, 164.03 (2C),

162.19, 160.62−159.32, 157.36, 138.61, 132.06, 128.44, 125.44,

122.38, 120.01, 119.32, 116.98, 97.25, 95.93, 94.36, 61.41, 60.72,

39.76, 31.64, 27.35, 24.16, 13.77. MS (ESI) m/z: 465.3 (M + H)⁺.

HRMS (ESI): Anal. Calcd for C₂₃H₂₆FN₈S [(M + H)⁺]: 465.1980.

Found: 465.1990.

4-(3-(1-Aminobutan-2-ylidene)azetidin-1-yl)-2-((5-bromopyri-

din-2-yl)thio)-6-ﬂuoro-N-methyl-9H-pyrimido[4,5-b]indol-8-amine

(42b). Compound 42b (33 mg, 57% yield) was prepared from 41b

(83 mg, 109 μmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine

hydrate (1.0 mL, 17.4 mmol) in the same manner as described for 26.

¹H NMR (400 MHz, DMSO-d₆): δ 8.70 (s, 1H), 8.11 (s, 1H), 7.97

(s, 1H), 6.90 (s, 1H), 6.32 (s, 1H), 5.79 (s, 1H), 5.04 (s, 2H), 4.87 (s,

2H), 3.16 (s, 2H), 2.85 (s, 3H), 2.02 (s, 2H), 0.99 (s, 3H). ¹³C NMR

(126 MHz, DMSO-d₆): δ 163.36, 158.45, 155.85, 154.34, 150.67,

139.91, 137.22, 134.29, 131.19, 123.09, 121.04, 119.59, 118.78, 95.86,

94.54d, (J = 23.3 Hz), 92.88, 60.32, 59.62, 41.88, 30.22, 22.87, 12.71.

MS (ESI) m/z: 528.2 (M + H)⁺. HRMS (ESI): Anal. Calcd for

C₂₃H₂₄BrFN₇S [(M + H)⁺]: 528.0976. Found: 528.0988.

1

white solid (32 mg, 30% yield). H NMR (500 MHz, DMSO-d₆): δ

11.69 (s, 1H), 8.72 (s, 2H), 6.88 (dd, J = 10.1, 1.3 Hz, 1H), 6.31 (d, J

= 10.8 Hz, 1H), 5.54 (d, J = 4.4 Hz, 1H), 5.49−5.43 (m, 1H), 5.09 (s,

2H), 4.92 (s, 2H), 4.79 (d, J = 4.4 Hz, 1H), 4.27 (d, J = 5.4 Hz, 1H),

2.85 (d, J = 4.8 Hz, 3H), 2.67 (s, 3H), 1.17 (d, J = 6.4 Hz, 3H). ¹³C

NMR (126 MHz, DMSO-d₆): δ 163.74, 161.91, 159.67, 158.84,

157.20, 151.19 (2C), 146.65, 136.97, 128.16, 127.37, 121.15, 118.92,

94.73, 94.40, 92.52, 64.96, 60.45, 30.27, 25.46, 24.08. MS (ESI) m/z:

434.3 (M − H)⁻. HRMS (ESI): Anal. Calcd for C₂₂H₂₁FN₇O₂[(M −

H)⁻]: 434.1746. Found: 434.1745.

2-(1-(6-Fluoro-8-(methylamino)-2-((2-methylpyrimidin-5-yl)-

oxy)-9H-pyrimido[4,5-b]indol-4-yl)azetidin-3-ylidene)propan-1-ol

(34b). Compound 34b (32 mg, 20% yield) was prepared from 11

(200 mg, 376 μmol), 32b (139 mg, 931 μmol), K₂CO₃(156 mg, 1.13

mmol), and TFA (2.0 mL, 26.9 mmol) in the same manner as

described for 34a. ¹H NMR (400 MHz, DMSO-d₆): δ 11.75 (s, 1H),

8.72 (s, 2H), 6.90 (d, J = 10.4 Hz, 1H), 6.30 (d, J = 12.2 Hz, 1H),

5.61 (s, 1H), 5.04 (s, 2H), 4.93 (s, 2H), 4.89−4.77 (m, 1H), 3.91 (s,

2H), 2.84 (d, J = 4.7 Hz, 3H), 2.67 (s, 3H), 1.56 (s, 3H). ¹³C NMR

(126 MHz, DMSO-d₆): δ 163.69, 161.90, 159.63, 158.81, 157.14,

151.13 (2C), 146.65, 136.96, 128.62, 122.48, 121.12, 118.94, 94.68,

94.36, 92.43, 62.70, 60.39, 59.85, 30.23, 25.44, 14.71. MS (ESI) m/z:

434.3 (M − H)⁻. HRMS (ESI): Anal. Calcd for C₂₂H₂₁FN₇O₂[(M −

H)⁻]: 434.1746. Found: 434.1739.

2-(1-(6-Fluoro-8-(methylamino)-2-((2-methylpyrimidin-5-yl)-

oxy)-9H-pyrimido[4,5-b]indol-4-yl)azetidin-3-ylidene)butan-1-ol

(34c). Compound 34c (44 mg, 31% yield) was prepared from 11 (170

mg, 317 μmol), 32c (112 mg, 880 μmol), K₂CO₃(132 mg, 958

μmol), and TFA (2.0 mL, 26.9 mmol) in the same manner as

described for 34a. ¹H NMR (500 MHz, DMSO-d₆): δ 11.69 (s, 1H),

8.72 (s, 2H), 6.91 (d, J = 8.7 Hz, 1H), 6.36−6.12 (m, 1H), 5.54 (d, J

= 4.2 Hz, 1H), 5.02 (d, J = 36.5 Hz, 4H), 4.78 (t, J = 5.6 Hz, 1H),

3.97 (d, J = 4.8 Hz, 2H), 2.85 (d, J = 4.8 Hz, 3H), 2.67 (s, 3H), 2.00

(dd, J = 14.6, 7.3 Hz, 2H), 1.01 (t, J = 7.6 Hz, 3H). ¹³C NMR (126

MHz, DMSO-d₆): δ 163.74, 161.94, 159.69, 158.83, 157.17, 151.18

(2C), 146.67, 136.96, 134.08, 122.53, 121.13, 118.97, 94.71, 94.44,

92.50, 60.75, 60.59, 59.78, 30.28, 25.46, 22.48, 12.77. MS (ESI) m/z:

448.3 (M − H)⁻. HRMS (ESI): Anal. Calcd for C₂₃H₂₃FN₇O₂[(M −

H)⁻]: 448.1903. Found: 448.1901.

