Structure-Based Design of Highly Potent Toll-like Receptor 7/8 Dual Agonists for Cancer Immunotherapy

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Structure-Based Design of Highly Potent Toll-like Receptor 7/8 Dual

Agonists for Cancer Immunotherapy

Zhisong Wang,^∥Yan Gao,^∥Lei He,^∥Shuhao Sun, Tingting Xia, Lu Hu, Licheng Yao, Liangliang Wang,

Dan Li, Hui Shi, and Xuebin Liao *

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ABSTRACT: Activation of the toll-like receptors 7 and 8 has emerged as a promising strategy for cancer immunotherapy. Herein,

we report the design and synthesis of a series of pyrido[3,2-d]pyrimidine-based toll-like receptor 7/8 dual agonists that exhibited

potent and near-equivalent agonistic activities toward TLR7 and TLR8. In vitro, compounds 24e and 25a signiﬁcantly induced the

secretion of IFN-α, IFN-γ, TNF-α, IL-1β, IL-12p40, and IP-10 in human peripheral blood mononuclear cell assays. In vivo,

compounds 24e, 24m, and 25a signiﬁcantly suppressed tumor growth in CT26 tumor-bearing mice by remodeling the tumor

microenvironment. Additionally, compounds 24e, 24m, and 25a markedly improved the antitumor activity of PD-1/PD-L1

blockade. In particular, compound 24e combined with the anti-PD-L1 antibody led to complete tumor regression. These results

demonstrated that TLR7/8 agonists (24e, 24m, and 25a) held great potential as single agents or in combination with PD-1/PD-L1

blockade for cancer immunotherapy.

broaden the clinical responses to immune checkpoint blocking

therapy.^7,9,10Many recent studies have shown that anti-PD-1

monotherapies might be insuﬃcient,^4,7,11which suggests that

researchers need to develop complementary treatment modal-

ities. A novel approach to improve the antitumor eﬀect of the

PD-1/PD-L1 blockade is to enhance tumor immunogenicity

via activation of toll-like receptors (TLRs), which can convert

the immunosuppressive tumor microenvironment into a T cell-

inﬂamed tumor microenvironment and then sensitize tumor

cells to immune attack.¹¹⁻¹⁴Accordingly, several TLR

agonists, including TLR1/2 agonist (Diprovocim),¹⁵TLR7

agonist (1V270),¹⁶and TLR9 agonist (SD-101),¹⁷have shown

synergistic eﬀects with anti-PD-1 antibodies to treat cancers

that are insensitive to the single anti-PD-1 antibody. Notably,

INTRODUCTION

■

Cancer immunotherapies are becoming powerful clinical

strategies to treat various cancers.¹⁻⁴Many immunotherapeu-

tic approaches are based on the blockade of immune inhibitory

receptors (IRs) found on activated T cells, such as

programmed cell death 1 (PD-1), T cell immunoglobulin

and mucin domain 3, and cytotoxic T-lymphocyte-associated

protein 4.^2,5The expression of IRs on the T cell surface is

sustained at high levels in a variety of tumor types or chronic

diseases, causing the progressive suppression of eﬀector

function of activated T cells.^6,7Thus, targeted blockade of

the negative immune regulatory checkpoints to unleash

activated T cells has emerged as a highly promising therapeutic

approach. In particular, multiple studies have demonstrated

that PD-1/PD-L1 blockade can induce substantial antitumor

immune responses and long-term remission in cancer

patients.^3,8

Received: February 1, 2021

Published: May 28, 2021

However, increasing clinical evidence has indicated that the

PD-1/PD-L1 blockade was only eﬀective in a subset of

carcinoma patients.^4,6Thus, a current focus in the ﬁeld of

cancer immunotherapy is on how to predict, enhance, and

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SD-101 combined with pembrolizumab has already been in

phase 2 clinical trials for treating advanced melanoma

(NCT03007732).

immunomodulating agents for use in diﬀerent therapeutic

areas, including viral infections (e.g., hepatitis B virus and

human immunodeﬁciency virus), autoimmune diseases, and

cancers.^26,27Nevertheless, the prospect of TLR7 and TLR8

agonist-based cancer immunotherapy is yet to be realized

because it is hampered either by the limited antitumor eﬃcacy

or adverse immune eﬀects.²⁸Recently, much eﬀort has been

devoted to the discovery of novel TLR7 and 8 agonists or

formulations with improved antitumor activities and reduced

toxicities.²⁹⁻³³For the purpose of extending scope of immuno-

oncology applications and enhancing opportunities of TLR7/8

agents for combination with or conjugation to immune

checkpoint antibodies, we developed a series of TLR7/8

dual agonists with signiﬁcant improvements of in vivo

activities, through elaborate studies of structure−activity

relationship (SAR) and structure−property relationship.

Human TLRs (hTLRs: 1−10) are a class of important

pattern recognition receptors in innate immunity that are

widely expressed on immune and epithelial cells.^18,19Although

most TLRs are expressed on the cell surface, TLR7 and 8 are

located in the endosome and mainly recognize uridine- and

guanosine-rich single-stranded RNA (ssRNA) within the

cytoplasm.^18,20Activation of TLR7 and 8 can induce cytokine

secretion through MyD88−NF-κB-dependent signaling cas-

cades, resulting in mobilization of innate and adaptive immune

responses.^12,13,20To note, TLR8 is widely distributed in more

subsets of immune cells than TLR7 (Figure 1).^21,22Due to

RESULTS AND DISCUSSION

■

Rational Design. TLRs 7 and 8 are phylogenetically close

and structurally similar members of the TLR family, sharing

high homology and similarity in sequences (Figure S1),

structures (Figure S2A,B), and functions (both sensing

ssRNA).^18,19,34In addition, the residues at active sites of

both receptors for binding small molecules are almost identical

(Figure S2C),^35,36thus we reasoned that generating similar

binding aﬃnities for both receptor isoforms was feasible. The

rationale for designing the novel TLR7/8 dual agonists with

close EC₅₀values was based on insights gained from

crystallographic studies of TLR7/8 and SAR studies on

pyridine- and pyrimidine-type TLR7/8 dual agonists (Figure

2).^24,37,38Starting from these structures, pyrimidin-2-amine

was chosen as the core unit, wherein the N-1 atom and the

NH₂group at the C-2 position were involved in critical

hydrogen bond interactions with residues Asp555/543 and

Thr586/574 of TLR7/8. The hydrogen bond donor and

acceptor pattern of pyrimidin-2-amine was ﬁxed, and then we

added an aromatic ring across the 5,6-bond to form a bicyclic

core system stacked on the side chain of Phe408/405 of

TLR7/8 by empirical structure optimization. As privileged

scaﬀolds, the motifs of quinazolin-2-amine and pyrido[3,2-

d]pyrimidin-2-amine, which feature high ligand eﬃciency and

drug-like properties, are present widely in drug or drug

candidates, particularly in antihepatitis C virus and anticancer

agents.^39,40Compared to the quinazolin-2-amine, pyrido[3,2-

Figure 1. Distributions of hTLR7 and hTLR8.

their heterogeneous distributions, simultaneous engagement of

TLR7 and 8 may compensate for one another to exert eﬀective

antitumor immune responses, resembling Bacillus Calmette−

Guerin as a multifunctional agent activating TLR2, TLR4, and

TLR9.²³Speciﬁcally, activation of TLR7 in plasmacytoid

dendritic cells (pDCs) leads to the secretion of type I

interferon-α (IFN-α) and engagement of TLR8 in monocytes

and myeloid DCs preferentially results in the production of

type II interferon (IFN-γ), tumor necrosis factor α (TNF-α),

and IL-12.^24,25These immunomodulatory cytokines are

important for inducing antitumor immune responses.

Given their crucial role in immunomodulation, TLR7 and 8

have been attractive targets in the design and development of

Figure 2. Structure-based design of novel TLR7/8 agonists.

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a

Scheme 1. Synthesis of Compounds 13a, 14a and 15a−g

a

Reagents and conditions: (i) NBS, acetonitrile, room temperature (rt), 30 min; (ii) chloroformamidine hydrochloride, methyl sulfone, 140 °C, 3

h; (iii) Ac₂O, 140 °C, 12 h; (iv) (COCl)₂, N,N-dimethylformamide (DMF), dioxane, rt, overnight; (v) alkylamines, diisopropylethylamine (DIEA),

tetrahydrofuran (THF), rt, 3 h; (vi) alkyl alcohols or alkylthiols, THF, NaH, 0 °C, 2 h; (vii) LiOH·H₂O, MeOH, rt, 3 h; (viii) Pd(PPh₃)₄, Cs₂CO₃,

dioxane/H₂O, boronic acids, 100 °C, 6 h; and (ix) benzyl bromides, Zn, Ni(PPh₃)₂Cl₂, THF, rt, 16 h.

a

Scheme 2. Synthesis of Compounds 17a−aa and 18

a

Reagents and conditions: (i) (2-bromoethyl)benzenes, Zn, Ni(PPh₃)₂Cl₂, THF, rt, 16 h; (ii) (E)-styrylboronic acid, Pd(PPh₃)₄, Cs₂CO₃,

dioxane/H₂O, 100 °C, 6 h; (iii) benzyl bromides, Zn, Ni(PPh₃)₂Cl₂, THF, rt, 16 h; (iv) benzyl bromides, Zn, Pd(amphos)Cl₂, THF, rt, 16 h; and

(v) LiOH·H₂O, THF/H₂O = 2:1, rt, 12 h.

d]pyrimidin-2-amine contained additional solubilizing function

and reduced hERG binding aﬃnity,^39,41we thus prioritized it

as the parent nucleus. Subsequently, a chlorine atom was

inserted at the C-6 position of N⁴-butylpyrido[3,2-d]-

pyrimidine-2,4-diamine to give compound 5 as a starting

point for further structural derivatization and optimization,

which displayed EC₅₀values of 2600 and 2000 nM for TLR7

and 8, respectively. Brieﬂy, our design strategy aimed to

decorate the novel scaﬀold architecture with the four desired

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a

Scheme 3. Synthesis of Compounds 21−25

a

Reagents and conditions: (i) LiAlH₄, THF, 0 °C, 2 h; (ii) SOCl₂, dichloromethane (DCM), 0 °C, 1 h; (iii) P(OEt)₃, 140 °C, 16 h; (iv) TMSBr,

DCM, 0 °C−rt, 5 h; (v) di-tert-butyl iminodicarboxylate, K₂CO₃, MeOH, 45 °C, 5 h; (vi) TFA, DCM, 0 °C, 3 h; (vii) NaN₃, dimethyl sulfoxide

(DMSO), rt, 10 h; (viii) trimethylamine, MeOH, reﬂux, 6 h; (ix) K₂CO₃, MeOH, reﬂux, 5 h; and (x) alkylamines, K₂CO₃, MeOH, 45 °C, 5 h.

a

Scheme 4. Synthesis of Compounds 26a−c

a

Reagents and conditions: (i) HATU, DIEA, alkylamines, DMF, rt, 3 h.

a

Scheme 5. Synthesis of Compounds 33−34 and 36−37

a

Reagents and conditions: (i) benzyl bromides, Zn, Ni(PPh₃)₂Cl₂, THF, rt, 16 h; (ii) LiAlH₄, THF, 0 °C, 2 h; (iii) SOCl₂, DCM, 0 °C, 1 h; and

(iv) alkylamines, K₂CO₃, MeOH, 45 °C, 5 h.

pharmacophore elements, the N-1 atom, C-2 amine, C-4 alkyl

group, and the pendant aromatic substituent (Figure 2).

Chemistry. The synthetic strategies developed for the

preparations of target compounds are illustrated in Schemes

1−5 and S1 −S3. Our synthetic routes started with

commercially available methyl 3-aminopicolinate 6. After

treatment with N-bromosuccinimide (NBS), 6 was brominated

to aﬀord bromide 7. The following cyclization of 7 with

chloroformamidine hydrochloride occurred to provide the key

intermediate 8. The amine group of 8 was then treated with

acetic anhydride at 140 °C to aﬀord the protected compound

9. Subsequently, chlorination of compound 9 with oxalyl

chloride aﬀorded 10, which rendered the potentials to be

modiﬁed through two heteroaryl C−Cl. Under the basic

conditions, the alkyl amine, alkyl alcohol, and alkyl thiol could

be installed at the C-4 position in dichloride 10 to aﬀord 11a−

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g. Using lithium hydroxyl, the acetyl group in 11a−g was

removed to aﬀord pyrimidin-2-amines 12a−g, which served as

the coupling partner for subsequent diversiﬁcations. Separately,

the Suzuki coupling of 12a−g with corresponding boronic

acids provided subseries 13a and 14a, while the Negishi

coupling with (bromomethyl)benzene formed subseries 15a−g

(Scheme 1). The preparation of compound 16a is described in

Scheme S1 of Supporting Information. Moreover, intermediate

12a underwent cross-coupling reactions to give the subseries

17a−aa, with diﬀerent aromatic substituents at the C-6

position of pyrido[3,2-d]pyrimidine core (Scheme 2).

Table 1. Exploration of the Core Scaﬀold and C-4

Substituent

a

With methyl esters 17u−w and 18 in hand, the following

attempts were made to decorate the phenyl moiety to expand

our chemical diversity (Scheme 3). In the presence of LiAlH₄,

esters 17u−w and 18 were converted to corresponding

alcohols 19a−f. Subsequently, chlorination with thionyl

chloride readily formed chlorides 20a−f. By treating with

triethyl phosphite, compound 20a was then converted into

phosphatide 21, and 21 was hydrolyzed by bromotrimethylsi-

lane (TMSBr) to aﬀord phosphoric acid 22. Nucleophilic

substitution of benzyl chloride 20a and 20c with NH(Boc)₂,

followed by triﬂuoroacetyl (TFA)-mediated deprotection

aﬀorded the primary amines 24a and 24g, respectively.

Compound 24b was obtained from 20a through a direct

azidation reaction. The intermediates 20a−f were treated with

the corresponding alkylamines in the presence of K₂CO₃to

aﬀord the desired compounds 24c−e, 24h−i, 24k−m, and

25a−c. Reaction of 20a with trimethylamine in methanol

provided compound 24f. Also, compound 20a was reﬂuxed in

methanol to give compound 24j. As shown in Scheme 4,

carboxylic acid 17aa was converted to desired amides 26a−c

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

hexaﬂuorophosphate (HATU)-mediated amidation with alkyl-

amines. In addition, starting from 12a, the synthesis

procedures for compounds 33−34 and 36−37 were the

same as those for compounds 24c−e (Scheme 5). The

preparation of compounds 27 and 35 is described in Schemes

S2 and S3 of Supporting Information, respectively.

Structure−Activity Relationship. Systematic SAR stud-

ies were performed in HEK293 cells stably cotransfected with

the hTLR7 or hTLR8 expression vector and a secreted

embryonic alkaline phosphatase (SEAP) reporter gene. We

focused our SAR campaign on investigating the core scaﬀolds,

the alkyl groups at the C-4 position, the aromatic substituents

at the C-6 position and, to a lesser extent, the linkers between

ring B and ring C. Initially, the installation of phenyl ring

substituents at the C-6 position of compound 5 without a

linker through Suzuki-coupling (13a, 14a) completely

abrogated TLR7/8 agonistic activities, presumably due to

steric clashes. Thus, further modiﬁcations incorporated at the

aryl ring of 13a were not pursued. Subsequently, benzyl was

coupled to compound 5 through Negishi-coupling to aﬀord

15a, which further improved both TLR7 and 8 activities

(EC₅₀: 1.18 and 0.95 μM, respectively) compared to lead

compound 5 (Table 1). However, the following replacement of

the C-8 carbon atom in compound 15a with nitrogen (16a)

resulted in signiﬁcant attenuation in both potencies and area

under curve in dose-escalation studies. Therefore, we further

identiﬁed the pyrido[3,2-d]pyrimidine as a core scaﬀold.

After the core scaﬀold was determined, we proceed to

investigate the alkyl substitution at the C-4 position of the

pyrido[3,2-d]pyrimidine ring (Table 1). Compared with

compound 15a, its 2-methoxyethyl analogue 15b showed

a

EC₅₀: concentration for 50% of the maximal eﬀect. EC₅₀values were

averaged when determined in three independent experiments, each

carried out in triplicate.

comparable agonistic activities toward both TLR7 and TLR8.

The replacement of the C-4 nitrogen atom in compound 15a

by O and S (15c−d) resulted in remarkable loss of potencies,

indicating that C-4 nitrogen is essential for eﬀective binding to

TLR7 and 8. Based on the result, we reasoned that the

heteroatoms at the C-4 position aﬀected the pK_aof the N-1

atom resulting from the opposite electronic eﬀect, which was

an underlying determinant of TLR7/8 binding aﬃnities in the

series. The pK_avalues of the N-1 atoms in 15a, 15c, and 15d

were predicted using program Epik implemented in Maestro

(Schrodinger 2019.01). The N-1 atoms of 15c and 15d

42

̈

cannot be eﬀectively protonated in endosome, where pH is

generally stable between pH 4 and pH 6. However, the same

atom in 15a could be protonated and a key ionic hydrogen

interaction with the negatively charged carboxyl group of Asp

555 [pK_a(Asp) = 2.77] of TLR7 is shown in Figure 3. The

hypothesis was supported by the ligand-binding model of

pteridinone core-based GS-9620 with TLR7 (Figure S3).⁴³

Furthermore, the N⁴-methyl analogue (15e) of 15a was also

bereft of activities without reducing the pK_avalue of the N-1

atom, suggesting that the sec-amine might serve as a hydrogen

bond donor and play a key role in the orientation of the N⁴-

butyl chain. In addition to the chain alkyl groups, we also

attempted structural modiﬁcations of this moiety with larger

substituents, such as the morpholine (15f) and piperidine

groups (15g). A signiﬁcant loss in activities of TLR7 and 8 was

observed, suggesting that this moiety was intolerant to cyclic

alkyl chains. Thus, n-butylamine at the C-4 position was ﬁxed

in the subsequent structural optimization.

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Figure 3. Schematic representation of interaction mode between the pyrimidin-2-amine scaﬀold and TLR7. The LRR domains of TLR7 and 8

responsible for ligand recognition are located within endosome.