2-(1-(6-Fluoro-8-(methylamino)-2-((2-methylpyrimidin-5-yl)-

oxy)-9H-pyrimido[4,5-b]indol-4-yl)azetidin-3-ylidene)-3-methylbu-

tan-1-ol (34d). Compound 15 (250 mg, 568 μmol) and triethylamine

(172 mg, 1.70 mmol) were dissolved in 5 mL of NMP. The BOP

reagent (353 mg, 795 μmol) was added into the solution at 0 °C

under an argon atmosphere and the reaction mixture was stirred for

30 min. Then, intermediate 32d (146 mg, 1.04 mmol) dissolved in 5

mL of NMP was added dropwise to the mixture. The reaction mixture

was heated to 50 °C and stirred for 1 h. The reaction mixture was

poured into 15 mL of water and a white precipitate appeared. After

ﬁltration and drying, the white solid was redissolved in TFA (2.0 mL,

26.9 mmol). The reaction mixture was allowed to stir at room

temperature for 2 min and then evaporated to dryness in vacuo. The

residue was adjusted to pH ∼8 by saturated solution of sodium

bicarbonate. The mixture was extracted with ethyl acetate and the

organic phase was washed with water and brine, dried over anhydrous

sodium sulfate (Na₂SO₄), and ﬁltered, and the ﬁltrate was

concentrated in vacuo. The residue was puriﬁed by chromatography

on silica gel with DCM/methanol (10:1) to aﬀord 34d as a white

6-Fluoro-4-(3-(1-hydroxybutan-2-ylidene)azetidin-1-yl)-8-(meth-

ylamino)-N-(2-methylpyrimidin-5-yl)-9H-pyrimido[4,5-b]indole-2-

carboxamide (47). Compound 46 (70 mg, 191 μmol) and

triethylamine (57.85 mg, 572 μmol) were dissolved in 5 mL of

NMP. The BOP reagent (101 mg, 229 μmol) was added into the

solution at 0 °C under an argon atmosphere and the reaction mixture

was stirred for 30 min. Then, intermediate 32c (70 mg, 308 μmol)

dissolved in 5 mL of NMP was added dropwise to the mixture. The

reaction mixture was heated to 50 °C and stirred for 1 h. The reaction

mixture was poured into 15 mL of water and a white precipitate

appeared. The white crude product was obtained after ﬁltration and

dryness. It was puriﬁed by recrystallization in acetonitrile and

1

methanol to aﬀord compound 47 as a white solid. H NMR (500

MHz, DMSO-d₆): δ 12.45 (s, 1H), 10.69 (s, 1H), 9.16 (s, 2H), 7.00

(d, J = 9.5 Hz, 1H), 6.76 (s, 1H), 6.37 (d, J = 11.9 Hz, 1H), 5.22 (s,

2H), 5.14 (s, 2H), 4.87 (s, 1H), 4.00 (s, 2H), 2.89 (d, J = 3.2 Hz,

3H), 2.64 (s, 3H), 2.05 (d, J = 6.9 Hz, 2H), 1.04 (t, J = 7.1 Hz, 3H).

¹³C NMR (126 MHz, DMSO-d₆): δ 162.78, 162.18, 158.43, 157.93,

154.82, 152.88 (2C), 148.66, 137.24, 133.39, 131.23, 122.58, 122.31,

117.75, 97.94, 93.84, 92.58, 60.42, 60.12, 59.59, 29.50, 25.00, 21.98,

1

solid (53 mg, 24% yield). H NMR (600 MHz, DMSO-d₆): δ 11.66

(s, 1H), 8.69 (s, 2H), 6.86 (d, J = 9.9 Hz, 1H), 6.27 (d, J = 10.6 Hz,

1H), 5.50 (d, J = 3.9 Hz, 1H), 5.02 (d, J = 38.3 Hz, 4H), 4.69 (t, J =

8661

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1

12.35. MS (ESI) m/z: 499.2 (M + Na)⁺. HRMS (ESI): Anal. Calcd

for C₂₄H₂₅FN₈NaO₂[(M + Na)⁺]: 499.1977. Found: 499.1975.

2-(1-(5,6-Diﬂuoro-8-(methylamino)-2-((2-methylpyrimidin-5-yl)-

oxy)-9H-pyrimido[4,5-b]indol-4-yl)azetidin-3-ylidene)butan-1-ol

(61). Compound 61 (53 mg, 21% yield) was prepared from 53a (300

mg, 545 μmol), 32c (84 mg, 660 μmol), K₂CO₃(225 mg, 1.63

mmol), and TFA (2.0 mL, 26.9 mmol) in the same manner as

described for 34a. ¹H NMR (500 MHz, DMSO-d₆): δ 11.82 (s, 1H),

8.70 (s, 2H), 6.73−6.52 (m, 1H), 6.39 (dd, J = 13.6, 6.7 Hz, 1H),

5.34 (d, J = 4.8 Hz, 1H), 4.47 (dd, J = 15.4, 4.3 Hz, 4H), 4.19 (d, J =

5.5 Hz, 2H), 2.81 (d, J = 4.9 Hz, 3H), 2.64 (s, 3H), 2.11 (q, J = 7.5

Hz, 2H), 0.92 (t, J = 7.6 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆):

δ 163.35, 162.64, 158.41, 157.39, 155.90, 150.73 (2C), 146.14,

135.98, 132.57, 132.49, 127.67, 120.27, 108.48, 92.73, 92.10, 76.82,

76.66, 35.03, 30.11, 24.95, 17.62, 12.74. MS (ESI) m/z: 468.3 (M +

H)⁺. HRMS (ESI): Anal. Calcd for C₂₃H₂₄F₂N₇O₂[(M + H)⁺]:

468.1954. Found: 468.1962.

yellow solid (30 mg, 21% yield). H NMR (500 MHz, DMSO-d₆): δ

11.99 (s, 1H), 8.74 (s, 2H), 8.31 (s, 3H), 7.00 (dd, J = 10.0, 1.7 Hz,

1H), 6.40 (dd, J = 11.8, 1.8 Hz, 1H), 5.15 (s, 2H), 4.91 (s, 2H),

3.95−3.83 (m, 1H), 2.68 (s, 3H), 1.67 (s, 3H), 1.31 (d, J = 6.7 Hz,

3H). ¹³C NMR (126 MHz, DMSO-d₆): δ 163.58, 161.73, 160.53,

159.65, 158.69, 157.24, 151.07 (2C), 146.65, 128.51, 125.46, 121.78,

119.21, 95.47, 94.75, 92.12, 59.87, 59.64, 47.66, 29.46, 25.34, 17.40,

12.20. MS (ESI) m/z: 452.2 (M + H)⁺. HRMS (ESI): Anal. Calcd for

C₂₃H₂₃D₃FN₈O [(M + H)⁺]: 452.2396. Found: 452.2396. The ee

value was 92.0%. Chiral HPLC retention time 29.28 min; column:

CHIRALPAK IA column (250 mm × 4.6 mm, 5 μm); column

temperature 30 °C; ﬂow rate 0.5 mL/min; detection UV 254 nm;

mobile phase: solvent A (80%) = methanol + 0.1% DEA, solvent B

(20%) = ethanol + 0.1% DEA; total run time 40 min.