To further deﬁne the optimal length of the alkyl spacer

between the aromatic substitutions (ring C) and the parent

nucleus, ethyl spacer analogue 17a with the same substituent as

15a was obtained by Negishi coupling. Although there was no

signiﬁcant decrease in agonistic activity toward TLR7,

compound 17a showed 1.6-fold reductions in TLR8-

stimulatory activity compared to 15a (Table 2). Furthermore,

the introduction of a methyl ester group at the opposite

position of the benzene ring of compound 17a resulted in a

signiﬁcant decrease of both TLR7 and TLR8 agonistic

activities (17b), probably caused by steric clashes. Notably,

when the rigid ethylene unit served as a linker, no signiﬁcant

activities to either TLR 7 or 8 were observed (17c).

Intriguingly, the insertion of a single methyl group into the

−CH₂− spacer of compound 15a brought about TLR8

selectivity with an EC₅₀value of 1127 nM (17d). The docking

model showed that the introduced methyl sterically clashed

with the Tyr356 residue of TLR7 (Figure. S4), and thus

perturbed a functional ligand−protein interaction. These

results indicated that methylene is an optimal linker between

the phenyl group and the parent nucleus for maintaining the

dual agonistic activities on TLR7 and TLR8.

Table 2. Exploration of the Pendant Substituent at the C-6

Position

Replacement of the phenyl ring of 15a by naphthalene

aﬀorded compound 17e (TLR7, EC₅₀= 2150 nM, Table 2),

which caused the dramatic decrease of TLR8 activity. This

result indicated that strategically organized terminal aromatic

groups were pivotal in conferring selectivity at TLR7 other

than modiﬁcations of the N⁴-substituent identiﬁed by previous

studies,^21,24presumably via increasing the entropic penalty

when binding to the active pocket of TLR8. We next

performed a SAR study to explore each position of the phenyl

ring of compound 15a by introducing diversiﬁed substituents

aiming to investigate the eﬀects of the steric and electronic

environment around the phenyl ring on the activity and

selectivity toward TLR7/8; the results are shown in Table 2.

As expected, the substituent position in the phenyl ring was

biologically relevant. For example, compound containing the

ortho-F-substituted phenyl ring (17f) was more potent in both

TLR7 and TLR8 activities than its meta- and para-substituted

analogues (17g, 17h). Compounds 17f and 17h showed

equivalent potencies on TLR7 and TLR8, however, compound

17g displayed a 2.4-fold selectivity over TLR8. The strongly

electron withdrawing triﬂuoromethyl-substituted 17i−k

showed signiﬁcant drops in potencies as compared to that of

the corresponding F-substituted derivatives 17f−h. In

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Table 3. Structural Derivatization and Optimization of 17w/aa

a

b

Calculated by the ACD/Percepta platform (http://www.ilab.acdlabs.com). Calculated by free online software (https://www.molinspiration.

com/).

particular, meta-CF₃analogue 17j almost lost both TLR7- and

8-stimulatory activities. In addition, the introduction of cyano

into the benzene ring of compound 15a had a less eﬀect on its

activities. Among them, the para-cyano analogue 17n exhibited

the most potent agonistic activities with EC₅₀values of 1.20

μM (TLR7) and 0.66 μM (TLR8), respectively.

The methyl analogues 17o−q showed a slight reduction in

the agonistic activities compared to 15a, and the position of

the substituent has little eﬀect on the agonistic activities.

Introduction of the methoxy group into the phenyl ring of

compound 15a aﬀorded regioisomers 17r−t, wherein 17r

showed a moderate TLR7 aﬃnity (EC₅₀= 630 nM) and ∼5.5-

fold target selectivity over TLR8, compound 17t showed more

potent and close activities toward TLR7 and 8 compared to

regioisomers 17r and 17s. We further explored the

consequences of incorporating the methyl ester group into

the phenyl ring, the para-analogue 17w exhibited the best

activities among the three regioisomers. The des-methyl

analogue 17aa was slightly more potent than 17w, however,

the ortho- and meta-carboxylic acid analogues (17y, 17z) were

feebly active. From the given data illustrated in examples 17f−

aa, it appeared that both the steric and electron eﬀects had

impacts on TLR7 and 8 activity and selectivity, and the

electron-donating substituents at the benzene ring may

contribute to improve ligand-binding aﬃnity as compared to

electron-withdrawing substituents, which further validated the

SAR trend.

The para-substituted methyl ester 17w and carboxylic acid

17aa were selected for further derivatization on account of

their equivalent potencies toward TLR7 and 8, with EC₅₀

values in the low micromolar and submicromolar range,

respectively (Table 3). Through hydrogenation−reduction of

17w, further optimization for potencies was achieved in benzyl

alcohol 19c with EC₅₀values of 300 nM (TLR7) and 124 nM

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Table 4. Structural Expansion of Compound 24e

(TLR8), respectively. To explore its potential as a cancer

vaccine adjuvant, the phosphonate group was incorporated

into compound 19c for adsorption to aluminum hydroxide.⁴⁴

First, the diethyl phosphonate analogue 21 was synthesized

from 20c, and its EC₅₀was 400 nM for TLR7 and 500 nM for

TLR8. However, the hydrolysis of 21 yielded des-diethyl

compound 22, which resulted in substantial loss in stimulatory

activities, because the unsubstituted phosphonic acid group

increased its aqueous equilibrium solubility and then restricted

its passive permeability. To acquire the optimal alkyl chain

spacer between the phosphonate and pharmacophore, a

diverse set of ethylene glycol and methylene units should be

further investigated.

amine moieties, including pyrrole (24k) and morpholine (24l).

Compared with 24e, although slight reductions in potencies

were observed in compounds 24k and 24l, their activities were

still potent enough to highlight the signiﬁcance of the basic

tertiary amine moieties. It has been reported that acidiﬁcation

and maturation of endosome is indispensable for ligands to

activate the TLR7 receptor.⁴⁵Compared with compound 24j

without a basic center, the tertiary amine derivatives 24e, 24k,

and 24l within endosome generated a high degree of

protonation due to endosomal acidiﬁcation, and therefore

reduced their own eﬄux.⁴⁵As a result, their residence time

within this cellular compartment was extended, which

potentially led to a higher concentration of such compounds

around TLR7/8 receptors.

The conversion of the hydroxyl group of 19c to a basic

primary amino group (24a) resulted in an 8-fold decrease in

TLR8 agonistic activity and a 4-fold decrease in TLR7-

stimulatory activity. The azidation of 24a (24b) further

induced the loss of agonistic activities, especially for TLR8-

stimulatory activity. Methylation of NH₂of 24a (24c) induced

a 2.4−5-fold increase in TLR7/8 activities relative to 24a, but

its strong electron-withdrawing triﬂuoromethyl analogue 24d

displayed markedly attenuated potencies relative to 24c.

Encouragingly, a dramatic enhancement of potencies was

achieved through further methylation of secondary amine in

24c. The compound 24e containing dimethylamine moiety

exhibited the most potent activities among the tested

compounds with near-equipotent TLR7 and TLR8 agonistic

activities (EC₅₀: 0.024 and 0.010 μM, respectively). However,

further methylation of tertiary amine 24e caused a more than

200-fold decrease in activities (24f). To investigate the eﬀects

of the substitution position of dimethylaminomethyl structure

at the phenyl ring, we synthesized the ortho- and meta-

regioisomers (24h, 24i). Compound 24h displayed a slightly

reduced and almost equal TLR7 and 8 aﬃnities relative to 24e

(EC₅₀: 70 and 59 nM, respectively). Compound 24i showed

equipotent but further reduced TLR7 and 8 activities, with

EC₅₀values of 110 nM for TLR7 and 102 nM for TLR8. These

results revealed that the p-substitution was the optimal

substitution pattern on the phenyl ring for potencies in the

series of TLR7/8 agonists.

Although the introduction of the basic group was important

to improve agonistic activities, we found that the agonistic

activities of TLR7/8 ligands were not positively correlated with

the strength of the base. For example, the primary amine 24a

with a higher basic pK_avalue (cpK_a, 9.2) was dramatically less

potent than tertiary amine 24e (cpK_a, 8.5). In addition, the

secondary amine 24c (cpK_a, 9.6) exhibited over a 30-fold

decrease in potency as compared to 24e. Within the tertiary

amine analogues, the pK_avalue of 24k (cpK_a, 9.0) was higher

than that of 24e, but 24e was more potent than 24k.

Moreover, compound 24m, with similar steric hindrance of the

substituent but reduced basicity relative to 24e, exhibited

comparable activities to compound 24e (EC₅₀: 27 nM for

TLR7 and 12 nM for TLR8, respectively).

Although a bit less potent than 24e, the N⁴-2-methoxyethyl

variant 25a was also signiﬁcantly active with EC₅₀values of 40

nM for TLR7 and 23 nM for TLR8, respectively. This result

suggested that the 2-methoxyethyl moiety was tolerated on the

alkyl side chain region in the series of TLR7/8 agonists. More

importantly, compared with 24e (clog P: 3.70; aqueous

solubility: 45 μg/mL), 25a showed an obvious increase in

hydrophilic eﬃciency (clog P: 2.24; aqueous solubility: 1029

μg/mL). As predicted, replacing the basic tertiary amines with

amide functionalities (26a−c) led to 3−5-fold decline in

potencies, but moved these analogs into a more hydrophilic

physicochemical space.

To further deﬁne the SARs, we preformed more structural

modiﬁcations on the compound 24e (Table 4). We

synthesized the quinazolin-2-amine counterpart 27, which

was designed through bioisosteric replacement of the N-5

atom of 24e by a CH unit. Compared to 24e, 27 showed over

The methoxymethyl derivative 24j was markedly 8-fold less

potent than 24e in cellular assays, which indicated the

requirement of the basic tertiary amine moieties to maintain

highly potent TLR7/8 agonism. The observation prompted us

to examine the potential of more sterically hindered cyclic

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Figure 4. SAR summary of pyrido[3,2-d]pyrimidine-based TLR7/8 dual agonists.

̈

Figure 5. Schrodinger-proposed binding modes for 24e (yellow) in the R848 binding site of macaca mulatta TLR7 (PDB ID: 5GMH; upper) and

hTLR8 (PDB ID: 3W3N; lower). The dimerization partners of proteins are colored red (protomer B) and green (protomer A), respectively. The

side views of TLR7 dimers and TLR8 dimers are presented in the left panel and the close-ups of 24e binding in the ligand-binding pockets are

presented in the middle and right panels. Hydrogen bonding, salt bridge, and π−π stacking are represented by black-, purple-, and brilliant blue-

dashed lines, respectively. The distance between the N-4 and N-5 atoms is 2.75 Å.

a 10-fold decrease in potencies and a 0.80 unit increase in the

clog P value, with an EC₅₀value of 300 nM for TLR7 and 128

nM for TLR8, respectively. We reasoned that the N-5 atom in

the scaﬀold formed a unique intramolecular hydrogen bond

(IMHB) with the adjacent −NH− group (N−N 2.75 Å),

which was supported by work reported by Aprahamian.⁴⁶The

IMHB resulted in the formation of pseudo-ﬁve-membered

ring, which would stabilize the coplanar orientation and then

make the n-butylamine side chain more productively bind into

the deep hydrophobic pocket, while at the same time reducing

the ligand entropy resulting from the conformation restric-

tions. Another possible reason for the increased activities of

24e was that the introduction of nitrogen into the aromatic

system led to a greater π−π stacking interaction energy with

Phe405/Phe408 at the bottom of the active pocket. The result

reinforced the importance of the nitrogen atom as a versatile

design element for lead optimization.

The ethyl spacer analogue 33 of 24e showed about 5-fold

reductions in activities compared to 24e, suggesting that the

aromatic substituent and the parent nucleus should be better

linked via a one-carbon unit rather than a zero- or two-carbon

unit. It is possible that the pendant substituent linked by one-

carbon unit interfered less with the preferential conformation

of the N⁴-alkyl chain bound to the active site, and to some

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Figure 6. Concentration−agonism curves for pyrido[3,2-d]pyrimidine analogues 24e and 25a in hTLR7 and 8−NF-κB−SEAP reporter gene assays

(A: hTLR7; C: hTLR8). EC₅₀values were estimated based on the ﬁtted curve (B: hTLR7; D: hTLR8). The normalized means standard

deviation determined on triplicates are shown. R848 was selected as a reference TLR7/8 dual agonist. High concentrations of test compounds

resulted in regression of NF-κB induction without detectable cytotoxicity (E: hTLR7; F: hTLR8).

extent, it might preferably interact with the surface of the

pocket to improve the binding aﬃnities. In addition, we

obtained compound 34 through extending the linker between

the dimethylamine and the benzene ring by one carbon length.

Diﬀerent from compound 33, 34 displayed comparable

agonistic activities to compound 24e. However, further moving

the NMe₂group away by introducing an ethoxy linker

(compound 35) resulted in an obvious decrease in potencies,

although it is not clear what the individual contributions of the

linker length and the O atom. We also attempted to introduce

additional substituents on the benzene ring of 24e. Methoxy

was introduced into the ortho- and meta-position of the

benzene ring to aﬀord compounds 36 and 37, respectively.

Compound 36 showed a slight increase in TLR7-stimulatory

activity, but a 2.6-fold decrease in TLR8 agonistic activity. The

TLR7 and TLR8 agonistic activities of compound 37 were

decreased by 2-fold and 5-fold, respectively. This result

suggested that an additional small substituent was tolerated

at the ortho-position of the benzene ring of compound 24e.

Taken together, through our SAR eﬀort, we discovered these

attributes below were pivotal for enhancing TLR7/8 agonistic

activities: (i) the basicity of the N-1 atom was crucial for

endosomal protonation and H-bond formation with Asp555/

543; (ii) introduction of tertiary basic amine was favorable to

enhance agonistic activities as a result of increased residence

time within endosome; (iii) introduction of the N-5 atom

formed an IMHB that mimicked the tricyclic TLR7/8 agonists

(e.g., R848) to stabilize the coplanar orientation and restrict

the ﬂexibility of the N⁴-alkyl side chain; and (iv) the optimal

spacer linking the bicyclic core and the pendant phenyl ring

was one methylene. Finally, we brieﬂy summarized our SAR

studies, as shown in Figure 4.

(Maestro 11.9).⁴⁷Analogous to the reference compound

R848 (Figure 5), the bicyclic ring system of 24e was involved

in π−π stacking arrangement with Phe408/405 in TLR7/8,

and the C-2 NH₂made a bifurcated hydrogen bond to strictly

conserved aspartate residue Asp555/543 in TLR7/8 and the

backbone carbonyl of Thr586/574 (TLR7/8) as a H donor.

The N-1 atom in 24e was protonated and then formed an

important ionic hydrogen bond with the crucial residue

Asp555/543 in TLR7/8, and the N-3 atom directly hydrogen

bound to Thr586/574. Additionally, the N⁴-butyl ﬁt well into

the deep hydrophobic pocket encompassed by the residues

Phe349/Thr406/Val381 in TLR7 (Phe346/Ile403/Gly376 in

TLR8). Diﬀerent from R848 without the pendant substituent,

the C-6-substituted ((dimethylamino)methyl)benzyl moiety

projected ipsilaterally with N⁴-butyl, extended across the

surface of neighboring protomer B.The highly optimized

dimethylamine eﬃciently engaged in a H-bonding interaction

with Gly354/Gln351, and thus resulted in more extensive

contacts and better target occupancies to TLR7 and 8, which

might confer additional binding aﬃnities to this compound.

This induced-ﬁt docking models for 24e in two receptors

revealed that the compound kept similar binding poses and

protein−ligand contacts in both isoforms, providing the

structural basis for the dual targeting activation. Likewise,

compounds 24m and 34 were docked into the active sites of

TLR7 and TLR8. As shown in Figure S5A,B, the cycloalkyl-

substituted tertiary amine in 24m could also form hydrogen

bonds with residues Gly354/Gln351of TLR7/8. As shown in

Figure S5C,D, compound 34 with an ethyl linker between the

dimethylamine and the benzene ring kept the H-bond

interaction between protonated dimethylamine and residues

Gly354/Gln351, to some extent, which explained why

compound 34 displayed comparable agonistic activities to

compound 24e.

Molecular Docking. 24e was “induced-ﬁt” docked into the

R848 binding sites identiﬁed in the MmTLR7 (PDB 5GMH)

̈

and hTLR8 (PDB 3W3N) using Schrodinger software

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Figure 7. Representative cytokine/chemokine induction from the 41-analyte multiplexed immunoassay. Human PBMCs cultured at 2 × 10⁶cells/

mL were stimulated for 12 h with various concentrations of TLR7/8 dual agonists. Means of duplicates are shown. EC_max: drug concentration for

maximum induction of cytokine.

Biological Evaluation. As shown in Figure 6, compounds

24e and 25a exhibited more potent activation of NF-κB in

HEK-Blue hTLR7 and 8 cells than R848. The compounds

were also evaluated in other homologous HEK-Blue hTLRs (2,

3, 4, and 9) and HEK-Blue Null2-k cells, and no NF-κB

activation was detected (Figure S6). To investigate the

cytokine/chemokine proﬁles induced by these TLR7/8 dual

agonists and the corresponding dose−response relationships,

human peripheral blood mononuclear cells (hPBMCs) from a

single donor were stimulated with escalating concentrations of

24e, 24l, 25a, 25c, and R848 (R848: 0.5−64 μg/mL; 24e, 24l,

25a, and 25c: 0.1−32 μg/mL) using a 41-analyte multiplexed

immunoassay platform. In line with their high potencies for

TLR7 and 8, this series of TLR7/8 agonists strongly induced

the production of systemic proinﬂammatory cytokines and

chemokines such as TNF-α, INF-γ, IL-1β, IL-6, IL-8, IL-12,

and MIP-1α (CCL3), as well as promoted production of IFN-

α2 and IFNα-regulated chemokine IP-10 (CXCL10). In

analogy with a previous study reported by David,⁴⁸these

TLR7/8 dual agonists induced a biphasic dose−response that

higher concentrations of compounds resulted in retrogressive

cytokine production.