(S)-4-(3-(3-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-N-

(methyl-d₃)-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]-

indol-8-amine (63e). Compound 63e (33 mg, 23% yield) was

prepared from 53b (150 mg, 280 μmol), 62 (101 mg, 392 μmol),

K₂CO₃(116 mg, 840 μmol), TFA (2.0 mL, 26.9 mmol), and 85%

hydrazine hydrate (1.0 mL, 17.4 mmol) in the same manner as

4-(3-(1-Aminobutan-2-ylidene)azetidin-1-yl)-5,6-diﬂuoro-N-

methyl-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-

8-amine (63a). Compound 63a (190 mg, 38% yield) was prepared

from 53a (300 mg, 545 μmol), 16k (168 mg, 654 μmol), K₂CO₃(225

mg, 1.63 mmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine

hydrate (1.0 mL, 17.4 mmol) in the same manner as described for

1

described for 63d. H NMR (500 MHz, DMSO-d₆): δ 8.71 (s, 2H),

6.92 (dd, J = 10.3, 2.0 Hz, 1H), 6.29 (dd, J = 12.0, 2.0 Hz, 1H), 5.54

(s, 1H), 5.07 (s, 2H), 4.87 (s, 2H), 3.54 (dd, J = 13.0, 6.5 Hz, 1H),

2.67 (s, 3H), 1.55 (s, 3H), 1.09 (d, J = 6.6 Hz, 3H). ¹³C NMR (126

MHz, DMSO-d₆): δ 163.16, 161.36, 160.11, 159.12, 158.28, 156.60,

150.60 (2C), 146.10, 136.43, 132.01, 120.89, 118.40, 94.07, 93.78,

91.91, 59.54, 59.16, 48.20, 28.92, 24.90, 20.91, 11.80. MS (ESI) m/z:

452.2 (M + H)⁺. HRMS (ESI): Anal. Calcd for C₂₃H₂₃D₃FN₈O [(M

+ H)⁺]: 452.2396. Found: 452.2399. The ee value was 95.8%. Chiral

HPLC retention time 23.41 min; column: CHIRALPAK IA column

(250 mm × 4.6 mm, 5 μm); column temperature 30 °C; ﬂow rate 0.5

mL/min; detection UV 254 nm; mobile phase: solvent A (80%) =

methanol + 0.1% DEA, solvent B (20%) = ethanol + 0.1% DEA; total

run time 40 min.

1

18k. H NMR (500 MHz, DMSO-d₆): δ 8.74 (s, 2H), 6.46 (dd, J =

13.4, 6.2 Hz, 1H), 5.47 (d, J = 4.5 Hz, 1H), 5.01 (s, 2H), 4.92 (s, 2H),

3.25 (s, 2H), 2.83 (d, J = 4.7 Hz, 3H), 2.68 (s, 3H), 2.07−2.01 (m,

2H), 0.98 (t, J = 7.5 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆): δ

163.95, 161.86, 161.71, 158.29, 157.64, 151.20 (2C), 146.58, 132.59,

129.23, 127.82, 121.51, 118.96, 109.15, 93.52, 92.58, 60.94, 60.38,

60.22, 30.65, 25.47, 22.63, 12.51. MS (ESI) m/z: 467.2 (M + H)⁺.

HRMS (ESI): Anal. Calcd for C₂₃H₂₅F₂N₈O [(M + H)⁺]: 467.2114.

Found: 467.2104.

4-(3-(1-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-N-(meth-

yl-d₃)-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-8-

amine (63b). Compound 63b (70 mg, 33% yield) was prepared from

53b (250 mg, 467 μmol), 16k (215 mg, 840 μmol), K₂CO₃(194 mg,

1.40 mmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine hydrate

(R)-4-(3-(3-Aminobutan-2-ylidene)azetidin-1-yl)-N-ethyl-6-ﬂuo-

ro-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-8-

amine (63f). Compound 63f (29 mg, 10% yield) was prepared from

53c (360 mg, 659 μmol), 16r (253 mg, 988 μmol), K₂CO₃(273 mg,

1.98 mmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine hydrate

1

(1.0 mL, 17.4 mmol) in the same manner as described for 18a. H

NMR (500 MHz, DMSO-d₆): δ 8.72 (s, 2H), 6.91 (dd, J = 10.3, 2.2

Hz, 1H), 6.29 (dd, J = 12.1, 2.2 Hz, 1H), 5.53 (s, 1H), 5.08 (s, 2H),

4.94 (s, 2H), 3.16 (s, 2H), 2.67 (s, 3H), 2.03 (q, J = 7.5 Hz, 2H), 1.00

(t, J = 7.6 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆): δ 163.72,

161.92, 159.65, 158.83, 157.15, 151.16 (2C), 146.66, 136.98, 134.85,

122.46, 121.14, 118.96, 94.70, 94.45, 92.46, 60.29, 59.66, 49.06, 42.04,

25.45, 22.84, 12.74. MS (ESI) m/z: 450.4 (M − H)⁻. HRMS (ESI):

Anal. Calcd for C₂₃H₂₁D₃FN₈O [(M − H)⁻]: 450.2251. Found:

450.2244.

1

(1.0 mL, 17.4 mmol) in the same manner as described for 18a. H

NMR (500 MHz, DMSO-d₆): δ 8.68 (d, J = 43.6 Hz, 2H), 6.91 (s,

1H), 6.32 (s, 1H), 5.52 (s, 1H), 5.08 (s, 2H), 4.88 (s, 2H), 3.60 (s,

3H), 2.67 (s, 3H), 1.57 (s, 3H), 1.24 (s, 3H), 1.12 (s, 3H). ¹³C NMR

(126 MHz, DMSO-d₆): δ 163.71, 161.91, 160.60, 159.66, 158.78,

157.14, 151.16 (2C), 146.65, 135.91, 131.59, 122.68, 121.09, 94.61,

94.31, 92.74, 59.93, 59.66, 48.60, 38.04, 25.46, 20.92, 14.58, 12.33.

MS (ESI) m/z: 463.2 (M + H)⁺. HRMS (ESI): Anal. Calcd for

C₂₄H₂₈FN₈O [(M + H)⁺]: 463.2365. Found: 463.2371. The ee value

was 95.2%. Chiral HPLC retention time 31.07 min; column:

CHIRALPAK IA column (250 mm × 4.6 mm, 5 μm); column

temperature 30 °C; ﬂow rate 0.5 mL/min; detection UV 254 nm;

mobile phase: solvent A (80%) = methanol + 0.1% DEA, solvent B

(20%) = ethanol + 0.1% DEA; total run time 40 min.

1-(5-((4-(3-(1-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-8-

((methyl-d₃)amino)-9H-pyrimido[4,5-b]indol-2-yl)oxy)pyrimidin-2-

yl)ethan-1-ol (65). Compound 65 (65 mg, 24% yield) was prepared

from 64 (340 mg, 573 μmol), 16k (176 mg, 687 μmol), K₂CO₃(237

mg, 1.72 mmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine

hydrate (1.0 mL, 17.4 mmol) in the same manner as described for

18a. ¹H NMR (500 MHz, DMSO-d₆): δ 8.82 (s, 2H), 6.92 (d, J = 8.9

Hz, 1H), 6.30 (d, J = 10.9 Hz, 1H), 5.56 (s, 1H), 5.11 (s, 2H), 4.96

(s, 2H), 4.86 (q, J = 6.4 Hz, 1H), 3.19 (d, J = 18.1 Hz, 2H), 2.06 (dd,

J = 14.1, 6.8 Hz, 2H), 1.47 (d, J = 6.5 Hz, 3H), 1.01 (t, J = 7.5 Hz,

3H). ¹³C NMR (126 MHz, DMSO-d₆): δ 167.86, 161.29, 159.09,

158.25, 156.47, 150.58 (2C), 146.58, 136.40, 132.66, 123.48, 120.56,

118.30, 94.13, 93.83, 91.88, 69.46, 59.65, 59.01, 40.65, 28.84, 22.64,

22.22, 12.05. MS (ESI) m/z: 482.3 (M + H)⁺. HRMS (ESI): Anal.

Calcd for C₂₄H₂₅D₃FN₈O₂[(M + H)⁺]: 482.2502. Found: 482.2513.

MIC Testing. The MICs of the target compounds against Gram-

positive and Gram-negative bacteria were determined using

4-(3-(3-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-N-(meth-

yl-d₃)-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-8-

amine (63c). Compound 63c (78 mg, 37% yield) was prepared from

53b (250 mg, 467 μmol), 16p (215 mg, 840 μmol), K₂CO₃(194 mg,

1.40 mmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine hydrate

1

(1.0 mL, 17.4 mmol) in the same manner as described for 18a. H

NMR (500 MHz, DMSO-d₆): δ 8.72 (s, 2H), 6.91 (dd, J = 10.3, 2.1

Hz, 1H), 6.29 (dd, J = 12.0, 2.0 Hz, 1H), 5.56 (s, 1H), 5.06 (s, 2H),

4.87 (s, 2H), 3.54 (dd, J = 13.1, 6.5 Hz, 1H), 2.67 (s, 3H), 1.55 (s,

3H), 1.09 (d, J = 6.6 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆): δ

163.21, 161.40, 159.14, 158.34, 156.65, 150.65 (2C), 146.16, 136.49,

132.15, 121.17, 120.65, 118.46, 94.21, 93.93, 91.95, 59.58, 59.21,

48.27, 26.31, 24.95, 21.01, 11.85. MS (ESI) m/z: 452.3 (M + H)⁺.

HRMS (ESI): Anal. Calcd for C₂₃H₂₃D₃FN₈O [(M + H)⁺]: 452.2396.

Found: 452.2398.

(R)-4-(3-(3-Aminobutan-2-ylidene)azetidin-1-yl)-6-ﬂuoro-N-

(methyl-d₃)-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]-

indol-8-amine (63d). Compound 63d was prepared from 53b (150

mg, 280 μmol), 16r (101 mg, 392 μmol), K₂CO₃(116 mg, 840

μmol), TFA (2.0 mL, 26.9 mmol), and 85% hydrazine hydrate (1.0

mL, 17.4 mmol) in the same manner as described for 18a. It was

redissolved in 5 mL of methanol and 5 mL of 4 M HCl methanol

solution. After being stirred for 20 min, it was evaporated to dryness

and the hydrochloride form of compound 63d was obtained as a pale-

8662

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

levoﬂoxacin, meropenem (provided by Sichuan Primed Bio-Tech

Group Co., Ltd.), GP-1 racemate, and ceﬁderocol (synthesized

according to published procedures^21,34) as reference compounds.

MIC values were determined using the broth microdilution protocol

according to the methods of the Clinical and Laboratory Standards

Institute (CLSI). All of the tested compounds except for levoﬂoxacin

and meropenem (dissolved in H₂O) were dissolved in DMSO to

prepare a stock solution with a concentration of 64 μg/mL, and serial

twofold dilutions were prepared from the stock solutions by the

addition of culture broth to reach concentrations ranging from 64 to

Fisher Scientiﬁc), Compound Discoverer 3.0 software (Thermo

Fisher Scientiﬁc). The relative peak areas are determined from

extracted ion chromatograms of liver microsome samples at 60 min.

Rat Pharmacokinetic Studies In Vivo. The pharmacokinetic

parameters of compounds 18k, 18m, 18o, 18p, 31c, and 61 were

subjected to PK studies on male SD rats weighing between 180 and

280 g with three animals in each group. These tested compounds (5%

DMSO + 5% EtOH + 40% PEG300 + 50% saline) were administered

intravenously at a dose of 4 mg/kg. Serial specimens (0.3 mL) were

collected via the retrobulbar vein 0.05, 0.25, 0.75, 2.0, 4.0, 8.0, and 24

h after administration and quantiﬁed by LC/MS/MS. The

pharmacokinetic parameters of compounds 63c (5 mg/kg), 63d (5

mg/kg), 18r (5 mg/kg), and 18r (20 mg/kg) were subjected to PK

studies on male SD rats weighing between 180 and 300 g with three

animals in each group. These tested compounds (5% DMSO + 5%

solutol + 90% saline) were administered intravenously. Serial

specimens (0.2 mL) were collected via the retrobulbar vein 0.083,

0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 24 h after administration and

quantiﬁed by LC/MS/MS. Pharmacokinetic parameters were

calculated from the mean plasma concentration by noncompartmental

analysis. The protocol for this study was reviewed and approved by

the Institutional Animal Care and Use Committee of the Shanghai

Institute of Materia Medica, Chinese Academy of Sciences (Shanghai,

China).