As detailed in Figure 7, 24e/l and 25a/c stimulation induced

a characteristic cytokine/chemokine proﬁle of TLR7 and 8

activation in hPBMCs. The potencies [EC_max: 0.1−0.5 μg/mL

for IL-1β, IL-6, IL-8, IL-10, IL-12p40, INF-γ, TNF-α, MIP-1α,

IP-10, monocyte chemoattractant protein-1 (MCP-1), and G-

CSF] of 24e markedly surpassed that of R848 (EC_max: 2−4

μg/mL). As we know, IFN-γ and IL-12 as key cytokine

signatures in the crosstalk between T cells and DCs were

required for antitumor immune responses to checkpoint

blockade.⁴⁹The series of compounds were found to potently

induce Th1-biasing cytokines IFN-γ and IL-12p40 with

remarkably low doses, which implied their potential to enhance

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Figure 8. Cytokine production by compounds 24e, 25a, and R848 (0.5 μg/mL) after 24 h incubation with hPBMCs (3 × 10⁶cells/mL) from ﬁve

healthy donors. One-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test was used for comparisons. *P < 0.05 and

****P < 0.0001.

the antitumor immune responses of PD-1/PD-L1 blockade. In

addition, these compounds signiﬁcantly triggered the secretion

of Th17-skewing cytokine IL-1β, proinﬂammatory cytokine IL-

6, anti-inﬂammatory cytokine IL-10, and VEGF (Figure S7). It

was reported that TLR7 or 8 activation-mediated upregulation

of PD-L1 on tumor cells was associated with production of

these cytokines.^23,24CXCL10 (IP-10), functioned as a major

chemoattractant for T helper 1 (Th-1) cells and natural killer

cells to attract their inﬁltrations into tumor microenviron-

ment,^50,51could also be signiﬁcantly induced by our

compounds. Most strikingly, these TLR7/8 agonists upregu-

lated the expression of MCP-1, which was known to be

induced by type I interferon.⁵²

These TLR7/8 agonists only induced relatively low levels of

IFN-α2 in the assay (Figure S8), which may be ascribed to the

low frequency (1−2%) of DCs population within hPBMCs.⁵³

Another possible reason was that cytokine induction may vary

from donor to donor. To further conﬁrm the cytokine proﬁles

of these compound-treated hPBMCs, compounds 24e, 25a,

and R848 were tested in hPBMCs from multiple donors. As

detailed in Figure 8, compounds 24e and 25a signiﬁcantly

induced the production of type I interferon (IFN-α) and type

II interferon (IFN-γ), as well as other cytokines described in

Figure 7.

model, which is a model for nonhypermutated/microsatellite-

instable colorectal cancer (MSI-CRC) and less immunogenic

than the MC38 model (hypermutated/MSI-CRC).⁵⁴When

the size of subcutaneous tumors reached a mean size of 100

mm³, the BALB/c mice were randomly sorted into eight

groups (n = 5), and intraperitoneally administered vehicle

control and indicated immunomodulators, respective to each

dose. As shown in Figure 9, anti-PD-1 antibodies alone did not

signiﬁcantly reduce tumor growth, but all the three TLR7/8

agonists (R848, 24e, and 25a) evidently delayed tumor growth

compared to anti-PD-1 antibody monotherapy. As a single

agent, 24e resulted in stronger tumor growth inhibition than

R848 (P < 0.01) and even R848 plus anti-PD-1 antibody

treatment, with a tumor growth inhibition (TGI) of >96.5%.

Compound 25a showed a comparable antitumoral therapeutic

eﬀects with R848. When combined with anti-PD-1 antibodies,

24e, 25a, and R848 induced further reductions in tumor

volume (TV) than did TLR agonist monotherapies. Notably,

mice treated with 24e in combination with anti-PD-1

antibodies had more pronounced reduced tumor sizes

compared to mice treated with either agent alone, to some

extent, achieved the regression of CT26 tumors. However,

initial synergistic analysis of the tumor size showed no

signiﬁcant synergistic eﬀect of 24e plus anti-PD-1 antibodies.

We then investigated the antitumor activity of half dose of 24e

as a single agent or in combination with anti-PD-1 antibodies.

The results showed that the combination of 24e (12.5 μg) and

anti-PD-1 antibodies synergistically suppressed the growth of

CT26 tumor (P = 0.003) (Figure S9).

The results of the multiplexed immunoassay showed that

our compounds can elicit strong immunostimulatory activities.

To further explore the in vivo activities of these TLR7/8

agonists on tumor growth, the most potent compound 24e in

terms of TLR7 agonistic activity and compound 25a with a

good balance between in vitro potencies and drug-like property

were investigated in the established CT26 tumor mouse

A moderate loss of body weight was observed in the mice

administered with 24e (approximately 0.9 g on day 4) or with

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Figure 9. In vivo antitumor eﬀects of single or combined treatment with 24e (25 μg, once weekly), 25a (25 μg), R848 (25 μg), and the anti-PD-1

antibody or the anti-PD-L1 antibody (200 μg, twice weekly). The time points for drug administration are indicated by vertical arrows (T: TLR

agonist; P: α-PD-1). TV (A,D−H, n = 5 per group), body weight (B,I), and survival rate (C, n = 10 per group) of CT26 tumor-bearing BALB/c

mice are shown. The values are presented as the mean standard error of the mean (SEM) (n = 5 or 10): *P < 0.05, **P < 0.01, ***P < 0.001,

and ****P < 0.0001.

24e plus anti-PD-1 antibodies (approximately 1.2 g on day 4)

(Figure 9B). The mice gradually regained the body weight on

day 4 postdrug administration. By contrast, neither 25a nor

R848 showed signiﬁcant weight loss when used alone or in

combination with anti-PD-1 antibodies. Furthermore, we also

evaluated the survival of treated mice (n = 10). As shown in

Figure 9C, TLR7/8 agonists in combination with anti-PD-1

antibodies generated a distinct survival beneﬁt relative to IgG

control (P < 0.001), and the 24e plus anti-PD-1 treatment

showed a signiﬁcant prolongation of survival compared with

R848 plus anti-PD-1 antibody combination (P < 0.05). Based

on synergistic analysis, there was an additive eﬀect on the

survival rate in the combination of anti-PD1 antibodies plus

TLR7/8 agonist 24e (see more details in S10).

combination treatment of 25a (50 μg) and anti-PD-1

antibodies showed enhancement of tumor suppression by a

TGI of 88.9%. Moreover, we also investigated the antitumor

activity of compound 24m with reduced basicity relative to

compound 24e. As shown in Figure 9E, 24m signiﬁcantly led

to the suppression of tumor growth, which was comparable to

the response to 24e. Compounds 24e (25 μg) and 25a (50 μg)

were further investigated for the antitumor eﬃcacy in

combination with anti-PD-L1 antibodies. As shown in Figure

9F, anti-PD-L1 antibodies did modestly reduce tumor growth a

TGI of 56.9%. Notably, TLR7/8 agonists combined with anti-

PD-L1 antibodies induced profound regression of established

tumors in mice. Compound 24e (25 μg) plus anti-PD-L1

treatment induced complete eradication of the treated tumor

in 2/5 mice (Figure 9G). Compound 25a (50 μg) plus anti-

PD-L1 treatment induced complete eradication of the treated

tumor in 1/5 mice (Figure 9H). Indeed, TLR7/8 agonists

When the administered dose was increased to 50 μg,

compound 25a resulted in signiﬁcant inhibition of tumor

growth with a TGI of 82.8% in a dose-dependent manner. The

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Figure 10. Flow cytometric analysis of cell populations in CD45⁺tumor-inﬁltrating lymphocytes, including CD11c⁺DCs (A), CD11b⁺F4/80⁺

(TAM) cells (B), CD11b⁺F4/80⁺MHCII⁺(M1) cells (C), CD11b⁺F4/80⁺CD206⁺(M2) cells (D), CD3⁺T cells (E), CD4⁺T cells (F), CD8⁺T

cells (G), and ratios of M1/M2 (H), CD8⁺/T_reg(CD4⁺CD25⁺FoxP3⁺) (I). Statistical data were pooled from BALB/c mice following indicated

treatments and displayed as means SEM (n = 5): *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001; NS, not statistically signiﬁcant.

combined with anti-PD-L1 antibodies led to further weight

loss compared to combined with anti-PD-1 antibodies

(approximately 1.9 g in the combination of 24e and anti-PD-

L1) (Figure 9I).

inﬁltration into the tumor microenvironment. TLR-activated

DCs can mediate antitumor responses not only through

antigen presentation and T cell priming but also through direct

cytotoxic eﬀects on tumor cells.⁵⁵

The mice treated with 24e, R848 monotherapies or 24e, and

R848 in combination with anti-PD-1 antibodies were selected

for immunoassay of the tumor microenvironment. Tumors

were harvested 23 days after tumor implantation, and single-

cell suspensions were antibodies stained and analyzed by

ﬂuorescence-activated cell sorting (FACS) to detect tumor-

inﬁltrating immune cells, including DCs, macrophages (M1

and M2), T_reg, CD4⁺, and CD8⁺T cells. Compared with the

anti-PD-1 antibody group, both TLR agonist monotherapies

and in combination with anti-PD-1 antibodies remarkably

increased inﬁltration of immune cells (Figure 10). Notably,

therapies involved 24e markedly induced inﬁltration of DCs

and led to more than a 2-fold increase in CD11c⁺DC

populations relative to treatment involving R848, demonstrat-

ing that this chemotype-based TLR7/8 agonists have

tremendous potential of triggering DC expansion and

The treatment involved compound 24e, either alone or in

combination with anti-PD-1 antibodies, dramatically evoked an

immunosuppressive M2-like (CD11b⁺F4/80⁺CD206⁺) status

to an inﬂammatory M1-like (CD11b⁺F4/80⁺MHCII⁺) pheno-

type switch in macrophages, and therefore induced raise of

M2/M1 TAM ratios (Figure 10). Moreover, TLR7/8 agonists

(24e, R848) as monotherapies or in combination with anti-

PD-1 antibodies signiﬁcantly increased the proportion of

inﬁltrated CD4⁺T helper type 1 (TH1) cells and CD8⁺T

cytotoxic lymphocytes, increasing the CD8⁺/regulatory T cell

(CD4⁺FoxP3⁺) ratio. Interestingly, 24e-treated mice exhibited

more CD4⁺T cells (P < 0.05) and higher T_eff/T_regratios (P <

0.05) compared to R848-treated mice, but there were no

signiﬁcant diﬀerences in CD8⁺T cell populations between the

two monotherapies (24e, R848) and between two combina-

tion regimens (24e or R848 plus anti-PD-1 antibodies).

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In addition to the percentages, we also analyzed FACS data

to examine the absolute counts of immune cells. As shown in

Figure S12, the statistical results for absolute counts and

percentages were generally consistent. Evaluation of the

synergistic eﬀects of the anti-PD-1 antibodies plus 24e showed

signiﬁcant synergy in the DC population (P = 0.043), although

there was no signiﬁcant synergy, a trend toward synergistic

eﬀects of the combination of anti-PD1 antibodies plus TLR7/8

agonist could be observed for CD4⁺T cells, CD8⁺T cells, and

the T_eff/T_regratio (Figure S12). Further analysis revealed that

there were statistically negative correlations between the

antitumor eﬀects and the immune cell densities within tumors

(Figure S13). Likewise, the strong negative correlations with

TVs were observed in M1/M2 ratios (r = −0.87, P < 0.0001)

and CD8⁺T/T_regratios (r = −0.79, P < 0.0001), respectively.

Taken together, these results suggested that TLR7/8 agonist

treatment could signiﬁcantly increase tumor-inﬁltrating

immune cells, thereby shaping the tumor microenvironment.

Safety Proﬁling. Upon the introduction of basic groups

into these TLR7/8 agonists, we assessed the risk of

phospholipidosis and hERG liability of compounds 24e,

24m, and 25a. To investigate the potential phospholipidosis

of candidate compounds, an in vitro screening assay was

performed in HepG2 cells with the HCS LipidTOX Red

phospholipidosis detection reagent. Compared with the vehicle

group, compound 24e at 3 μM induced a moderate

phospholipidosis in HepG2 cells (P < 0.01) (Figures S14

and S15). Compared with 24e, 24m containing reduced

basicity exhibited mitigated phospholipidosis (P < 0.05, 3 μM).

Signiﬁcantly, compound 25a with reduced hydrophobicity

induced no signiﬁcant phospholipidosis at a concentration of 3

μM as compared to the vehicle group. These results suggested

that reducing basicity or hydrophobicity may lower the risk of

phospholipidosis in the series of TLR7/8 dual agonists.

Compounds 24e, 24m, and 25a were evaluated for hERG

liability in a stable HEK293 cell line expressing the hERG

channel. Compound 24e demonstrated clear hERG inhibition

(63.22% inhibition at 3 μM) (Table 5). Compared with 24e,

mortality of mice (24e: 100 mg/kg; 25a: 120 mg/kg; and

R848: 120 mg/kg). Compound 24e caused 20.3% body weight

loss of one mouse at a dose of 60 mg/kg, which was also

counted as death. In accordance with their TLR7 agonistic

potencies, compounds 24e (17.6%) and 25a (15.3%) resulted

in more body weight loss than R848 (8.7%) at the same dose

(60 mg/kg). R848-treated mice gradually regained body

weight after day 3 postdrug administration. Compound 24e-

and 25a-treated mice gradually regained body weight after day

4 postdrug administration. As shown in Figure 11G, the

swollen spleen would gradually return to normal after the drug

administration was stopped. As summarized in Figure 11H, the

TLR7/8 agonists caused obvious white discharge from the eyes

and mild diarrhea in some mice. Additionally, high doses of

TLR7/8 agonist administration showed decreased locomotor

activity in most of the mice. However, these adverse eﬀects are

transient and reversible, gradually disappearing 24 h after

administration. According to these results, we determined the

maximum tolerated dose (MTD) (Figure 11I). Taken

together, systematic administration of high doses of TLR7/8

agonists resulted in immune-related adverse eﬀects mediated

through mouse TLR7.

In the subacute toxicity study, TLR7/8 agonists induced

signiﬁcant body weight loss (R848: 1.12 g; 24e: 2.19 g; and

25a: 1.73 g) and splenomegaly in TLR7 potency-dependent

manner (Figure 12). Except for one mouse in the 24e-

treatment group with white discharge from the eyes, all mice

had no signiﬁcant adverse eﬀects. Mice gradually regained the

body weight on days 3 and 4 after drug administration in 25a-

and 24e-treated mice, respectively. As shown in Figure 12D,

hematoxylin and eosin (H&E) staining analysis of tissues from

mice treated with 25a and 24e exhibited no marked

pathological damage.

CONCLUSIONS

■

In summary, an extensive SAR exploration of the pyrido[3,2-

d]pyrimidin-2-amine core led to the discovery of highly potent

dual TLR7/TLR8-active compounds,⁵⁶which extended the

range of permissible chemical structures that concurrently

activate both TLR7 and TLR8. A detailed examination of

activation in hPBMCs indicated that these dual TLR7/8

agonists potently induced the prototypical secretion of

proinﬂammatory cytokines and chemokines. As a single

agent, compounds 24e, 24m, and 25a showed remarkable

antitumor eﬀects in a CT26 colon carcinoma model.

Furthermore, these TLR7/8 agonists obviously improved the

antitumor immune responses of immune checkpoint blockade.

Especially, the combination of compound 24e with anti-PD-L1

antibodies led to complete tumor regression. Mechanisms

underlying the notable antitumor eﬀects were associated with

the TLR7/8 agonist-mediated remodeling of the tumor

microenvironment. Our results provided a rationale for

combining TLR7/8 agonists with immune checkpoint

inhibitors to improve the antitumor immune responses,

particularly in settings where the immune checkpoint blockade

alone was not eﬀective.

Table 5. Evaluation of hERG Inhibitory Activity of 24e,

24m, and 25a

compd

24e

24m

25a

cisapride

hERG (inhibition % at 3 μM) hERG (inhibition % at 10 μM)

63.22 2.81%

69.03 0.93%

N.T.

89.33 0.16%

89.23 0.95%

14.80 2.96%

a

b

13.1 2.2 nM

a

b

Not tested. IC₅₀value.

24m did not show decay in hERG inhibition despite reduced

basicity. A signiﬁcant drop oﬀ in hERG inhibition was achieved

in compound 25a by lowering overall lipophilicity (14.8%

inhibition at 10 μM). Based on these results, compound 25a

showed a favorable safety proﬁle with a lower hERG binding

aﬃnity and risk of phospholipidosis.

To further investigate the safety of compounds 24e and 25a,

acute and subacute toxicity studies were conducted in healthy

8-week-old BALB/c mice, and compound R848 was selected

as a reference compound (Figure 11). In acute toxicity studies,

66 BALB/c mice were randomly divided into eleven groups,

and six animals (half male and half female) in each group were

intraperitoneally administered single doses of TLR7/8

agonists. The high doses of TLR7/8 agonists quickly led to

To explore the therapeutic potential of TLR7/8 agonist in

multiple tumor models insensitive to immune checkpoint

blockade, we performed extensive antitumor activity and in-

depth mechanistic studies on compound 24e. We discovered

that 24e was involved in epigenetic regulation (histone 3 lysine

4 demethylase KDM5A upregulation) of the tumor and DCs.

TLR7 activation-mediated CXCL9 and CXCL10 secretion

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Figure 11. In vivo acute toxicity studies of TLR7/8 agonists. Survival rate (A−C), body weight (D−F), spleen weight at the end of the

experimental period (G), and adverse reactions (H) (NAR: no obvious adverse reactions; AR1: white discharge from the eyes; and AR2: white

discharge from the eyes and mild diarrhea). (I) MTD of compounds 24e, 25a, and R848.

played a critical role in the induction of immune cell

inﬁltration.⁵⁷It should be noted that TLR8-dependent

transduction is not embodied in mouse because mTLR8

cannot sense small ligands.⁵⁸Thus, to deﬁne the synergistic

eﬀects of dual activation of TLR7 and 8, these TLR7/8

agonists should be further investigated in a model system with

functional TLR8 signaling.

Indeed, TLR7/8 agonists might cause on-target adverse

eﬀects. To relieve the adverse eﬀects of TLR7/8 agonists,

lower doses or frequencies of regimens are preferred for

systemic administration. To our delight, the remarkable

antitumor activity at a relatively low dose allowed these

TLR7/8 agonists to act as payloads for antibody−drug

conjugates (ADCs). To avoid systemic exposure of TLR7/8

agonists, we have developed an immuno TLR7/8 agonist−

anti-PD-L1 antibody conjugate (ADC) for targeted delivery of

compound 24e, which showed a signiﬁcant enhancement of

antitumor activities compared to the combination of 24e and

anti-PD-L1 antibodies along with a remarkable improvement

of the safety index (manuscript in submission).