In Vivo Neutropenic Mouse Thigh Infection Experiments.

Kunming mice (half male and half female) weighing between 18 and

22 g were used in this study with 8 mice in each administration group

and 13 mice in the vehicle control group. The animals were housed at

20−25 °C and 40−70% relative humidity, with food and water

available ad libitum throughout the study. The protocol for this study

was reviewed and approved by the Institutional Animal Care and Use

Committee of Sichuan Primed Bio-Tech Group Co., Ltd. Mice were

rendered neutropenic (neutrophils, less than 100/mm³) by injection

of cyclophosphamide intraperitoneally 4 days (150 mg/kg body

weight) and 1 day (100 mg/kg) before thigh infection. Thigh

infection in mice was carried out by intramuscular injection of 0.1 mL

of inoculum of MDR A. baumannii clinical isolate into the right

posterior thigh muscle (inoculum was prepared by inoculating single

colony into fresh MHB media the day prior to thigh infection and

cultured at 37 °C for 6 h. The overnight culture (the bacterial

suspension) was further diluted to a concentration of 10⁷cfu/mL in

fresh MHB media prior to use. Two hours post thigh infection,

compound 18r was administered at a dose of 10, 20, and 30 mg/kg

via intravenous tail injection, and levoﬂoxacin in the positive control

group was administered intravenously at a dose of 30 mg/kg. Two

hours post thigh infection, the vehicle control group was administered

intravenously equal volumes of saline, and 5 mice were sacriﬁced by

euthanasia. The thigh muscles were removed for cfu determination as

the data at 0 h. All other mice were sacriﬁced by euthanasia after 24 h

of therapy, and the thigh muscles were removed. Thigh muscles were

transferred into 1.5 mL sterile EP tubes and homogenized in 1 mL of

saline for subsequent cfu determination.

0.008 μg/mL. The tested organisms were incubated in Mueller-

̈

Hinton broth medium at 35−37 °C for 18−20 h, and then, the MIC

values were determined.

hERG K⁺Channel Inhibition Assay. Whole-cell recordings were

performed using automated QPatch (Sophion). The cells were

voltage-clamped at a holding potential of −80 mV. The hERG current

was activated by depolarizing at +20 mV for 5 s, after which the

current was brought back to −50 mV and kept for 5 s to remove the

inactivation and to observe the deactivating tail current. The

maximum amount of tail current was used to determine hERG

current amplitude. Compound stock solutions (10 or 30 mM in

DMSO) were prepared before the experiments. The stock solutions

were diluted to test concentrations upon use. After achieving a break-

in (whole-cell) conﬁguration, the cells were recorded for 120 s to

assess current stability. The voltage protocol described above was

then applied to the cells every 20 s throughout the procedure. Only

stable cells with recording parameters above threshold were allowed

for subsequent drug additions. External solution containing 0.1%

DMSO (vehicle) was applied to the cells to establish the baseline. The

current was allowed to stabilize for 3 min before the test compound

was added. The cells were kept in the test solution until the

compound’s eﬀect reached a steady state for a minimum of 3 min.

Drug washout was performed with an external solution until the

recovery of the current reached a steady state. Cisapride was used as

the positive control. Data were analyzed using Assay software

provided by Sophion, XLFit, or GraphPad Prism 6.0.

Molecular Docking. Small molecules were prepared using the

̈

LigPrep (version 2.4, Schrodinger, LLC, New York, NY, 2010). The

protonation states of molecules were generated with Epik.³⁵The

crystal structure of GP-1 in complex with DNA Gyrase B (PDB id:

4KFG) was used for protein preparation and grid generation before

docking. The docking procedure was performed using the Glide

̈

module of Maestro software (Glide, version 6; Schrodinger LLC, New

York, NY, 2010) with the standard precision mode (SP). The binding

interaction was analyzed and displayed by PyMOL (version 1.8).

Metabolic Stability Assay. The assay was performed using liver

microsomes from rats and humans. The test compounds (ﬁnal

concentration of 0.1 μM in 0.01% DMSO with 0.005% bovine serum

albumin) were incubated with live microsomes [0.33 mg/mL in 0.1 M

tris(hydroxymethyl)-aminomethane/hydrochloric acid buﬀer (pH

7.4), cofactor MgCl₂(5 mM), and reduced nicotinamide adenine

dinucleotide phosphate (NADPH, 1 mM)] at 37 °C for 60 min.

Aliquots were sampled at 0, 7, 17, 30, and 60 min, respectively, and

methanol (cold in 4 °C) was added to terminate the reaction. After

centrifugation (4000 rpm, 5 min), samples were then analyzed by

liquid chromatography/tandem mass spectrometry (LC−MS/MS).

The metabolic stability of the compounds is presented as the in vitro

half-life (T_1/2), clearance (Cl_int), and metabolic bioavailability (MF %)

in rat and human liver microsomes as previously described.^36,37

Metabolite Identiﬁcation Assay. The assay was performed

using liver microsomes from male SD rats. The test compound was

preincubated in an incubation mixture consisted of 1 mg of

microsomal protein/mL rat liver microsomes, 2 mM NADP, and 5

mM MgCl₂in a total volume of 200 μL of potassium phosphate buﬀer

(100 mM, pH 7.4) for 5 min at 37 °C. Aliquots were sampled at 0, 30,

and 60 min, respectively, and 400 μL of cold acetonitrile (0.1% formic

acid) was added to terminate the reaction. After centrifugation for 15

min at 16,000g, samples were then analyzed by UHPLC-MS/MS.

Data are acquired using Xcalibur v4.1 software (Thermo Fisher

Scientiﬁc) and processed using Xcalibur v4.1 software (Thermo

Cellular Cytotoxicity Assay. HEK293 cells and L02 cells were

plated in the 96-well plates at a density of 1 × 10⁴cells per well for 48

h. Then, the cells were incubated with the test articles at diﬀerent

concentrations (1−200 μM) for another 48 h (n = 6). A Cell

Counting Kit 8 (CCK 8) purchased from Yeasen Biotech Co., Ltd.