EXPERIMENTAL SECTION

■

Chemistry. Unless otherwise noted, all reagents were obtained

commercially and used without further puriﬁcation. All anhydrous

solvents such as acetonitrile, dioxane, DCM, and THF were puriﬁed

using solvent puriﬁcation systems. Chromatographic puriﬁcations

were performed by ﬂash chromatography using silica gel (200−300

mesh). Purity for all ﬁnal compounds was conﬁrmed to be >95% by

Shimadzu LCMS2020 using a Shimadzu C18 3 μm 150 × 2 mm

reverse-phase column with H₂O−CH₃CN gradients and a Shimadzu

2020 electrospray ionization quadrupole time-of-ﬂight accurate mass

spectrometer (a mass accuracy of 5 ppm) operating in the positive ion

acquisition mode. The yields of the products included referred to

1

isolated yields. The H NMR spectra were obtained on a Bruker 400

MHz spectrometer, and chemical shifts are reported in ppm relative to

the residual protiated solvent as the internal standard. ¹³C NMR

spectra were obtained at 100.6 MHz on a Bruker 400 MHz

instrument, and chemical shifts were recorded relative to the solvent

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Figure 12. In vivo subacute toxicity studies of TLR7/8 agonists. (A) Body weight of the treated and control groups was recorded every day. (B,C)

Weight (B) and photographs (C) of spleen from treated and control groups. (D) Organs from treated and vehicle groups were stained with H&E

to detect the possible organ toxicity. Photographs were obtained under magniﬁcation (scale bar represents 50 μM).

resonance. Both ¹H NMR and ¹³C NMR chemical shifts are reported

in parts per million downﬁeld from tetramethylsilane. High-resolution

mass spectra (HRMS) were recorded on a hybrid quadrupole-

orthogonal acceleration-TOF mass spectrometer (Xevo G2 Q-Tof,

Waters Micromass) with an ESI source.

N-(6-Chloro-4-hydroxypyrido[3,2-d]pyrimidin-2-yl)acetamide

(9). A solution of 2-amino-6-chloropyrido[3,2-d]pyrimidin-4(4aH)-

one (1960 mg, 10 mmol) and acetic anhydride (25 mL) was heated at

140 °C for 12 h and cooled down to 0 °C, then ﬁltered to give a crude

product. The residue was puriﬁed with column chromatography to

yield 9 as a white solid (1737 mg, 73.0% yield). ¹H NMR (400 MHz,

DMSO-d₆): δ 12.29 (s, 1H), 11.78 (s, 1H), 7.93 (d, J = 8.6 Hz, 1H),

7.81 (d, J = 8.6 Hz, 1H), 2.20 (s, 3H). ¹³C NMR (100 MHz, DMSO-

d₆): δ 179.2, 162.8, 152.9, 151.7, 150.9, 143.2, 141.4, 135.4, 29.1. MS

(ESI): calcd for C₉H₈ClN₄O₂[M + H]⁺, 239.03; found, 239.4.

N-(4,6-Dichloropyrido[3,2-d]pyrimidin-2-yl)acetamide (10). To a

solution of N-(6-chloro-4-hydroxy-pyrido[3,2-d]pyrimidin-2-yl)-

acetamide (1193 mg, 5.0 mmol) in dry dioxane (30 mL) was

added DMF (35 μL), after cooling to 0 °C, oxalyl chloride (514 μL,

6.0 mmol) was added dropwise. The reaction mixture was then

warmed to rt and stirred overnight. After the reaction was completed,

the resulting mixture was concentrated under reducing pressure and

used without puriﬁcation.

General Procedure A for the Synthesis of 11a−b and 11e−

g. To a solution of N-(4,6-dichloropyrido[3,2-d]pyrimidin-2-yl)-

acetamide (10, 5 mmol) in dry THF (12 mL) were added the

corresponding amine (7.5 mmol) and DIEA (10.0 mmol). The

resulting mixture was stirred at rt for 3 h at which point the reaction

was monitored by liquid chromatography (LC)-mass spectrometry.

The mixture was diluted with ethyl acetate and washed with saturated

brine. The organic phase was dried over Na₂SO₄and then

concentrated in vacuo. The residue was puriﬁed by ﬂash

chromatography on silica gel (EtOAc in the Hex gradient) to yield

the titled compounds.

Methyl 3-Amino-6-bromopicolinate (7). To a solution of methyl

3-aminopicolinate (1526 mg, 10 mmol) in 20 mL acetonitrile at 0 °C

was added 1-bromo-2,5-pyrrolidinedione (1780 mg, 11 mmol). The

mixture was stirred at rt for 0.5 h, quenched by dropwise addition of

saturated aqueous solution of NH₄Cl, and then extracted with ethyl

acetate (2 × 50 mL). The combined organic layers were dried over

anhydrous Na₂SO₄, ﬁltered oﬀ, and concentrated under reduced

pressure. The crude residue was puriﬁed by ﬂash chromatography on

silica gel (EtOAc/cyclohexane 1:20 to 1:5) to aﬀord the titled

1

compound as a yellow solid (2531 mg, 94.0% yield). H NMR (400

MHz, CDCl₃): δ 7.34 (d, J = 8.8 Hz, 1H), 6.95 (d, J = 8.8 Hz, 1H),

3.95 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 167.2, 146.5, 132.8,

128.1, 127.6, 127.0, 52.7. MS (ESI): calcd for C₇H₇BrN₂O₂[M +

H]⁺, 230.97, 232.97; found, 231.2, 233.2.

2-Amino-6-chloropyrido[3,2-d]pyrimidin-4(4a-H)-one (8). Meth-

yl 3-amino-6-bromopicolinate (620 mg, 2.7 mmol) and chloroforma-

midine hydrochloride (345 mg, 3 mmol) were added to dimethyl

sulfone (5076 mg, 54 mmol), and the mixture was heated at 140 °C

for 3 h. After the reaction was completed, 100 mL water was added to

the reaction mixture after cooling to rt. A suspension was formed with

the help of ultrasonic vibration. A brown solid was collected after

ﬁltration and washed with H₂O and acetone, giving the crude product.

The crude product was puriﬁed by silica gel chromatography

(CH₂Cl₂/MeOH 1:20 to 1:9) to aﬀord the titled compound as a

white solid (343 mg, 65.0% yield). ¹H NMR (400 MHz, DMSO-d₆):

δ 7.79 (t, J = 7.8 Hz, 1H), 7.72 (t, J = 7.6 Hz, 1H), 7.52 (s, 1H). ¹³C

NMR (100 MHz, DMSO-d₆): δ 159.0, 152.4, 144.8, 133.9, 133.7,

130.3. MS (ESI): calcd for C₇H₆ClN₄O [M + H]⁺, 197.02; found,

197.20.

General Procedure B for the Synthesis of 11c−d. To a

solution of NaH (0.5 mmol) (60% dispersion in mineral oil) in dry

butanol or butanethiol (0.2 M) was added N-(4,6-dichloropyrido[3,2-

d]pyrimidin-2-yl)acetamide (10, 0.1 mmol). The resulting mixture

was stirred at rt for 2 h at which point the reaction was monitored by

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LC-mass spectrometry. The resulting mixture was diluted with ethyl

acetate and washed with saturated brine. The organic phase was dried

over Na₂SO₄and then concentrated in vacuo. The residue was

puriﬁed by ﬂash chromatography on silica gel (EtOAc in Hex

gradient) to yield the titled compounds.

N-(4-(Butylamino)-6-chloropyrido[3,2-d]pyrimidin-2-yl)-

acetamide (11a). ¹H NMR (400 MHz, CDCl₃): δ 8.10 (s, 1H), 7.90

(d, J = 8.8 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 7.05 (t, J = 6.3 Hz, 1H),

3.60 (td, J = 13.2, 5.9 Hz, 2H), 2.63 (s, 3H), 1.75−1.69 (m, 2H), 1.46

(m, 2H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ

171.9, 159.5, 154.9, 146.5, 144.6, 137.7, 129.3, 129.2, 40.8, 31.2, 25.4,

20.2, 13.8. MS (ESI): calcd for C₁₃H₁₇ClN₅O [M + H]⁺, 294.1;

found, 294.3.

(3 equiv) in a glovebox. The reaction mixture was left to stir at rt for 3

h. The mixture was ﬁltrated to aﬀord zinc reagents. The ﬁltrate was

added to a solution of N⁴-butyl-6-chloropyrido[3,2-d]pyrimidine-2,4-

diamine (12a) (0.5 mmol, 1 equiv) and Pd(amphos)Cl₂(0.05 mmol,

0.1 equiv), and the mixture was stirred overnight at rt. After

completion of the reaction, the mixture was ﬁltered oﬀ and

concentrated under reduced pressure. After puriﬁcation by column

chromatography on silica gel (EtOAc in Hex gradient), the pure

products were obtained.

General Procedure G for the Synthesis of 17y−aa. To a

solution of compounds 17u−w (0.5 mmol, 1 equiv) in a 15 mL mixed

solvent (THF/H₂O = 2:1) was added lithium hydroxide mono-

hydrate (1.5 mmol, 3 equiv). The reaction mixture was left to stir at rt

for 12 h. After completion of the reaction, the pH of the reaction

mixture was adjusted to 5−6 with 2 N HCl (aq) and ﬁltrated oﬀ, and

the residue was washed with water, ethanol, and diethyl ether. The

target compounds were obtained by vacuum drying of the residue.

6-Benzyl-N⁴-butylpyrido[3,2-d]pyrimidine-2,4-diamine (15a).

General Procedure C for the Synthesis of 12a−f. To a

solution of 11a−f (0.5 mmol, 1 equiv) in MeOH (10 mL) was added

LiOH·H₂O (1.5 mmol, 3 equiv). The reaction mixture was stirred at

rt for 3 h at which point the reaction was monitored by thin-layer

chromatography. The resulting mixture was diluted with ethyl acetate

and washed with water and saturated brine, the organic portion was

dried over Na₂SO₄, and the solvent was removed under reduced

pressure. The crude product was puriﬁed by ﬂash chromatography on

silica gel (EtOAc in the Hex gradient) to yield the corresponding

compounds 12a−f.

1

White solid. Yield: 85.0%. H NMR (400 MHz, CDCl₃): δ 7.56 (d,

J = 8.6 Hz, 1H), 7.36−7.16 (m, 6H), 7.06 (s, 1H), 5.02 (s, 2H), 4.17

(s, 2H), 3.56 (td, J = 13.2, 6.4 Hz, 2H), 1.80−1.58 (m, 2H), 1.46 (dd,

J = 14.9, 7.4 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H). ¹³C NMR (100 MHz,

CDCl₃): δ 160.5, 160.4, 154.6, 144.8, 139.5, 133.2, 129.2, 128.7,

128.2, 127.9, 126.5, 44.4, 40.3, 31.6, 20.3, 14.0. HRMS (ESI): calcd

for C₁₈H₂₂N₅[M + H]⁺, 308.1875; found, 308.1882.

N⁴-Butyl-6-chloropyrido[3,2-d]pyrimidine-2,4-diamine (12a).

1

Yellow solid. Yield: 92.0%. H NMR (400 MHz, CDCl₃): δ 7.62

6-Benzyl-N⁴-(2-methoxyethyl)pyrido[3,2-d]pyrimidine-2,4-dia-

mine (15b). White solid. Yield: 81.0%. ¹H NMR (400 MHz, CDCl₃):

δ 7.60 (d, J = 8.6 Hz, 1H), 7.55 (t, J = 5.8 Hz, 1H), 7.34−7.21 (m,

6H), 5.68 (s, 2H), 4.17 (s, 2H), 3.79 (m, 2H), 3.65 (t, J = 5.2 Hz,

2H), 3.43 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 160.3, 159.0,

155.6, 142.2, 139.1, 131.44, 129.1, 128.6, 128.3, 127.5, 126.5, 70.8,

58.9, 44.3, 40.4. HRMS (ESI): calcd for C₁₇H₂₀N₅O [M + H]⁺,

310.1668; found, 310.1690.

(d, J = 8.6 Hz, 1H), 7.41 (d, J = 8.8 Hz, 1H), 6.84 (t, J = 6.0 Hz, 1H),

5.14 (s, 2H), 3.56 (td, J = 13.2, 5.8 Hz, 2H), 1.75−1.63 (m, 2H), 1.46

(m, 2H), 0.98 (t, J = 7.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ

160.8, 159.5, 145.6, 143.5, 135.7, 128.7, 128.4, 40.3, 31.4, 20.2, 13.8.

MS (ESI): calcd for C₁₁H₁₅ClN₅[M + H]⁺, 252.1; found, 252.3.

General Procedure D for the Synthesis of 13a, 14a, and 17c.

A mixture of N⁴-butyl-6-chloropyrido[3,2-d]pyrimidine-2,4-diamine

(12a) (0.5 mmol, 1 equiv), Pd(PPh₃)₄(0.025 mmol, 0.05 equiv),

CsCO₃(1 mmol, 2 equiv), and the corresponding boronic acid (0.6

mmol, 1.2 equiv) in 15 mL of dioxane was heated at 100 °C under N₂

for 6 h. The reaction mixture was then ﬁltered and the solvent was

removed under reduced pressure. The residue was puriﬁed by ﬂash

chromatography on silica gel (EtOAc in the Hex gradient) to the

desired products 13a, 14a and 17c.

6-Benzyl-4-butoxypyrido[3,2-d]pyrimidin-2-amine (15c). White

1

solid. Yield: 89.0%. H NMR (400 MHz, CDCl₃): δ 7.67 (d, J = 8.6

Hz, 1H), 7.33−7.23 (m, 6H), 5.07 (s, 2H), 4.61 (t, J = 7.2 Hz, 2H),

4.31 (s, 2H), 1.97−1.91 (m, 2H), 1.52 (m, 2H), 1.02 (t, J = 7.4 Hz,

3H). ¹³C NMR (100 MHz, CDCl₃): δ 166.9, 159.0, 157.4, 150.3,

147.5, 139.2, 133.3, 129.3, 128.7, 128.7, 128.5, 126.6, 67.7, 44.8, 30.6,

19.2, 14.0. HRMS (ESI): calcd for C₁₈H₂₁N₄O [M + H]⁺, 309.1715;

found, 309.1724.

N⁴-Butyl-6-phenylpyrido[3,2-d]pyrimidine-2,4-d-iamine (13a).

1

Yellow solid. Yield: 85.0%. H NMR (400 MHz, CDCl₃): δ 8.00

6-Benzyl-4-(butylthio)pyrido[3,2-d]pyrimidin-2-amine (15d).

(d, J = 7.8 Hz, 2H), 7.90 (d, J = 8.7 Hz, 1H), 7.72 (d, J = 8.7 Hz, 1H),

7.47 (t, J = 7.4 Hz, 2H), 7.40 (t, J = 7.1 Hz, 1H), 7.21 (br s, 1H), 5.35

(s, 2H), 3.59 (dd, J = 13.4, 6.7 Hz, 2H), 1.76−1.65 (m, 2H), 1.46 (dt,

J = 14.7, 7.4 Hz, 2H), 0.98 (t, J = 7.3 Hz, 3H). ¹³C NMR (100 MHz,

CDCl₃): δ 160.5, 160.5, 150.8, 145.1, 138.8, 133.0, 128.8, 128.8,

128.5, 126.7, 125.1, 40.3, 31.5, 20.2, 13.9. HRMS (ESI): calcd for

C₁₇H₂₀N₅[M + H]⁺, 294.1719; found, 294.1725.

1

Yellow solid. Yield: 85.0%. H NMR (400 MHz, CDCl₃): δ 7.67

(d, J = 8.7 Hz, 1H), 7.36 (d, J = 8.7 Hz, 1H), 7.35−7.19 (m, 5H),

5.23 (s, 2H), 4.29 (s, 2H), 3.25 (t, J = 7.3 Hz, 2H), 1.80 (m, 2H),

1.56 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃):

δ 175.3, 158.5, 157.0, 144.1, 134.0, 135.0, 133.8, 129.3, 128.8, 128.7,

126.6, 44.7, 30.9, 28.8, 22.2, 13.8. HRMS (ESI): calcd for C₁₈H₂₁N₄S

[M + H]⁺, 325.1487; found, 325.1488.

N⁴-Butyl-6-(4-methoxyphenyl)pyrido[3,2-d]pyrimidine-2,4-dia-

mine (14a). Yellow solid. Yield: 90.0%. ¹H NMR (400 MHz, CDCl₃):

δ 8.02−7.93 (m, 2H), 7.88 (d, J = 8.8 Hz, 1H), 7.71 (d, J = 8.8 Hz,

1H), 7.16 (t, J = 5.8 Hz, 1H), 7.06−6.97 (m, 2H), 4.92 (s, 2H), 3.88

(s, 3H), 3.60 (td, J = 7.3, 5.9 Hz, 2H), 1.79−1.66 (m, 2H), 1.49 (dq, J

= 14.6, 7.3 Hz, 2H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C NMR (100 MHz,

CDCl₃): δ 160.5, 160.4, 160.2, 150.7, 144.9, 133.2, 131.5, 128.4,

128.0, 124.6, 114.2, 55.4, 40.3, 31.6, 20.3, 13.9. HRMS (ESI): calcd

for C₁₈H₂₂N₅O [M + H]⁺, 324.1824; found, 324.1827.

6-Benzyl-N⁴-butyl-N⁴-methylpyrido[3,2-d]pyrimidine-2,4-dia-

mine (15e). Yellowish solid. Yield: 87.0%. ¹H NMR (400 MHz,

CDCl₃): δ 7.62 (d, J = 8.6 Hz, 1H), 7.33−7.16 (m, 6H), 5.15 (s, 2H),

4.14 (s, 3H), 3.45−3.32 (m, 2H), 1.76−1.62 (m, 2H), 0.93 (t, J = 7.4

Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 159.9, 158.6, 153.4, 146.2,

139.6, 132.5, 130.2, 129.0, 128.6, 126.8, 126.4, 53.1, 44.6, 39.7, 29.7,

20.13, 14.1. HRMS (ESI): calcd for C₁₉H₂₄N₅[M + H]⁺, 322.2032;

found, 322.2036.

6-Benzyl-4-morpholinopyrido[3,2-d]pyrimidin-2-amine (15f).

General Procedure E for the Synthesis of 15a−g, 17a−b,

17d−e, 17l−x, and 18. To a suspension of intermediates 12a−g

(0.5 mmol, 1 equiv) in 20 mL THF (5 mL, 0.1 M) were added

corresponding 1-bromoalkylbenzene (0.5 mmol, 1 equiv), zinc

powder (98.1 mg, 1.5 mmol), and Ni(PPh₃)₂Cl₂(32.7 mg, 0.05

mmol) in a glovebox. The reaction mixture was stirred at rt for 16 h.

After completion of the reaction, the mixture was ﬁltered and the

solvent was removed under reduced pressure. The residue was

puriﬁed by ﬂash chromatography on silica gel (EtOAc in Hex

gradient) to the desired products.

1

Yellow solid. Yield: 84.0%. H NMR (400 MHz, CDCl₃): δ 7.68

(d, J = 8.6 Hz, 1H), 7.37 (d, J = 8.6 Hz, 1H), 7.33 (dd, J = 8.2, 6.4 Hz,

2H), 7.28−7.22 (m, 3H), 5.39 (s, 2H), 4.30 (t, J = 4.8 Hz, 4H), 4.19

(s, 2H), 3.74 (t, J = 4.8 Hz, 4H). ¹³C NMR (100 MHz, CDCl₃): δ

178.2, 159.8, 159.8, 158.6, 153.8, 146.4, 139.2, 132.7, 129.8, 129.4,

128.5, 127.2, 126.4, 67.1, 44.2. HRMS (ESI): calcd for C₁₈H₂₀N₅O

[M + H]⁺, 322.1668; found, 322.1664.

6-Benzyl-4-(piperidin-1-yl)pyrido[3,2-d]pyrimidin-2-amine (15g).

Yellow solid. Yield: 82.0%. ¹H NMR (400 MHz, CDCl₃): δ 7.60 (d, J

= 8.6 Hz, 1H), 7.32−7.19 (m, 6H), 4.78 (s, 2H), 4.21 (t, J = 5.2 Hz,

4H), 4.15 (s, 2H), 1.75−1.65 (m, 2H), 1.65−1.56 (m, 4H). ¹³C NMR

(100 MHz, CDCl₃): δ 160.3, 159.4, 153.2, 147.9, 139.8, 133.7, 130.6,

General Procedure F for the Synthesis of 17f−k. To a

suspension of corresponding benzyl bromide or benzyl chloride (0.5

mmol, 1 equiv) in 20 mL THF (5 mL, 0.1 M) was added zinc powder

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129.4, 128.6, 126.8, 126.4, 49.0, 44.7, 26.5, 25.1. HRMS (ESI): calcd

for C₁₉H₂₂N₅[M + H]⁺, 320.1875; found, 320.1880.

N⁴-Butyl-6-(2-(triﬂuoromethyl)benzyl)pyrido[3,2-d]pyrimidine-

1

2,4-diamine (17i). Yellow solid. Yield: 75.0%. H NMR (400 MHz,

N⁴-Butyl-6-phenethylpyrido[3,2-d]pyrimidine-2,4-diamine (17a).

DMSO-d₆): δ 7.72 (d, J = 8.0 Hz, 1H), 7.59 (m, 2H), 7.48 (d, J = 8.7

Hz, 2H), 7.46−7.42 (m, 2H), 7.23 (d, J = 8.6 Hz, 1H), 6.26 (s, 2H),

4.31 (s, 2H), 3.47−3.42 (m, 2H), 1.60−1.53 (m, 2H), 1.35−1.26 (m,

2H), 0.90 (t, J = 7.3 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ

160.8, 160.0, 152.0, 145.3, 138.1, 133.0, 132.9, 132.7, 128.1, 127.9,

127.8, 127.5, 126.3 (q, J = 11.0 Hz), 124.6 (q, J = 273.8 Hz), 39.9,

31.4, 27.2, 20.1, 14.2. HRMS (ESI): calcd for C₁₉H₂₁F₃N₅[M + H]⁺,

376.1749; found, 376.1744.

1

Yellowish solid. Yield: 70.0%. H NMR (400 MHz, CDCl₃): δ 7.56

(d, J = 8.6 Hz, 1H), 7.29−7.22 (m, 3H), 7.18 (m, 3H), 7.05 (br s,

1H), 5.08 (s, 2H), 3.56 (td, J = 13.2, 6.0 Hz, 2H), 3.14 (m, 2H), 3.08

(m, 2H), 1.69 (m, 2H), 1.46 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H). ¹³C

NMR (100 MHz, CDCl₃): δ 160.3, 160.1, 155.2, 141.5, 132.5, 128.5,

128.4, 128.1, 127.8, 126.0, 40.2, 39.4, 35.7, 31.5, 20.2, 13.9. HRMS

(ESI): calcd for C₁₉H₂₄N₅[M + H]⁺, 322.2032; found, 322.2028.

(E)-N⁴-Butyl-6-styrylpyrido[3,2-d]pyrimidine-2,4-diamine (17c).

N⁴-Butyl-6-(3-(triﬂuoromethyl)benzyl)pyrido[3,2-d]pyrimidine-

1

2,4-diamine (17j). Yellow solid. Yield: 81.0%. H NMR (400 MHz,

1

Yellow solid. Yield: 88.0%. H NMR (400 MHz, MeOD): δ 7.67

CDCl₃): δ 7.63 (d, J = 8.6 Hz, 1H), 7.55 (s, 1H), 7.50−7.42 (m, 3H),

7.31 (d, J = 8.6 Hz, 1H), 7.08 (t, J = 6.0 Hz, 1H), 5.56 (s, 2H), 4.23

(s, 2H), 3.56 (td, J = 13.6, 6.8 Hz, 2H), 1.68 (m, 2H), 1.46 (m, 2H),

1.01 (t, J = 7.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 160.2,

159.8, 153.7, 143.1, 140.1, 132.4, 129.2, 129.0, 127.9, 127.8, 125.9 (q,

J = 4 Hz), 125.5 (q, J = 271 Hz), 123.4 (q, J = 3 Hz), 43.8, 40.3, 31.3,

20.2, 13.8. HRMS (ESI): calcd for C₁₉H₂₁F₃N₅[M + H]⁺, 376.1749;

found, 376.1765.

(t, J = 8.0 Hz, 1H), 7.61−7.44 (m, 4H), 7.27 (t, J = 7.2 Hz, 2H),

7.20−7.14 (m, 2H), 3.49 (td, J = 12.4, 6.2 Hz, 2H), 1.66−1.55 (m,

2H), 1.35 (dt, J = 13.9, 7.0 Hz, 2H), 0.89 (t, J = 7.3 Hz, 3H). ¹³C

NMR (100 MHz, CDCl₃): δ 160.3, 160.3, 149.6, 145.3, 136.7, 132.6,

132.0, 128.8, 128.5, 128.3, 128.0, 127.0, 125.9, 40.3, 31.5, 20.3, 13.9.

HRMS (ESI): calcd for C₁₉H₂₂N₅[M + H]⁺, 320.1875; found,

320.1879.

N⁴-Butyl-6-(1-phenylethyl)pyrido[3,2-d]pyrimidine-2,4-diamine

N⁴-Butyl-6-(4-(triﬂuoromethyl)benzyl)pyrido[3,2-d]pyrimidine-

1

(17d). White solid. Yield: 89.0%. H NMR (400 MHz, CDCl₃): δ

2,4-diamine (17k). Yellow solid. Yield: 75.0%. H NMR (400 MHz,

7.54 (d, J = 8.6 Hz, 1H), 7.31−24 (m, 5H), 7.20 (t, J = 6.8 Hz, 1H),

7.08 (s, 1H), 5.01 (s, 2H), 4.31 (d, J = 7.2 Hz, 1H), 3.57 (td, J = 13.2,

6.8 Hz, 2H), 1.79−1.61 (m, 5H), 1.47 (td, J = 14.8, 7.4 Hz, 2H), 1.00

(t, J = 7.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 160.5, 160.5,

158.5, 145.1, 144.8, 133.04, 128.6, 127.8, 127.3, 126.5, 47.0, 40.3,

31.6, 20.9, 20.3, 14.0. HRMS (ESI): calcd for C₁₉H₂₄N₅[M + H]⁺,

322.2032; found, 322.2032.

CDCl₃): δ 7.60 (d, J = 8.6 Hz, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.36 (d,

J = 8.0 Hz, 2H), 7.28 (d, J = 8.6 Hz, 1H), 7.01 (t, J = 6.0 Hz, 1H),

4.94 (s, 2H), 4.23 (s, 2H), 3.57 (td, J = 13.6, 6.8 Hz, 2H), 1.71 (m,

2H), 1.47 (dq, J = 14.6, 7.4 Hz, 2H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C

NMR (100 MHz, CDCl₃): δ 160.2, 160.0, 153.6, 144.1, 143.4, 132.9,

129.4, 128.9 (q, J = 32.3 Hz), 128.1, 127.8, 125.5 (q, J = 17.0 Hz),

44.0, 40.3, 31.4, 20.2, 13.8. HRMS (ESI): calcd for C₁₉H₂₁F₃N₅[M +

H]⁺, 376.1749; found, 376.1755.

N⁴-Butyl-6-(naphthalen-2-ylmethyl)pyrido[3,2-d]pyrimidine-2,4-

diamine (17e). Yellow solid. Yield: 78.0%. ¹H NMR (400 MHz,

CDCl₃): δ 7.82−7.77 (m, 3H), 7.68 (s, 1H), 7.58 (d, J = 8.6 Hz, 1H),

7.48−7.40 (m, 2H), 7.38 (dd, J = 8.6, 1.6 Hz, 1H), 7.31 (d, J = 8.6

Hz, 1H), 7.09 (t, J = 6.8 Hz, 1H), 5.11 (s, 2H), 4.34 (s, 2H), 3.57 (td,

J = 13.2, 5.8 Hz, 2H), 1.69 (m, 2H), 1.44 (m, 2H), 0.98 (t, J = 7.4 Hz,

3H). ¹³C NMR (100 MHz, CDCl₃): δ 160.3, 160.2, 154.5, 144.4,

136.9, 133.6, 132.9, 132.2, 128.2, 128.1, 128.0, 127.7, 127.6, 127.4,

126.1, 125.6, 44.5, 40.3, 31.5, 20.2, 13.9. HRMS (ESI): calcd for

C₂₂H₂₄N₅[M + H]⁺, 358.2032; found, 358.2029.

2-((2-Amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6-yl)methyl)-

1

benzonitrile (17l). White solid. Yield: 73.0%. H NMR (400 MHz,

CDCl₃): δ 7.68 (d, J = 8.6 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.51 (t, J

= 7.6 Hz, 1H), 7.39−7.27 (m, 3H), 7.19−7.09 (m, 1H), 5.09 (s, 2H),

4.38 (s, 2H), 3.55 (td, J = 13.6, 6.8 Hz, 2H), 1.70 (m, 2H), 1.46 (m,

2H), 0.98 (t, J = 7.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 160.4,

160.2, 152.0, 144.6, 143.2, 133.2, 133.1, 132.9, 130.2, 128.4, 127.5,

127.1, 118.5, 112.9, 42.4, 40.2, 31.3, 20.2, 13.9. HRMS (ESI): calcd

for C₁₉H₂₁N₆[M + H]⁺, 333.1828; found, 333.1826.

N⁴-Butyl-6-(2-ﬂuorobenzyl)pyrido[3,2-d]pyrimidine-2,4-diamine

(17f). White solid. Yield: 73.0%. ¹H NMR (400 MHz, CDCl₃): δ 7.57

(d, J = 8.6 Hz, 1H), 7.30 (d, J = 8.6 Hz, 1H), 7.24−7.16 (m, 2H),

7.11−7.00 (m, 3H), 5.11 (s, 2H), 4.20 (s, 2H), 3.53 (td, J = 13.6, 6.8

Hz, 2H), 1.67 (m, 2H), 1.43 (m, 2H), 0.97 (t, J = 7.4 Hz, 3H). ¹³C

NMR (100 MHz, CDCl₃): δ 160.7 (d, J = 246.6 Hz), 159.9, 153.0,

144.1, 132.6, 131.0 (d, J = 4.4 Hz), 128.4 (d, J = 8.1 Hz), 128.3,

128.1, 127.6, 126.3 (d, J = 16.0 Hz), 124.1 (d, J = 3.6 Hz), 115.1 (d, J

= 22.0 Hz), 107.8, 39.9, 36.8, 36.7, 31.4, 31.1, 19.9, 13.6. HRMS

(ESI): calcd for C₁₈H₂₁FN₅[M + H]⁺, 326.1781; found, 326.1770.

N⁴-Butyl-6-(3-ﬂuorobenzyl)pyrido[3,2-d]pyrimidine-2,4-diamine

3-((2-Amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6-yl)methyl)-

1

benzonitrile (17m). White solid. Yield: 80.0%. H NMR (400 MHz,

MeOD): δ 7.72 (s, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.56 (m, 2H), 7.46

(m, 2H), 4.24 (s, 2H), 3.57 (t, J = 7.1 Hz, 2H), 1.78−1.61 (m, 2H),

1.50−1.39 (m, 2H), 0.99 (t, J = 7.3 Hz, 3H). ¹³C NMR (100 MHz,

MeOD): δ 161.9, 161.6, 155.1, 144.9, 142.8, 135.0, 133.7, 132.8,

131.3, 130.7, 129.2, 129.1, 119.8, 113.5, 44.2, 41.2, 32.5, 21.3, 14.3.

HRMS (ESI): calcd for C₁₉H₂₁N₆[M + H]⁺, 333.1828; found,

333.1833.

4-((2-Amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6-yl)methyl)-

1

benzonitrile (17n). White solid. Yield: 68.0%. H NMR (400 MHz,

CDCl₃): δ 7.78 (d, J = 8.6 Hz, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.40−

7.33 (m, 4H), 6.37−6.00 (m, 2H), 4.26 (s, 2H), 3.62−3.56 (m, 2H),

1.69 (td, J = 13.6, 6.0 Hz, 2H), 1.48−1.41 (m, 2H), 1.00 (t, J = 7.4

Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 159.9, 157.9, 154.5, 144.2,

139.2, 132.4, 130.2, 129.8, 128.6, 127.2, 110.7, 44.0, 40.7, 31.1, 20.1,

13.8. HRMS (ESI): calcd for C₁₉H₂₁N₆[M + H]⁺, 333.1828; found,

333.1826.

1

(17g). White solid. Yield: 77.0%. H NMR (400 MHz, CDCl₃): δ

7.60 (d, J = 8.6 Hz, 1H), 7.52 (t, J = 5.5 Hz, 1H), 7.35 (d, J = 8.5 Hz,

1H), 7.27−7.20 (dd, J = 14.4, 7.2 Hz, 2H), 7.00 (d, J = 7.6 Hz, 1H),

6.90−6.87 (m, 2H), 4.16 (s, 2H), 3.59 (td, J = 13.2, 5.8 Hz, 2H),

1.72−1.64 (m, 2H), 1.47−1.37 (m, 2H), 0.97 (t, J = 7.4 Hz, 3H). ¹³C

NMR (100 MHz, CDCl₃): δ 162.9 (d, J = 246.1 Hz), 160.0, 157.5,

156.0, 141.1 (d, J = 7.3 Hz), 138.8, 130.1 (d, J = 8.3 Hz), 129.6,

128.9, 127.0, 124.7 (d, J = 2.8 Hz), 115.9 (d, J = 21.3 Hz), 113.5 (d, J

= 21.0 Hz), 43.7, 40.7, 31.1, 20.1, 13.7. HRMS (ESI): calcd for

C₁₈H₂₁FN₅[M + H]⁺, 326.1781; found, 326.1796.

N⁴-Butyl-6-(2-methylbenzyl)pyrido[3,2-d]pyrimidine-2,4-dia-

mine (17o). White solid. Yield: 83.0%. ¹H NMR (400 MHz, CDCl₃):

δ 7.66 (d, J = 8.5 Hz, 1H), 7.31 (t, J = 6.0 Hz, 1H), 7.26 (d, J = 8.7

Hz, 1H), 7.19−7.10 (m, 4H), 5.84 (s, 1H), 4.19 (s, 2H), 3.58 (td, J =

13.2, 6.4 Hz, 2H), 2.27 (s, 3H), 1.68 (m, 2H), 1.45 (m, 2H), 0.98 (t, J

= 7.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 160.1, 158.3, 155.8,

139.9, 137.0, 136.7, 130.5, 130.4, 130.0, 128.2, 127.2, 127.0, 126.2,

41.9, 40.5, 31.2, 20.1, 19.8, 13.8. HRMS (ESI): calcd for C₁₉H₂₄N₅

[M + H]⁺, 322.2032; found, 322.2030.

N⁴-Butyl-6-(4-ﬂuorobenzyl)pyrido[3,2-d]pyrimidine-2,4-diamine

1

(17h). White solid. Yield: 75.0%. H NMR (400 MHz, CDCl₃): δ

7.60 (d, J = 8.6 Hz, 1H), 7.29 (d, J = 8.6 Hz, 1H), 7.26−7.19 (m,

2H), 7.07 (t, J = 5.4 Hz, 1H), 7.05−6.96 (m, 2H), 4.97 (s, 2H), 4.17

(s, 2H), 3.59 (td, J = 13.4, 5.8 Hz, 2H), 1.72 (m, 2H), 1.54−1.42 (m,

2H), 1.02 (t, J = 7.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 161.6

(d, J = 243 Hz), 160.2, 160.1, 154.4, 144.3, 135.0 (d, J = 4.0 Hz),

133.0, 130.5 (d, J = 8.0 Hz), 128.0, 127.7, 115.5 (d, J = 8.4 Hz), 43.4,

40.2, 31.4, 20.2, 13.9. HRMS (ESI): calcd for C₁₈H₂₁FN₅[M + H]⁺,

326.1781; found, 326.1802.