(Shanghai, China) was used for the cytotoxicity assay with 10 μL of

CCK 8 being added to each well for 2 h. The absorbance was

measured using an automatic microplate reader (Biotek, Winooski,

VT, USA) at a wavelength of 450 nm. The CC₅₀values for each

compound were calculated using GraphPad Prism 7.0 software

(GraphPad Software Inc., La Jolla, CA, USA) and are shown as the

mean value.

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00621.

8663

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

ABBREVIATIONS

More MIC data and details of tested compounds for

Tables 1, 3, and 4; more MIC data and details of tested

compounds for Table 5; metabolite identiﬁcation of

compound 18k in rat liver microsomes; proposed

metabolic pathways of compound 18k; preparation

and characterization data of all intermediates; NMR

spectra and HRMS data of ﬁnal compounds; and HPLC

traces for lead compounds (PDF)

■

AMR, antimicrobial resistance; G⁺, Gram-positive; G⁻, Gram-

negative; ESBLs, extended-spectrum β-lactamases; mCPBA, 3-

chloroperoxybenzoic acid; Phth, phthaloyl; MeI, methyl

iodide; PPh₃, triphenylphosphine; HATU, 2-(7-azabenzotria-

zol-1-yl)-N,N,N′,N′-tetramethyluronium hexaﬂuorophosphate;

CS₂, carbon disulﬁde; DEA, diethylamine; MDR, multidrug-

resistant; MSSA, methicillin-sensitive Staphylococcus aureus;

MSSE, methicillin-sensitive Staphycoccus epidermidis; MRSE,

methicillin-resistant Staphylococcus epidermidis; Eco, Escherichia

coli; Kpn, Klebsiella pneumoniae; Aba, Acinetobacter baumannii;

Pae, Pseudomonas aeruginosa; MF, metabolic bioavailability;

SD, Sprague−Dawley; PK, pharmacokinetics; cfu, colony-

forming units; PDB, Protein Data Bank

Molecular formula strings (CSV)

AUTHOR INFORMATION

■

Corresponding Author

Yushe Yang − State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China; School of Pharmacy,

University of Chinese Academy of Sciences, Beijing 100049,

China; orcid.org/0000-0002-1007-2492; Phone: +86-

21-50806786; Email: ysyang@mail.shcnc.ac.cn

REFERENCES

■

(1) Watkins, R. R.; Bonomo, R. A. Overview: Global and Local

Impact of Antibiotic Resistance. Infect. Dis. Clin. North Am. 2016, 30,

313−322.

(2) O’Neill, J.; Grande-Bretagne; Trust, W. Antimicrobial Resist-

ance: Tackling a Crisis for the Health and Wealth of Nations. Review

on Antimicrobial Resistance, 2014.

(3) Shrivastava, R.; Chng, S.-S. Lipid Trafficking across the Gram-

Negative Cell Envelope. J. Biol. Chem. 2019, 294, 14175−14184.

(4) Li, X.-Z.; Plésiat, P.; Nikaido, H. The Challenge of Efflux-

Mediated Antibiotic Resistance in Gram-Negative Bacteria. Clin.

Microbiol. Rev. 2015, 28, 337−418.

(5) Zgurskaya, H. I. An Old Problem in a New Light: Antibiotic

Permeation Barriers. ACS Infect. Dis. 2020, 6, 3090−3091.

(6) Kingwell, K. New Antibiotic Hits Gram-Negative Bacteria. Nat.

Rev. Drug Discovery 2018, 17, 785.

Authors

Qidi Kong − State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China; School of Pharmacy,

University of Chinese Academy of Sciences, Beijing 100049,

China

Wei Pan − State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences,

Shanghai 201203, China

Heng Xu − School of Pharmaceutical Science and Technology,

Hangzhou Institute for Advanced Study, University of

Chinese Academy of Sciences, Hangzhou 310024, China

Yaru Xue − State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences,

Shanghai 201203, China

Bin Guo − State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences,

Shanghai 201203, China

(7) Lewis, K. The Science of Antibiotic Discovery. Cell 2020, 181,

29−45.

̀

(8) Schillaci, D.; Spano, V.; Parrino, B.; Carbone, A.; Montalbano,

A.; Barraja, P.; Diana, P.; Cirrincione, G.; Cascioferro, S.

Pharmaceutical Approaches to Target Antibiotic Resistance Mecha-

nisms. J. Med. Chem. 2017, 60, 8268−8297.

(9) Tacconelli, E.; Carrara, E.; Savoldi, A.; Harbarth, S.; Mendelson,

M.; Monnet, D. L.; Pulcini, C.; Kahlmeter, G.; Kluytmans, J.; Carmeli,

Y.; Ouellette, M.; Outterson, K.; Patel, J.; Cavaleri, M.; Cox, E. M.;

Houchens, C. R.; Grayson, M. L.; Hansen, P.; Singh, N.;

Theuretzbacher, U.; Magrini, N.; Aboderin, A. O.; Al-Abri, S. S.;

Awang Jalil, N.; Benzonana, N.; Bhattacharya, S.; Brink, A. J.; Burkert,

F. R.; Cars, O.; Cornaglia, G.; Dyar, O. J.; Friedrich, A. W.; Gales, A.

C.; Gandra, S.; Giske, C. G.; Goff, D. A.; Goossens, H.; Gottlieb, T.;

Guzman Blanco, M.; Hryniewicz, W.; Kattula, D.; Jinks, T.; Kanj, S.

S.; Kerr, L.; Kieny, M.-P.; Kim, Y. S.; Kozlov, R. S.; Labarca, J.;

Laxminarayan, R.; Leder, K.; Leibovici, L.; Levy-Hara, G.; Littman, J.;

Malhotra-Kumar, S.; Manchanda, V.; Moja, L.; Ndoye, B.; Pan, A.;

Xin Meng − State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences,

Shanghai 201203, China

Cheng Luo − State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China; School of Pharmaceutical

Science and Technology, Hangzhou Institute for Advanced

Study, University of Chinese Academy of Sciences, Hangzhou

310024, China; orcid.org/0000-0003-3864-8382

Ting Wang − Department of Microbiology, Sichuan Primed

Bio-Tech Group Company, Limited, Chengdu 610041,

Sichuan Province, China

Paterson, D. L.; Paul, M.; Qiu, H.; Ramon-Pardo, P.; Rodríguez-Bano,

̃

J.; Sanguinetti, M.; Sengupta, S.; Sharland, M.; Si-Mehand, M.; Silver,

L. L.; Song, W.; Steinbakk, M.; Thomsen, J.; Thwaites, G. E.; van der

Meer, J. W.; Van Kinh, N.; Vega, S.; Villegas, M. V.; Wechsler-Fördös,

A.; Wertheim, H. F. L.; Wesangula, E.; Woodford, N.; Yilmaz, F. O.;

Zorzet, A. Discovery, Research, and Development of New Antibiotics:

The Who Priority List of Antibiotic-Resistant Bacteria and Tuber-

culosis. Lancet Infect. Dis. 2018, 18, 318−327.