N⁴-Butyl-6-(3-methylbenzyl)pyrido[3,2-d]pyrimidine-2,4-dia-

mine (17p). White solid. Yield: 83.0%. ¹H NMR (400 MHz, CDCl₃):

δ 7.57 (d, J = 8.6 Hz, 1H), 7.28 (d, J = 8.8 Hz, 1H), 7.19 (t, J = 7.4

Hz, 1H), 7.12 (s, 1H), 7.12−7.04 (m, 3H), 5.19 (s, 2H), 4.13 (s, 2H),

3.56 (td, J = 13.2, 6.6 Hz, 2H), 2.31 (s, 3H), 1.70 (m, 2H), 1.45 (m,

7525

https://doi.org/10.1021/acs.jmedchem.1c00179

J. Med. Chem. 2021, 64, 7507−7532

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

2H), 0.98 (t, J = 7.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 160.4,

160.3, 154.9, 144.3, 139.4, 138.3, 132.9, 129.9, 128.6, 128.1, 128.0,

127.3, 126.2, 44.4, 40.3, 31.6, 21.5, 20.3, 14.0. HRMS (ESI): calcd for

C₁₉H₂₄N₅[M + H]⁺, 322.2032; found, 322.2034.

2-((2-Amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6-yl)methyl)-

benzoic Acid (17y). ¹H NMR (400 MHz, MeOD): δ 7.60 (d, J = 7.2

Hz, 1H), 7.53 (d, J = 8.6 Hz, 1H), 7.48 (d, J = 8.6 Hz, 1H), 7.32−

7.25 (m, 3H), 4.59 (s, 2H), 3.64 (t, J = 7.3 Hz, 2H), 1.76−1.67 (m,

2H), 1.47−1.37 (m, 2H), 0.99 (t, J = 7.3 Hz, 3H). ¹³C NMR (100

MHz, MeOD): δ 160.4, 159.2, 155.6, 140.3, 136.4, 133.8, 131.1,

129.6, 129.3, 128.5, 126.8, 126.4, 125.7, 42.0, 41.2, 31.1, 20.4, 13.4.

HRMS (ESI): calcd for C₁₉H₂₂N₅O₂[M + H]⁺, 352.1773; found,

352.1785.

N⁴-Butyl-6-(4-methylbenzyl)pyrido[3,2-d]pyrimidine-2,4-dia-

mine (17q). White solid. Yield: 85.0%. ¹H NMR (400 MHz, CDCl₃):

δ 7.59 (d, J = 8.6 Hz, 1H), 7.29 (d, J = 8.6 Hz, 1H), 7.18−7.11 (m,

5H), 5.10 (s, 2H), 4.14 (s, 2H), 3.57 (td, J = 13.2, 5.8 Hz, 2H), 2.32

(s, 3H), 1.71 (m, 2H), 1.48 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C

NMR (100 MHz, CDCl₃): δ 160.3, 159.8, 155.3, 143.6, 136.2, 136.1,

132.5, 129.3, 128.9, 127.9, 127.8, 43.9, 40.3, 31.5, 21.0, 20.2, 13.9.

HRMS (ESI): calcd for C₁₉H₂₄N₅[M + H]⁺, 322.2032; found,

322.2034.

3-((2-Amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6-yl)methyl)-

1

benzoic Acid (17z). White solid. Yield: 75.0%. H NMR (400 MHz,

methanol-d₄): δ 7.98 (s, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.78 (d, J = 8.6

Hz, 1H), 7.66 (d, J = 8.6 Hz, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.42 (t, J

= 7.8 Hz, 1H), 4.33 (s, 2H), 3.70 (td, J = 13.2, 7.4 Hz, 2H), 1.76 (m,

2H), 1.45 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H). ¹³C NMR (100 MHz,

methanol-d₄): δ 168.4, 160.0, 156.0, 154.9, 139.2, 133.3, 133.2, 131.1,

129.9, 129.3, 128.5, 127.7, 126.7, 125.6, 43.0, 40.6, 30.6, 19.8, 12.8.

HRMS (ESI): calcd for C₁₉H₂₂N₅O₂[M + H]⁺, 352.1773; found,

352.1772.

N⁴-Butyl-6-(2-methoxybenzyl)pyrido[3,2-d]pyrimidine-2,4-dia-

mine (17r). White solid. Yield: 86.0%. ¹H NMR (400 MHz, MeOD):

δ 7.47 (d, J = 8.6 Hz, 1H), 7.29 (d, J = 8.6 Hz, 1H), 7.20 (t, J = 7.8

Hz, 1H), 7.12 (d, J = 7.2 Hz, 1H), 6.93 (d, J = 8.2 Hz, 1H), 6.86 (t, J

= 7.4 Hz, 1H), 4.14 (s, 2H), 3.77 (s, 3H), 3.52 (t, J = 6.9 Hz, 2H),

1.70−1.59 (m, 2H), 1.41 (m, 2H), 0.96 (t, J = 7.3 Hz, 3H). ¹³C NMR

(100 MHz, MeOD): δ 161.8, 161.5, 158.8, 156.5, 145.0, 132.6, 131.7,

129.1, 129.0, 128.9, 128.7, 121.6, 111.7, 55.9, 41.2, 39.2, 32.4, 21.2,

14.2. HRMS (ESI): calcd for C₁₉H₂₄N₅O [M + H]⁺, 338.1981; found,

338.1977.

4-((2-Amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6-yl)methyl)-

1

benzoic Acid (17aa). H NMR (400 MHz, MeOD): δ 7.95 (d, J =

8.0 Hz, 2H), 7.72 (d, J = 8.6 Hz, 1H), 7.65 (d, J = 8.6 Hz, 1H), 7.42

(d, J = 7.8 Hz, 2H), 4.34 (s, 2H), 3.69 (t, J = 7.2 Hz, 2H), 1.78−1.68

(m, 2H), 1.51−1.39 (m, 2H), 1.0 (t, J = 7.4 Hz, 3H). ¹³C NMR (101

MHz, MeOD): δ 172.1, 160.1, 157.8, 154.93, 144.1, 136.2, 133.3,

129.8, 129.4, 128.8, 126.4, 125.5, 43.2, 40.6, 30.6, 19.8, 12.8. HRMS

(ESI): calcd for C₁₉H₂₂N₅O₂[M + H]⁺, 352.1773; found, 352.1796.

(4-((2-Amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6-yl)-

methyl)phenyl)methanol (19c). Lithium aluminum hydride (75.9

mg, 2.0 mmol) was weighted out and introduced into a 25 mL oven

dried ﬂask, and the residues were rinsed with dry THF (2 mL) at 0

°C. A solution of 17w (182.7 mg, 0.5 mmol) was added using a

dropping funnel to dry THF. After 2 h, the reaction was quenched by

ordinal addition of water (76 μL), 10% sodium hydroxide solution

(76 μL), and water (228 μL). The mixture was ﬁltrated and

concentrated under reduced pressure. After puriﬁcation by column

chromatography on silica gel (CH₂Cl₂/MeOH 20:1 to 10:1), the pure

product 19c (91.0 mg, 52.0% yield) was obtained as a yellow solid. ¹H

NMR (400 MHz, CDCl₃): δ 7.44 (d, J = 8.6 Hz, 1H), 7.31 (d, J = 7.9

Hz, 2H), 7.21 (d, J = 5.1 Hz, 2H), 7.19 (d, J = 5.7 Hz, 1H), 7.07 (t, J

= 5.9 Hz, 1H), 4.94 (s, 2H), 4.67 (s, 2H), 4.15 (s, 2H), 3.55 (td, J =

13.2, 5.8 Hz, 2H), 1.73−1.65 (m, 2H), 1.49−1.41 (m, 2H), 0.99 (t, J

= 7.3 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 160.3, 160.2, 154.6,

144.4, 139.6, 138.7, 133.0, 129.2, 128.0, 127.7, 127.4, 64.8, 44.0, 40.3,

31.5, 20.2, 13.9. MS (ESI): calcd for C₁₉H₂₃N₅O [M + H]⁺, 338.2;

found, 338.3.

N⁴-Butyl-6-(3-methoxybenzyl)pyrido[3,2-d]pyrimidine-2,4-dia-

mine (17s). White solid. Yield: 82.0%. ¹H NMR (400 MHz, MeOD):

δ 7.52 (d, J = 8.6 Hz, 1H), 7.36 (d, J = 8.6 Hz, 1H), 7.18 (t, J = 7.8

Hz, 1H), 6.79 (m, 3H), 4.91 (s, 7H), 4.13 (s, 2H), 3.74 (s, 3H), 3.54

(t, J = 7.1 Hz, 2H), 1.79−1.57 (m, 2H), 1.55−1.31 (m, 2H), 0.97 (t, J

= 7.3 Hz, 3H). ¹³C NMR (100 MHz, MeOD): δ 160.3, 160.2, 159.9,

155.0, 143.4, 140.9, 131.2, 129.2, 127.8, 127.4, 121.0, 114.4, 111.4,

54.2, 43.6, 39.8, 31.0, 19.8, 12.8. HRMS (ESI): calcd for C₁₉H₂₄N₅O

[M + H]⁺, 338.1981; found, 338.1976.

N⁴-Butyl-6-(4-methoxybenzyl)pyrido[3,2-d]pyrimidine-2,4-dia-

mine (17t). White solid. Yield: 79.0%. ¹H NMR (400 MHz, CDCl₃):

δ 7.56 (d, J = 8.6 Hz, 1H), 7.25 (d, J = 8.6 Hz, 1H), 7.16 (d, J = 8.3

Hz, 2H), 7.07 (t, J = 6.0 Hz, 1H), 6.85 (d, J = 8.3 Hz, 2H), 4.90 (s,

2H), 4.11 (s, 2H), 3.78 (s, 3H), 3.57 (td, J = 13.3, 6.7 Hz, 2H), 1.76−

1.65 (m, 2H), 1.53−1.41 (m, 2H), 1.0 (t, J = 7.6 Hz, 1H). ¹³C NMR

(100 MHz, CDCl₃): δ 160.3, 160.2, 158.3, 155.1, 144.5, 133.0, 131.5,

130.0, 128.0, 127.7, 114.0, 55.3, 43.4, 40.2, 31.5, 20.2, 13.9. HRMS

(ESI): calcd for C₁₉H₂₄N₅O [M + H]⁺, 338.1981; found, 338.1994.

Methyl2-((2-amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6-yl)-

methyl)benzoate (17u). ¹H NMR (400 MHz, CDCl₃): δ 7.92 (d, J =

7.7 Hz, 1H), 7.57 (d, J = 8.6 Hz, 1H), 7.44 (t, J = 7.4 Hz, 1H), 7.36−

7.26 (m, 3H), 7.06 (br s, 1H), 5.02 (s, 2H), 4.58 (s, 2H), 3.81 (s,

3H), 3.52 (dd, J = 13.2, 6.7 Hz, 2H), 1.75−1.62 (m, 2H), 1.43 (dq, J

= 14.5, 7.3 Hz, 2H), 0.98 (t, J = 7.3 Hz, 3H). ¹³C NMR (100 MHz,

CDCl₃): δ 169.0, 160.2, 159.9, 154.1, 143.8, 139.7, 132.5, 131.9,

131.7, 131.0, 130.5, 128.1, 127.8, 126.7, 52.1, 42.0, 40.3, 31.3, 20.2,

13.9. HRMS (ESI): calcd for C₂₀H₂₄N₅O₂[M + H]⁺, 366.1930;

found, 366.1935.

N⁴-Butyl-6-(4-(chloromethyl)benzyl)pyrido[3,2-d]pyrimidine-2,4-

diamine Thionylchloride (20c). Thionyl chloride (182 μL, 2.5 mmol)

was slowly added to a solution of 19c (675 mg, 2.0 mmol) in dry

CH₂Cl₂. The reaction mixture was left to stir at 0 °C for 1 h.

Compound 20c was obtained after evaporation in vacuo and used

without further puriﬁcation.

Methyl3-((2-amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6-yl)-

1

methyl)benzoate (17v). White solid. Yield: 86.0%. H NMR (400

Diethyl(4-((2-amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6-yl)-

methyl)benzyl)phosphonate (21). Compound 20c (92.0 mg, 0.25

mmol) was added to triethyl phosphite (815 μL, 0.32 mmol). The

reaction mixture was heated to reﬂux for 16 h. The solution was then

cooled to rt and concentrated under reduced pressure. The residue

was diluted with diethyl ether (2 × 50 mL) and 2 N NaOH solution

(2 × 20 mL), the combined organic layer was dried over Na₂SO₄,

concentrated, and puriﬁed by ﬂash chromatography on silica gel

(EtOAc/cyclohexane 1:2 to 4:1) to give 21 as a yellowish solid, 68.0%

yield. ¹H NMR (400 MHz, CDCl₃): δ 7.56 (d, J = 8.6 Hz, 1H), 7.27

(d, J = 5.2 Hz, 1H), 7.23 (d, J = 6.4 Hz, 2H), 7.19 (d, J = 8.2 Hz, 2H),

7.08 (t, J = 6.0 Hz, 1H), 5.05 (s, 2H), 4.15 (s, 2H), 4.01 (m, 4H),

3.57 (td, J = 13.0, 6.5 Hz, 2H), 3.21 (d, J = 21.2 Hz, 1H), 1.79−1.60

(m, 2H), 1.47 (dd, J = 14.6, 7.2 Hz, 2H), 1.24 (t, J = 7.0 Hz, 6H),

0.99 (t, J = 7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 160.3,

160.1, 154.6, 144.3, 138.0 (d, J = 3.8 Hz), 132.9, 130.0 (d, J = 6.5

Hz), 129.7 (d, J = 9.2 Hz), 129.2 (d, J = 3.0 Hz), 128.0, 127.8, 62.1

(d, J = 6.7 Hz), 43.9, 40.2, 33.3 (d, J = 138.2 Hz), 31.5, 20.2, 16.4 (d,

MHz, MeOD): δ 7.96 (s, 1H), 7.86 (d, J = 7.7 Hz, 1H), 7.54 (d, J =

8.5 Hz, 2H), 7.45−7.37 (m, 2H), 4.24 (s, 2H), 3.88 (s, 3H), 3.56 (t, J

= 7.0 Hz, 2H), 1.73−1.62 (m, 2H), 1.50−1.37 (m, 2H), 0.98 (t, J =

7.2 Hz, 3H). ¹³C NMR (100 MHz, MeOD): δ 167.1, 160.1, 156.9,

156.5, 139.7, 136.2, 133.6, 130.3, 129.7, 128.9, 128.6, 127.4, 127.2,

126.7, 51.3, 43.0, 40.4, 30.7, 19.8, 12.8. HRMS (ESI): calcd for

C₂₀H₂₄N₅O₂[M + H]⁺, 366.1930; found, 366.1931.

Methyl4-((2-amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6-yl)-

methyl)benzoate (17w). Yellowish solid. Yield: 89.0%. ¹H NMR

(400 MHz, CDCl₃): δ 7.99 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.6 Hz,

1H), 7.33 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.6 Hz, 1H), 7.03 (s, 1H),

5.01 (s, 1H), 4.23 (s, 2H), 3.91 (s, 3H), 3.57 (td, J = 13.2, 6.0 Hz,

2H), 1.76−1.65 (m, 2H), 1.46 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C

NMR (100 MHz, CDCl₃): δ 167.1, 160.5, 160.4, 153.5, 144.9, 144.9,

133.4, 129.9, 129.2, 128.5, 128.4, 127.8, 52.1, 44.3, 40.3, 31.6, 20.3,

13.9. HRMS (ESI): calcd for C₂₀H₂₄N₅O₂[M + H]⁺, 366.1930;

found, 366.1913.

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J = 6.0 Hz), 13.9. ³¹P NMR (162 MHz, CDCl₃): δ 26.50. HRMS

(ESI): calcd for C₂₃H₃₃N₅O₃P [M + H]⁺, 458.2321; found, 458.2320.

(4-((2-Amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6-yl)-

methyl)benzyl)phosphonic Acid (22). To a solution of 21 (173 mg,

0.378 mmol) in DCM (5 mL) was added TMSBr (300 μL, 2.27

mmol). The reaction mixture was stirred at rt for 5 h. When the

reaction was completed, the solvent was removed under reduced

pressure, and the residue was stirred in methanol (5 mL) for 1 h at rt.

The reaction mixture was concentrated and recrystallized (diethyl

2H), 2.36 (s, 3H), 1.68 (dd, J = 15.1, 7.4 Hz, 2H), 1.50−1.40 (m,

2H), 0.99 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, MeOD): δ 160.6,

160.2, 154.9, 143.9, 138.6, 137.0, 131.5, 128.7, 128.4, 127.8, 127.5,

54.6, 43.2, 39.8, 34.1, 31.1, 19.8, 12.8. HRMS (ESI): calcd for

C₂₀H₂₇N₆[M + H]⁺, 351.2297; found, 351.2301.

N⁴-Butyl-6-(4-(((2,2,2-triﬂuoroethyl)amino)methyl)benzyl)-

pyrido[3,2-d]pyrimidine-2,4-diamine (24d). Yellow solid. Yield:

1

70.0%. H NMR (400 MHz, CDCl₃): δ 7.61 (d, J = 8.6 Hz, 1H),

7.30 (d, J = 8.6 Hz, 1H), 7.26 (d, J = 7.6 Hz, 2H), 7.22 (d, J = 7.8 Hz,

2H), 7.20−7.12 (m, 1H), 5.24 (s, 2H), 4.17 (s, 2H), 3.88 (s, 2H),

3.58 (td, J = 13.6, 6.8 Hz, 2H), 3.17 (dd, J = 18.8, 9.2 Hz, 2H), 1.74−

1.68 (m, 2H), 1.49−1.44 (m, 2H), 0.99 (t, J = 7.3 Hz, 3H). ¹³C NMR

(100 MHz, CDCl₃): δ 160.2, 159.4, 155.2, 143.8, 138.3, 137.4, 131.9,

131.6, 129.2, 128.4, 128.1, 127.8, 122.5 (q, J = 226.2 Hz), 52.6, 49.5

(q, J = 62.0 Hz), 43.9, 40.4, 31.4, 20.2, 13.9. HRMS (ESI): calcd for

C₂₁H₂₆F₃N₆[M + H]⁺, 419.2171; found, 419.2181.