Shuhua Zhang − Department of Microbiology, Sichuan

Primed Bio-Tech Group Company, Limited, Chengdu

610041, Sichuan Province, China

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00621

(10) Hiasa, H. DNA Topoisomerases as Targets for Antibacterial

Agents. In DNA Topoisomerases: Methods and Protocols; Drolet, M.,

Ed.; Springer New York: New York, NY, 2018; pp 47−62.

(11) Mayer, C.; Janin, Y. L. Non-Quinolone Inhibitors of Bacterial

Type Iia Topoisomerases: A Feat of Bioisosterism. Chem. Rev. 2014,

114, 2313−2342.

(12) Laponogov, I.; Sohi, M. K.; Veselkov, D. A.; Pan, X.-S.;

Sawhney, R.; Thompson, A. W.; McAuley, K. E.; Fisher, L. M.;

Sanderson, M. R. Structural Insight into the Quinolone −DNA

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The authors thank ﬁnancial support from the National Natural

Science Foundation of China (no. 81872726).

8664

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Cleavage Complex of Type Iia Topoisomerases. Nat. Struct. Mol. Biol.

2009, 16, 667−669.

(26) Keating, T. A.; Lister, T.; Verheijen, J. C. New Antibacterial

Agents: Patent Applications Published in 2011. Pharm. Pat. Anal.

2014, 3, 87−112.

(13) Stokes, S. S.; Vemula, R.; Pucci, M. J. Advancement of Gyrb

Inhibitors for Treatment of Infections Caused by Mycobacterium

Tuberculosis and Non-Tuberculous Mycobacteria. ACS Infect. Dis.

2020, 6, 1323−1331.

̌

(27) Gjorgjieva, M.; Tomasic, T.; Barancokova, M.; Katsamakas, S.;

̌

Ilas, J.; Tammela, P.; Peterlin Masic, L.; Kikelj, D. Discovery of

Benzothiazole Scaffold-Based DNA Gyrase B Inhibitors. J. Med. Chem.

2016, 59, 8941−8954.

(14) Bisacchi, G. S.; Manchester, J. I. A New-Class Antibacterial 

Almost. Lessons in Drug Discovery and Development: A Critical

Analysis of More Than 50 Years of Effort toward Atpase Inhibitors of

̌

(28) Tiz, D. B.; Skok, Z.; Durcik, M.; Tomasic, T.; Masic, L. P.; Ilas,

J.; Zega, A.; Draskovits, G.; Révész, T.; Nyerges, A.; Pál, C.; Cruz, C.

D.; Tammela, P.; Zigon, D.; Kikelj, D.; Zidar, N. An Optimised Series

́

̌

DNA Gyrase and Topoisomerase Iv. ACS Infect. Dis. 2015, 1, 4−41.

̌

of Substituted N-Phenylpyrrolamides as DNA Gyrase B Inhibitors.

Eur. J. Med. Chem. 2019, 167, 269−290.

(29) Bensen, D.; Chen, Z.; Finn, J.; Lam, T. T.; Lee, S. J.; Li, X.;

Phillipson, D. W.; Tari, L. W.; Trzoss, M.; Zhang, J. Tricyclic Gyrase

Inhibitors. U.S. Patent 9,732,083 B2, 2017.

(30) Grant, E. B.; Macielag, M. J.; Xu, X.; Paget, S. D.; Weidner-

Wells, M. A. 7-Amino Alkylidenyl-Heterocyclic Quinolones and

Naphthyridones. WO 2005033108 A1, 2005.

(31) McGarry, D. H.; Cooper, I. R.; Walker, R.; Warrilow, C. E.;

Pichowicz, M.; Ratcliffe, A. J.; Salisbury, A.-M.; Savage, V. J.; Moyo,

E.; Maclean, J.; Smith, A.; Charrier, C.; Stokes, N. R.; Lindsay, D. M.;

Kerr, W. J. Design, Synthesis and Antibacterial Properties of

Pyrimido[4,5-B]Indol-8-Amine Inhibitors of DNA Gyrase. Bioorg.

Med. Chem. Lett. 2018, 28, 2998−3003.

(32) Gant, T. G. Using Deuterium in Drug Discovery: Leaving the

Label in the Drug. J. Med. Chem. 2014, 57, 3595−3611.

(33) Kong, Q.; Yang, Y. Recent Advances in Antibacterial Agents.

Bioorg. Med. Chem. Lett. 2021, 35, 127799.

(34) Aoki, T.; Yoshizawa, H.; Yamawaki, K.; Yokoo, K.; Sato, J.;

Hisakawa, S.; Hasegawa, Y.; Kusano, H.; Sano, M.; Sugimoto, H.;

Nishitani, Y.; Sato, T.; Tsuji, M.; Nakamura, R.; Nishikawa, T.;

Yamano, Y. Cefiderocol (S-649266), a New Siderophore Cepha-

losporin Exhibiting Potent Activities against Pseudomonas Aeruginosa

and Other Gram-Negative Pathogens Including Multi-Drug Resistant

Bacteria: Structure Activity Relationship. Eur. J. Med. Chem. 2018,

155, 847−868.

(35) Shelley, J. C.; Cholleti, A.; Frye, L. L.; Greenwood, J. R.;

Timlin, M. R.; Uchimaya, M. Epik: a software program for pK a

prediction and protonation state generation for drug-like molecules. J.

Comput.-Aided Mol. Des. 2007, 21, 681−691.

(36) Obach, R. S. Prediction of Human Clearance of Twenty-Nine

Drugs from Hepatic Microsomal Intrinsic Clearance Data: An

Examination of in Vitro Half-Life Approach and Nonspecific Binding

to Microsomes. Drug Metab. Dispos. 1999, 27, 1350.

̌

(15) Skok, Z.; Barancoková, M.; Benek, O.; Cruz, C. D.; Tammela,

̌

P.; Tomasic, T.; Zidar, N.; Masic, L. P.; Zega, A.; Stevenson, C. E. M.;

Mundy, J. E. A.; Lawson, D. M.; Maxwell, A.; Kikelj, D.; Ilas, J.

̌

Exploring the Chemical Space of Benzothiazole-Based DNA Gyrase B

Inhibitors. ACS Med. Chem. Lett. 2020, 11, 2433−2440.

(16) Dennie, J.; Vandell, A. G.; Inoue, S.; Gajee, R.; Pav, J.; Zhang,

G.; Zamora, C.; Masuda, N.; Uchiyama, M.; Yamada, M.; Senaldi, G.

A Phase I, Single-Ascending-Dose Study in Healthy Subjects to Assess

the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of

Ds-2969b, a Novel Gyrb Inhibitor. J. Clin. Pharmacol. 2018, 58,

1557−1565.

(17) Vandell, A. G.; Inoue, S.; Dennie, J.; Nagasawa, Y.; Gajee, R.;

Pav, J.; Zhang, G.; Zamora, C.; Masuda, N.; Senaldi, G. Phase 1 Study

to Assess the Safety, Tolerability, Pharmacokinetics, and Pharmaco-

dynamics of Multiple Oral Doses of Ds-2969b, a Novel Gyrb

Inhibitor, in Healthy Subjects. Antimicrob. Agents Chemother. 2018,

62, No. e02537-17.

̌

(18) Durcik, M.; Tomasic, T.; Zidar, N.; Zega, A.; Kikelj, D.; Masic,

̌

L. P.; Ilas, J. Atp-Competitive DNA Gyrase and Topoisomerase Iv

Inhibitors as Antibacterial Agents. Expert Opin. Ther. Pat. 2019, 29,

171−180.

(19) Basarab, G. S.; Hill, P. J.; Garner, C. E.; Hull, K.; Green, O.;

Sherer, B. A.; Dangel, P. B.; Manchester, J. I.; Bist, S.; Hauck, S.;

Zhou, F.; Uria-Nickelsen, M.; Illingworth, R.; Alm, R.; Rooney, M.;

Eakin, A. E. Optimization of Pyrrolamide Topoisomerase Ii Inhibitors

toward Identification of an Antibacterial Clinical Candidate

(Azd5099). J. Med. Chem. 2014, 57, 6060−6082.

(20) Tari, L. W.; Li, X.; Trzoss, M.; Bensen, D. C.; Chen, Z.; Lam,

T.; Zhang, J.; Lee, S. J.; Hough, G.; Phillipson, D.; Akers-Rodriguez,

S.; Cunningham, M. L.; Kwan, B. P.; Nelson, K. J.; Castellano, A.;

Locke, J. B.; Brown-Driver, V.; Murphy, T. M.; Ong, V. S.; Pillar, C.

M.; Shinabarger, D. L.; Nix, J.; Lightstone, F. C.; Wong, S. E.;

Nguyen, T. B.; Shaw, K. J.; Finn, J. Tricyclic Gyrb/Pare (Tribe)

Inhibitors: A New Class of Broad-Spectrum Dual-Targeting

Antibacterial Agents. PLoS One 2013, 8, No. e84409.

(21) Finn, J.; Tari, L. W.; Chen, Z.; Zhang, J.; Phillipson, D.; Lee, S.

J.; Trzoss, M.; Bensen, D.; Li, X.; Teng, M.; Ong, V.; Borchardt, A. J.;

Lam, T. T. Tricyclic Gyrase Inhibitors. WO 2015038661 A1, 2015.

(22) Dey, F.; Hu, Y.; Liu, Y.; Lin, X.; Shen, H.; Shi, H.; Tan, X.;

Vercruysse, M.; Yan, S.; Zhou, C.; Zhou, M. Novel Pyrido[2,3-

B]Indole Compounds for the Treatment and Prophylaxis of Bacterial

Infection. WO 2018178041 A1, 2018.

(37) Lau, Y. Y.; Krishna, G.; Yumibe, N. P.; Grotz, D. E.; Sapidou,

E.; Norton, L.; Chu, I.; Chen, C.; Soares, A. D.; Lin, C. C. The Use of

in Vitro Metabolic Stability for Rapid Selection of Compounds in

Early Discovery Based on Their Expected Hepatic Extraction Ratios.

Pharm. Res. 2002, 19, 1606−1610.

(23) Hu, Y.; Shi, H.; Zhou, M.; Ren, Q.; Zhu, W.; Zhang, W.; Zhang,

Z.; Zhou, C.; Liu, Y.; Ding, X.; Shen, H. C.; Yan, S. F.; Dey, F.; Wu,

W.; Zhai, G.; Zhou, Z.; Xu, Z.; Ji, Y.; Lv, H.; Jiang, T.; Wang, W.; Xu,

Y.; Vercruysse, M.; Yao, X.; Mao, Y.; Yu, X.; Bradley, K.; Tan, X.

Discovery of Pyrido[2,3-B]Indole Derivatives with Gram-Negative

Activity Targeting Both DNA Gyrase and Topoisomerase Iv. J. Med.

Chem. 2020, 63, 9623−9649.

(24) Ho, S. Y.; Wang, W.; Ng, F. M.; Wong, Y. X.; Poh, Z. Y.; Tan,

S. W. E.; Ang, S. H.; Liew, S. S.; Joyner Wong, Y. S.; Tan, Y.; Poulsen,

A.; Pendharkar, V.; Sangthongpitag, K.; Manchester, J.; Basarab, G.;

Hill, J.; Keller, T. H.; Cherian, J. Discovery of Dual Gyrb/Pare

Inhibitors Active against Gram-Negative Bacteria. Eur. J. Med. Chem.

2018, 157, 610−621.

(25) Richter, M. F.; Drown, B. S.; Riley, A. P.; Garcia, A.; Shirai, T.;

Svec, R. L.; Hergenrother, P. J. Predictive Compound Accumulation

Rules Yield a Broad-Spectrum Antibiotic. Nature 2017, 545, 299−

304.

8665

https://doi.org/10.1021/acs.jmedchem.1c00621

J. Med. Chem. 2021, 64, 8644−8665

Article Doi

Design, Synthesis, and Biological Evaluation of Novel Pyrimido[4,5-b]indole Derivatives against Gram-Negative Multidrug-Resistant Pathogens

DOI: 10.1021/acs.jmedchem.1c00621

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.1c00621

Full text of DOI:10.1021/acs.jmedchem.1c00621

Products guided by the article

R&D Labs maybe for 849701-76-4

Relevant to this article

Hot Product