1

ether) to give 22 as a white solid, 88.0% yield. H NMR (400 MHz,

DMSO-d₆): δ 12.14 (s, 1H), 9.44 (t, J = 6.2 Hz, 1H), 7.81 (d, J = 8.6

Hz, 1H), 7.69 (d, J = 8.6 Hz, 1H), 7.27 (d, J = 7.8 Hz, 2H), 7.18 (d, J

= 8.0 Hz, 2H), 4.19 (s, 2H), 3.60 (td, J = 16.4, 6.8 Hz, 2H), 2.92 (d, J

= 21.2 Hz, 2H), 1.67 (m, 2H), 1.37 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H).

¹³C NMR (100 MHz, DMSO-d₆): δ 159.9, 158.1, 154.6, 137.0, 133.8,

132.2 (d, J = 8.8 Hz), 130.0 (d, J = 6.4 Hz), 129.9 (d, J = 6.2 Hz),

129.0, 126.6, 126.5, 43.3, 40.9, 35.2 (d, J = 132.8 Hz), 30.9, 20.1, 14.2.

³¹P NMR (162 MHz, DMSO): δ 21.27. HRMS (ESI): calcd for

N⁴-Butyl-6-(4-((dimethylamino)methyl)benzyl)pyrido[3,2-d]-

1

pyrimidine-2,4-diamine (24e). White solid. Yield: 72.0%. H NMR

C₁₉H₂₅N₅O₃P [M + H]⁺, 402.1695; found, 402.1684.

(400 MHz, CDCl₃): δ 7.56 (d, J = 8.6 Hz, 1H), 7.28 (d, J = 8.6 Hz,

1H), 7.24 (d, J = 7.8 Hz, 2H), 7.19 (d, J = 7.8 Hz, 2H), 7.05 (br s,

1H), 4.84 (s, 2H), 4.16 (s, 2H), 3.57 (td, J = 13.6, 6.8 Hz, 2H), 3.39

(s, 2H), 2.23 (s, 6H), 1.70 (m, 2H), 1.47 (m, 2H), 0.99 (t, J = 7.4 Hz,

3H). ¹³C NMR (100 MHz, CDCl₃): δ 160.4, 160.2, 154.7, 144.6,

138.1, 137.1, 131.5, 129.3, 129.0, 128.1, 127.8, 64.1, 45.4, 44.0, 40.2,

31.5, 20.2, 13.9. HRMS (ESI): calcd for C₂₁H₂₉N₆[M + H]⁺,

365.2454; found, 365.2446.

General Procedure H for the Synthesis of 24a and 24g. To a

stirred solution of chlorides 20a/c (0.5 mmol, 1 equiv) in MeOH (10

mL) were added di-tert-butyl iminodicarboxylate (1.5 mmol, 3 equiv)

and K₂CO₃(1.5 mmol, 3 equiv), the reaction mixture was stirred at

45 °C for 5 h. The reaction mixture was then cooled to rt,

concentrated, diluted with ethyl acetate, and washed with brine (2 ×

30 mL). The combined organic layer was dried by Na₂SO₄,

concentrated, and used without further puriﬁcation (23a/b). To

10% TFA in dry DCM was added 23a/b. The reaction mixture was

stirred at 0 °C for 3 h. The reaction mixture was concentrated in

vacuo, and the titled compound was obtained after puriﬁcation by

ﬂash chromatography on silica gel (CH₂Cl₂in the MeOH gradient).

6-(4-(Aminomethyl)benzyl)-N⁴-butylpyrido[3,2-d]pyrimidine-2,4-

diamine (24a). White solid. Yield: 40.0%. ¹H NMR (400 MHz,

MeOD): δ 7.78 (d, J = 8.7 Hz, 1H), 7.64 (d, J = 8.7 Hz, 1H), 7.43

(m, 4H), 4.30 (s, 2H), 4.10 (s, 2H), 3.73 (t, J = 7.2 Hz, 2H), 1.77−

1.69 (m, 2H), 1.52−1.38 (m, 2H), 1.00 (t, J = 7.4 Hz, 2H). ¹³C NMR

(101 MHz, MeOD): δ 160.9, 159.0, 155.7, 140.9, 134.1, 132.3, 130.3,

130.2, 130.0, 127.1, 126.4, 43.9, 43.5, 41.5, 31.5, 20.6, 13.6. HRMS

(ESI): calcd for C₁₉H₂₅N₆[M + H]⁺, 337.2141; found, 337.2127.

6-(4-(Azidomethyl)benzyl)-N⁴-butylpyrido[3,2-d]pyrimidine-2,4-

diamine (24b). Sodium azide (39.0 mg, 0.6 mmol) was dissolved in

DMSO (1 mL) and the solution was degassed with nitrogen for 15

min, followed by addition of compound 20c (177.9 mg, 0.5 mmol).

The reaction mixture was stirred for 10 h at rt. Water (1 mL) was

added slowly to quench the reaction (exothermic process), and the

mixture was stirred until it cooled to rt. The mixture was poured into

water (15 mL) and extracted four times with ethyl ether (30 mL).

The organic layer was separated, washed with brine, dried with

Na₂SO₄, ﬁltered, and concentrated in vacuo to aﬀord 24b as a white

1-(4-((2-Amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6-yl)-

methyl)phenyl)N,N,N-trimethylmethanaminium Chloride (24f). To

a solution of N⁴-butyl-6-(4-(chloromethyl)benzyl)pyrido[3,2-d]-

pyrimidine-2,4-diamine (20c, 173 mg, 0.50 mmol) in methanol (10

mL), trimethylamine (3.2 M in methanol, 156 μL, 0.5 mmol) was

added. The reaction mixture was reﬂuxed for 6 h. The reaction

mixture was then cooled to rt, concentrated, and recrystallized (EA)

1

to aﬀord 24f as a white solid. Yield: 85.0%. H NMR (400 MHz,

DMSO-d₆): δ 7.75 (t, J = 6.0 Hz, 1H), 7.55−7.39 (m, 6H), 6.25 (s,

2H), 4.51 (s, 2H), 4.20 (s, 2H), 3.49 (td, J = 13.8, 5.8 Hz, 2H), 3.01

(s, 9H), 1.61 (m, 2H), 1.36 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H). ¹³C

NMR (100 MHz, methanol-d₄): δ 160.7, 160.2, 153.0, 145.3, 142.6,

133.4, 132.8, 130.9, 129.8, 128.2, 128.0, 126.8, 67.9, 52.2, 52.1, 43.5,

+

31.4, 20.2, 14.3. HRMS (ESI): calcd for C₂₂H₃₁N₆[M]⁺, 379.2610;

found, 379.2608.

6-(2-(Aminomethyl)benzyl)-N⁴-butylpyrido[3,2-d]pyrimidine-2,4-

diamine (24g). White solid. Yield: 47.0%. ¹H NMR (400 MHz,

MeOD): δ 7.51 (d, J = 8.7 Hz, 1H), 7.38 (d, J = 8.7 Hz, 1H), 7.31−

7.12 (m, 4H), 4.18 (s, 2H), 3.82 (s, 2H), 3.56 (t, J = 7.3 Hz, 2H),

1.68 (dd, J = 14.5, 7.5 Hz, 2H), 1.43 (dd, J = 14.9, 7.5 Hz, 2H), 0.99

(t, J = 7.3 Hz, 2H). ¹³C NMR (101 MHz, MeOD): δ 160.6, 160.2,

154.8, 143.9, 140.4, 140.0, 131.5, 128.6, 128.4, 128.0, 127.8, 127.5,

125.5, 44.7, 43.5, 39.8, 31.1, 19.8, 12.8. HRMS (ESI): calcd for

C₁₉H₂₅N₆[M + H]⁺, 337.2141; found, 337.2148.

1

solid, 73.0% yield. H NMR (400 MHz, CDCl₃): δ 7.52 (d, J = 8.6

N⁴-Butyl-6-(2-((dimethylamino)methyl)benzyl)pyrido[3,2-d]-

pyrimidine-2,4-diamine (24h). Yellowish solid. Yield: 67.0%. ¹H

NMR (400 MHz, CDCl₃): δ 7.54 (d, J = 8.6 Hz, 1H), 7.28 (d, J = 8.6

Hz, 1H), 7.30−7.21 (m, 4H), 7.04 (br s, 1H), 4.95 (s, 2H), 4.34 (s,

2H), 3.56 (dd, J = 13.2, 6.7 Hz, 2H), 3.39 (s, 2H), 2.19 (s, 6H),

1.70−1.66 (m, 2H), 1.48−1.42 (m, 2H), 0.98 (t, J = 7.3 Hz, 3H). ¹³C

NMR (100 MHz, CDCl₃): δ 160.3, 160.1, 155.0, 144.2, 138.6, 137.4,

132.7, 130.6, 130.5, 127.9, 127.8, 127.4, 126.5, 62.2, 45.5, 40.9, 40.2,

31.4, 20.2, 13.9. HRMS (ESI): calcd for C₂₁H₂₉N₆[M + H]⁺,

365.2454; found, 365.2457.

Hz, 1H), 7.21 (d, J = 10.6 Hz, 5H), 7.01 (t, J = 5.7 Hz, 1H), 5.00 (s,

2H), 4.25 (s, 2H), 4.12 (s, 2H), 3.50 (td, J = 13.2, 5.8 Hz, 2H), 1.70−

1.59 (m, 2H), 1.47−1.33 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H). ¹³C NMR

(100 MHz, CDCl₃): δ 160.4, 160.3, 154.34, 144.4, 139.7, 133.7,

133.0, 129.6, 128.6, 128.2, 127.9, 54.6, 44.0, 40.4, 31.6, 20.3, 14.0.

LC−MS (ESI): calcd for C₁₉H₂₃N₈[M + H]⁺, 363.20; found, 363.40.

General Procedure I for the Synthesis of 24c−f,h−i,k−m,

25a−c, 33−34, and 36−37. To a stirred solution of chlorides 20a−

f, 31a, or 32a−c (0.5 mmol, 1 equiv) in MeOH (10 mL) were added

the corresponding alkylamines (1.5 mmol, 3 equiv) and K₂CO₃(1.5

mmol, 3 equiv), the reaction mixture was stirred at 45 °C for 5 h. The

reaction mixture was then cooled to rt, concentrated, diluted with

ethyl acetate, and washed with brine (2 × 30 mL), and the combined

organic layer was dried by Na₂SO₄. The titled compounds were

obtained after puriﬁcation by ﬂash chromatography on silica gel

(CH₂Cl₂in the MeOH gradient).

N⁴-Butyl-6-(3-((dimethylamino)methyl)benzyl)pyrido[3,2-d]-

pyrimidine-2,4-diamine (24i). Yellowish solid. Yield: 63.0%. ¹H

NMR (400 MHz, CDCl₃): δ 7.56 (d, J = 8.6 Hz, 1H), 7.29−7.22 (m,

3H), 7.14 (t, J = 8.0 Hz, 2H), 7.08 (br s, 1H), 4.87 (s, 2H), 4.17 (s,

2H), 3.57 (dd, J = 13.2, 6.7 Hz, 2H), 3.39 (s, 2H), 2.23 (s, 6H),

1.74−1.67 (m, 2H), 1.52−1.42 (m, 2H), 1.00 (t, J = 7.3 Hz, 3H). ¹³C

NMR (100 MHz, MeOD): δ 162.0, 161.6, 156.3, 151.0, 145.2, 141.2,

138.9, 132.8, 131.4, 129.6, 129.4, 129.2, 128.9, 64.8, 45.4, 44.9, 41.2,

32.5, 21.2, 14.2. HRMS (ESI): calcd for C₂₁H₂₉N₆[M + H]⁺,

365.2454; found, 365.2447.

N⁴-Butyl-6-(4-((methylamino)methyl)benzyl)pyrido[3,2-d]-

1

pyrimidine-2,4-diamine (24c). White solid. Yield: 65.0%. H NMR

(400 MHz, MeOD): δ 7.52 (d, J = 8.6 Hz, 1H), 7.36 (d, J = 8.6 Hz,

1H), 7.26 (m, 4H), 4.17 (s, 2H), 3.66 (s, 2H), 3.56 (t, J = 7.1 Hz,

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N⁴-Butyl-6-(4-(methoxymethyl)benzyl)pyrido[3,2-d]pyrimidine-

2,4-diamine (24j). To a solution of 20c (35 mg, 0.1 mmol) in

methanol (5 mL) was added K₂CO₃(41 mg, 0.3 mmol). The reaction

mixture was reﬂuxed for 5 h. After the reaction was completed, the

resulting mixture was cooled to rt and then ﬁltered oﬀ. The ﬁltrate

was concentrated to aﬀord the crude product. The titled compound

was obtained after puriﬁcation by ﬂash chromatography on silica gel

(CH₂Cl₂in the MeOH gradient). White solid. Yield: 55.0%. ¹H NMR

(400 MHz, CDCl₃): δ 7.65 (d, J = 8.6 Hz, 1H), 7.38 (d, J = 8.6 Hz,

1H), 7.31−7.25 (m, 4H), 7.16 (t, J = 6.0 Hz, 1H), 6.30 (s, 2H), 4.47

(s, 2H), 4.27 (s, 2H), 3.58 (td, J = 13.4, 6.6 Hz, 2H), 3.35 (s, 3H),

1.70 (m, 2H), 1.46 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C NMR (100

MHz, CDCl₃): δ 160.2, 159.0, 155.5, 141.4, 138.4, 135.7, 131.5,

129.7, 129.1, 128.3, 127.5, 73.0, 58.2, 40.8, 40.4, 31.2, 20.0, 13.8.

HRMS (ESI): calcd for C₂₀H₂₆N₅O [M + H]⁺, 352.2137; found,

352.2145.

3.79 (dd, J = 10.8, 5.6 Hz, 2H), 3.70 (t, J = 4.6 Hz, 4H), 3.66 (t, J =

5.3 Hz, 2H), 3.47 (s, 2H), 3.44 (s, 3H), 2.43 (t, J = 4.6 Hz, 4H). ¹³C

NMR (100 MHz, CDCl₃): δ 160.4, 160.0, 154.9, 144.6, 138.3, 135.9,

133.0, 129.5, 128.9, 128.0, 127.9, 71.0, 67.0, 63.1, 58.9, 53.6, 44.0,

40.2. HRMS (ESI): calcd for C₂₂H₂₉N₆O₂[M + H]⁺, 409.2352;

found, 409.2360.

General Procedure J for the Synthesis of 26a−c. To a

solution of compound 17aa (0.5 mmol, 1 equiv) in DMF (5 mL)

were added HATU (0.75 mmol, 1.5 equiv), DIEA (1 mmol, 2 equiv),

and the corresponding alkylamine (0.75 mmol, 1.5 equiv), and the

reaction mixture was stirred at rt for 3 h. The resulting mixture was

then diluted with ethyl acetate and washed with water and saturated

brine. The combined organic layer was dried over sodium sulfate, and

the solvent was removed. The residue was puriﬁed by ﬂash

chromatography on silica gel (CH₂Cl₂in the MeOH gradient)

yielding the products.

N⁴-Butyl-6-(4-(pyrrolidin-1-ylmethyl)benzyl)pyrido[3,2-d]-

4-((2-Amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6-yl)methyl)-

1

pyrimidine-2,4-diamine (24k). White solid. Yield: 76.0%. H NMR

N,N-dimethylbenzamide (26a). White solid. Yield: 72.0%. H NMR

(400 MHz, MeOD): δ 7.52 (d, J = 8.6 Hz, 1H), 7.37 (d, J = 8.6 Hz,

1H), 7.35−7.19 (m, 4H), 4.17 (s, 2H), 3.62−3.50 (m, 4H), 2.52 (m,

4H), 1.79 (m, 4H), 1.68−1.60 (m, 2H), 1.60−1.32 (m, 2H), 0.99 (t, J

= 7.3 Hz, 3H). ¹³C NMR (100 MHz, MeOD): δ 154.8, 143.9, 138.7,

136.0, 131.5, 129.3, 128.6, 127.8, 59.7, 53.4, 43.2, 39.8, 31.1, 22.7,

19.8, 12.8. HRMS (ESI): calcd for C₂₃H₃₁N₆[M + H]⁺, 391.2610;

found, 391.2610.

(400 MHz, MeOD): δ 7.57 (d, J = 8.7 Hz, 1H), 7.45 (d, J = 8.6 Hz,

1H), 7.41−7.34 (m, 4H), 4.24 (s, 2H), 3.60 (t, J = 7.2 Hz, 2H), 3.08

(s, 3H), 2.98 (s, 3H), 1.72−1.64 (m, 2H), 1.48−1.39 (m, 2H), 1.00

(d, J = 7.4 Hz, 2H). ¹³C NMR (100 MHz, MeOD): δ 172.3, 160.2,

159.3, 155.1, 141.6, 141.5, 134.0, 130.2, 128.8, 128.2, 127.3, 127.0,

43.3, 40.0, 38.7, 34.3, 31.0, 19.8, 12.8. HRMS (ESI): calcd for

C₂₁H₂₇N₆O [M + H]⁺, 379.2246; found, 379.2249.

N⁴-Butyl-6-(4-(morpholinomethyl)benzyl)pyrido[3,2-d]-

pyrimidine-2,4-diamine (24l). Yellowish solid. Yield: 69.0%. ¹H

NMR (400 MHz, CDCl₃): δ 7.65 (d, J = 8.5 Hz, 1H), 7.32 (d, J = 8.7

Hz, 1H), 7.27 (d, J = 7.6 Hz, 2H), 7.35 (s, 1H), 7.18 (d, J = 7.8 Hz,

2H), 5.83 (s, 2H), 4.16 (s, 2H), 3.70 (t, J = 4.7 Hz, 4H), 3.58 (td, J =

13.4, 6.8 Hz, 2H), 3.47 (s, 2H), 2.43 (t, J = 4.7 Hz, 4H), 1.80−1.66

(m, 2H), 1.56−1.46 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H). ¹³C NMR

(100 MHz, CDCl₃): δ 160.2, 158.8, 155.7, 141.0, 137.8, 136.1, 131.0,

129.5, 128.9, 128.4, 127.3, 67.0, 63.1, 53.6, 43.9, 40.5, 31.3, 20.2, 13.8.

HRMS (ESI): calcd for C₂₃H₃₁N₆O [M + H]⁺, 407.2559; found,

407.2558.

(4-((2-Amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6-yl)-

methyl)phenyl)(pyrrolidin-1-yl)methanone (26b). White solid.

1

Yield: 89.0%. H NMR (400 MHz, CDCl₃): δ 7.57 (d, J = 8.6 Hz,

1H), 7.47 (d, J = 8.2 Hz, 2H), 7.29 (d, J = 8.6 Hz, 1H), 7.25 (d, J =

8.0 Hz, 2H), 7.10 (s, 1H), 5.02 (s, 2H), 4.20 (s, 2H), 3.63 (t, J = 6.9

Hz, 2H), 3.56 (td, J = 13.2, 6.0 Hz, 2H), 1.94 (m, 2H), 1.85 (m, 2H),

1.76−1.63 (m, 2H), 1.53−1.36 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H). ¹³C

NMR (100 MHz, CDCl₃): δ 169.7, 160.4, 160.0, 154.4, 144.0, 141.3,

135.6, 132.8, 129.0, 128.1, 128.1, 127.6, 49.8, 46.3, 44.2, 40.4, 31.6,

26.5, 24.6, 20.3, 14.0. HRMS (ESI): calcd for C₂₃H₂₉N₆O [M + H]⁺,

405.2403; found, 405.2394.

6-(4-((2-Oxa-6-azaspiro[3.3]heptan-6-yl)methyl)benzyl)-N⁴-

butylpyrido[3,2-d]pyrimidine-2,4-diamine (24m). ¹H NMR (400

MHz, CDCl₃): δ 7.56 (d, J = 8.6 Hz, 1H), 7.26 (d, J = 8.6 Hz, 1H),

7.18 (m, 4H), 7.08 (br s, 1H), 5.09 (s, 2H), 4.72 (s, 4H), 4.15 (s,

2H), 3.57 (td, J = 13.2, 5.9 Hz, 2H), 3.50 (s, 2H), 3.35 (s, 4H), 1.73−

1.66 (m, 2H), 1.51−1.42 (m, 2H), 1.00 (t, J = 7.3 Hz, 3H). ¹³C NMR

(100 MHz, CDCl₃): δ 160.3, 160.1, 154.7, 144.2, 138.3, 136.0, 132.8,

129.1, 128.7, 128.0, 127.9, 81.4, 63.7, 63.2, 44.0, 40.3, 39.1, 31.5, 20.2,

13.9. HRMS (ESI): calcd for C₂₄H₃₁N₆O [M + H]⁺, 419.2559; found,

419.2561.

(4-((2-Amino-4-(butylamino)pyrido[3,2-d]pyrimidin-6yl)methyl)-

phenyl)(morpholino) Methanone (26c). Yellow solid. Yield: 35.0%.

¹H NMR (400 MHz, MeOD): δ 7.54 (d, J = 8.6 Hz, 1H), 7.44 (d, J =

8.6 Hz, 1H), 7.41 (d, J = 7.8 Hz, 2H), 7.37 (d, J = 7.8 Hz, 2H), 4.24

(s, 2H), 3.72−3.45 (m, 10H), 1.73−1.65 (m, 2H), 1.46 (dt, J = 14.9,

7.4 Hz, 2H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, MeOD):

δ 171.1, 160.3, 160.2, 154.4, 143.3, 142.0, 133.1, 131.2, 128.9, 128.8,

127.9, 127.1, 66.4, 43.3, 39.8, 31.0, 19.8, 12.8. HRMS (ESI): calcd for

C₂₃H₂₉N₆O₂[M + H]⁺, 421.2353; found, 421.2364.

N⁴-Butyl-6-(4-((dimethylamino)methyl)phenethyl)pyrido[3,2-d]-

1

6-(4-((Dimethylamino)methyl)benzyl)-N⁴-(2-methoxyethyl)-

pyrido[3,2-d]pyrimidine-2,4-diamine (25a). White solid. Yield:

pyrimidine-2,4-diamine (33). White solid. Yield: 65.0%. H NMR

(400 MHz, CDCl₃): δ 7.55 (d, J = 8.6 Hz, 1H), 7.26 (d, J = 8.6 Hz,

1H), 7.21 (d, J = 7.6 Hz, 2H), 7.15 (d, J = 7.6 Hz, 2H), 7.06 (t, J = 6.0

Hz, 1H), 5.07 (s, 2H), 3.56 (td, J = 13.4, 6.0 Hz, 2H), 3.38 (s, 2H),

3.14−3.04 (m, 4H), 2.23 (s, 6H), 1.74−1.66 (m, 2H), 1.49−1.43 (m,

2H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 160.2,

160.1, 155.1, 144.6, 140.2, 136.5, 132.6, 129.2, 128.3, 128.1, 127.7,

64.1, 45.3, 40.3, 39.5, 35.3, 31.5, 20.2, 13.9. HRMS (ESI): calcd for

C₂₂H₃₁N₆[M + H]⁺, 379.2610; found, 379.2622.

1

68.0%. H NMR (400 MHz, CDCl₃): δ 7.58 (d, J = 8.6 Hz, 1H),

7.40 (t, J = 5.8 Hz, 1H), 7.29 (d, J = 8.6 Hz, 1H), 7.26 (d, J = 7.9 Hz,

2H), 7.21 (d, J = 7.9 Hz, 2H), 5.06 (s, 2H), 4.17 (s, 2H), 3.79 (td, J =

13.2, 5.4 Hz, 2H), 3.65 (t, J = 5.4 Hz, 2H), 3.44 (s, 3H), 3.42 (s, 2H),

2.25 (s, 6H). ¹³C NMR (100 MHz, CDCl₃): δ 160.3, 159.7, 155.1,

144.0, 138.2, 136.6, 132.6, 129.5, 129.0, 128.0, 127.9, 71.0, 63.9, 59.0,

45.2, 44.0, 40.2. HRMS (ESI): calcd for C₂₀H₂₇N₆O [M + H]⁺,

367.2246; found, 367.2239.

N⁴-Butyl-6-(4-(2-(dimethylamino)ethyl)benzyl)pyrido[3,2-d]-

1

N⁴-(2-Methoxyethyl)-6-(4-(pyrrolidin-1-ylmethy-l)benzyl)pyrido-

pyrimidine-2,4-diamine (34). H NMR (400 MHz, CDCl₃): δ 7.56

1

[3,2-d]pyrimidine-2,4-diamine (25b). Yellow solid. Yield: 70.0%. H

(d, J = 8.6 Hz, 1H), 7.28 (d, J = 8.6 Hz, 1H), 7.19−7.12 (m, 4H),

7.10 (t, J = 5.5 Hz, 1H), 5.09 (s, 2H), 4.14 (s, 2H), 3.56 (td, J = 13.2,

7.1 Hz, 2H), 2.76 (dd, J = 9.5, 6.6 Hz, 2H), 2.53 (dd, J = 9.5, 6.7 Hz,

2H), 2.30 (s, 6H), 1.75−1.64 (m, 2H), 1.46 (dq, J = 14.6, 7.3 Hz,

2H), 0.99 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 160.2,

160.0, 154.8, 144.1, 138.4, 137.0, 132.7, 129.1, 128.8, 127.9, 127.8,

76.7, 61.5, 45.4, 43.9, 40.2, 33.8, 31.4, 20.2, 13.9. HRMS (ESI): calcd

for C₂₂H₃₁N₆O [M + H]⁺, 379.2610; found, 379.2619.

NMR (400 MHz, CDCl₃): δ 7.57 (d, J = 8.6 Hz, 1H), 7.37 (s, 1H),

7.30 (m, 3H), 7.21 (d, J = 8.0 Hz, 2H), 5.01 (s, 2H), 4.17 (s, 2H),

3.79 (td, J = 10.8, 5.6 Hz, 2H), 3.66 (t, J = 5.7 Hz, 2H), 3.64 (s, 2H),

3.44 (s, 3H), 2.59 (t, J = 6.4 Hz, 4H), 1.82 (m, 4H). ¹³C NMR (100

MHz, CDCl₃): δ 160.4, 159.85, 155.0, 144.2, 138.3, 136.3, 132.7,

129.4, 129.0, 123.0, 128.0, 71.0, 60.1, 59.0, 54.0, 44.0, 40.2, 23.4.

HRMS (ESI): calcd for C₂₂H₂₉N₆O [M + H]⁺, 393.2403; found,

393.2398.

N⁴-Butyl-6-(4-((dimethylamino)methyl)-2-methoxybenzyl)-

pyrido[3,2-d]pyrimidine-2,4-diamine (36). ¹H NMR (400 MHz,

CDCl₃): δ 7.54 (d, J = 8.6 Hz, 1H), 7.28 (d, J = 8.6 Hz, 1H), 7.07 (m,

2H), 6.89 (s, 1H), 6.81 (dd, J = 7.6, 1.5 Hz, 1H), 4.90 (s, 2H), 4.16

(s, 2H), 3.82 (s, 3H), 3.55 (dt, J = 7.2, 5.9 Hz, 2H), 3.40 (s, 2H), 2.25

N⁴-(2-Methoxyethyl)-6-(4-(morpholinomethyl)benzyl)pyrido[3,2-

d]pyrimidine-2,4-diamine (25c). Yellow solid. Yield: 71.0%. ¹H

NMR (400 MHz, CDCl₃): δ 7.56 (d, J = 8.6 Hz, 1H), 7.36 (s, 1H),

7.28 (m, 3H), 7.21 (d, J = 7.8 Hz, 2H), 4.97 (s, 2H), 4.16 (s, 2H),

7528

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Article

(s, 6H), 1.74−1.64 (m, 2H), 1.46 (dq, J = 14.7, 7.4 Hz, 2H), 0.98 (t, J

= 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 160.3, 160.0, 157.4,

155.0, 144.3, 134.0, 132.7, 130.3, 128.0, 127.8, 126.6, 121.2, 111.1,

64.5, 55.5, 45.5, 40.2, 38.2, 31.5, 20.2, 13.9. HRMS (ESI): calcd for

C₂₂H₃₁N₆O [M + H]⁺, 395.2559; found, 395.2556.

replaced with fresh medium 1 day prior to injection. CT26 cells (1.0

× 10⁵) with a viability of more than 80% were implanted

subcutaneously into the right sides of 6−8 weeks old female BALB/

c mice, 5 or 10 mice were used per group. Mice were treated when

tumor’s volume reached 100 mm³(V = 1/2ab²: a represents the long

diameter; b represents the short diameter of tumor). 24e, 24m, 25a or

R848 was administered once per week. IgG isotype control antibody,

anti-PD-1 antibodies, or anti-PD-L1 antibodies (200 μg per injection)

were injected intraperitoneally twice per week. Tumors were

measured every 3 days, and then the tumor growth curve of each

group was drawn using Prism software (GraphPad software version

7.0). Mice that had no visible or palpable tumors that could be

measured on consecutive measurement days were considered as

“complete regressions”. Animals were euthanized if they exhibited

signs of distress or when the total tumor size reached 2000 mm³.

Tumor-Inﬁltrating Immune Cell Isolation and Flow Cytom-

etry Analysis. To further clarify the mechanism by which TLR7/8

agonist 24e, R848 exerted antitumor immunity, we detected the

inﬁltrating immune cells in tumor tissue by ﬂow cytometry. Tumors

separated from mice were minced with scissors and then digested with

a tumor dissociation kit (MACS, cat # 130-095-929) and ground

using a gentle MACS octo dissociator instrument to generate single-

cell suspensions. The cells were washed and then stained with

antibodies for 15 min in the dark and then detected by ﬂow

cytometry. For intracellular staining, cells were further permeabilized

using a ﬁxation and permeabilization kit (BD, cat # 554714) and

stained by antibodies (PE-cy7-CD3, FITCCD4, PE-FOXP3, Percp-

CD8, APC-CD25, bv410-CD11c, bv510-CD11b, APC-cy7-MHCII,

PE-cy594-F4/80, and APC-CD206). Data were acquired using the

LSRII ﬂow cytometer (BD Biosciences) and analyzed using FlowJo

software (FlowJo 7.6).

N⁴-Butyl-6-(4-((dimethylamino)methyl)-3-methoxybenzyl)-

pyrido[3,2-d]pyrimidine-2,4-diamine (37). ¹H NMR (400 MHz,

CDCl₃): δ 7.57 (d, J = 8.6 Hz, 1H), 7.28 (d, J = 8.6 Hz, 1H), 7.21 (d,

J = 7.6 Hz, 1H), 7.07 (t, J = 5.8 Hz, 1H), 6.81 (d, J = 7.6 Hz, 1H),

6.75 (s, 1H), 4.92 (s, 2H), 4.16 (s, 2H), 3.78 (s, 3H), 3.57 (td, J =

13.5, 6.8 Hz, 2H), 3.45 (s, 2H), 2.28 (s, 6H), 1.70 (dt, J = 14.9, 7.4

Hz, 2H), 1.47 (dd, J = 15.0, 7.5 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H). ¹³C

NMR (101 MHz, CDCl₃): δ 160.3, 160.1, 158.0, 154.6, 144.5, 139.8,

133.0, 131.2, 128.0, 127.8, 124.5, 120.9, 111.3, 57.6, 55.5, 45.3, 44.4,

40.2, 31.5, 20.2, 13.9. HRMS (ESI): calcd for C₂₂H₃₁N₆O [M + H]⁺,

395.2559; found, 395.2562.

HEK-Blue hTLR-7- and 8-Speciﬁc Reporter Gene Assays (NF-

κB Induction). Compounds were screened for agonistic activities

using reporter cell lines speciﬁc for human TLR2, TLR3, TLR4,

TLR7, TLR8, or TLR9.^23,45,50SEAP reporter HEK293 cells

expressing the corresponding hTLRs gene were maintained in a

growth medium in the presence of appropriate selection antibiotics.

The SEAP reporter gene is stably expressed under the control of NF-

kB and AP-1-inducible promoter. Stimulation with TLR ligands

induced activation of NF-kB and AP-1, followed by the production of

SEAP, and the levels of SEAP can be determined using HEK-Blue

detection. Reporter cells were incubated at a corresponding density

according to operating instruction in a volume of 180 μL/well, in ﬂat-

bottom 96-well plates, and treated with escalating concentrations of

stimuli. After incubation for 6−16 h, SEAP was determined using a

spectrophotometer at 620 nm. As HEK293 cells express endogenous

levels of TLR3, TLR5, and NOD1, HEK-Blue Null2-k cells that do

not express TLR7 and TLR8 were selected as the parental cell line of

HEK-Blue TLR7, 8 cells.

Multiplexed Immunoassays for Cytokines in hPBMCs.

hPBMCs were isolated from human peripheral blood of healthy

volunteers by Ficoll−Hypaque gradient centrifugation, with informed

consent and following the guidelines approved by the Human Subjects

Experimentation Committee of Tsinghua University. The hPBMCs (2.0

× 10⁶cells/mL) from one donor were stimulated for 12 h with

escalating concentrations of test compounds or 24 h with one

concentration of test compounds. After centrifugation, supernatants

were separated and then determined in duplicates using the human

cytokine/chemokine magnetic bead panel kit (catalog # HCYTMAG-

60K-PX41, Merck Millipore, Billerica, USA) according to the

technical guidelines and protocol. The analytes below were

simultaneously quantiﬁed using a Luminex instrument: VEGF,

TNF-β, TNF-α, MIP-1β, MIP-1α, MCP-1, IP-10, IL-17A, IL-15,

IL-13, IL-12 (p70), IL-12 (p40), GM-CSF, G-CSF, IL-10, IL-8, IL-7,

IL-6, IL-5, IL-4, IL-3, IL-2, IL-1ra, IL-1β, IL-1α, IFN-γ, IFN-α2,

eotaxin, and EGF. The hPBMCs (3.0 × 10⁶cells/mL) from ﬁve

donors were stimulated for 24 h with one concentration of test

compounds. After centrifugation, supernatants were separated and

then determined in duplicate using the human cytokine/chemokine

magnetic bead panel kit (catalog # HCYTMAG-60K-10, Merck

Millipore, Billerica, USA). The analytes below were quantiﬁed using a

Luminex instrument: TNF-α, MIP-1α, MCP-1, IP-10, IL-12 (p40),

IL-10, IL-6, IL-1β, IFN-γ, and IFN-α2.

Statistical Analysis. One way ANOVA with Tukey’s multiple

comparison test was performed through GraphPad Prism (version

8.0.2) for comparisons of multiple groups. Survival analysis was

performed using Kaplan−Meier curves and evaluated with log-rank

Mantel−Cox tests. The synergy statistics was performed with SPSS

software. The data were displayed as means SEM, unless otherwise

noted. Statistic diﬀerences were denoted as follows: *P < 0.05, **P <

0.01, ***P < 0.001, ****P < 0.0001, and NS not signiﬁcant.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00179.

■

sı

Homology and similarity analysis between TLR7 and 8;

agonistic activities of compounds 24e, 25a, and 26a in

other homologous TLRs; supplementary multiplexed

immunoassays for cytokines; synergy analysis of tumor

size and the survival rate; representative ﬂow cytometric

analysis of tumor-inﬁltrating immune cells; detection of

absolute counts of tumor-inﬁltrating immune cells and

synergy analysis; relationships between the TVs and the

amounts of immune cells; phospholipidosis detection of

24e, 24m, and 25a; synthesis of compounds 16a, 27,

and 35; NMR spectra of target compounds; and HPLC

traces of compounds 5, 24e, 24k, 24m, 25a, and 34

(PDF)

Tumor Treatment Experiments. BALB/c mice were housed

according to the rules of Tsinghua University Laboratory Animal

Research Center, Animal Care and Use Committee. All animals were

maintained under pathogen-free conditions and cared for in

accordance with the International Association for Assessment and

Accreditation of Laboratory Animal Care policies and certiﬁcation.

CT26 cells were cultured in Dulbecco’s modiﬁed Eagle’s medium

containing 10% fetal bovine serum and cultured in a 5% CO₂

incubator at 37 °C. The cells are routinely passaged and digested

for 1−3 min by 0.25% trypsin ethylenediaminetetraacetic acid (Gibco,

cat # 25200-056, a cell passaging ratio of 1:3, and cell density less than

80% passage). Cells were passaged 3 days prior to inoculation and

Molecular formula strings (CSV)

Molecular docking for GS-9620 into TLR7 and

molecular docking for 17d, 24e, 24m, and 34, into

TLR7 and TLR8 (ZIP)

AUTHOR INFORMATION

Corresponding Author

■

Xuebin Liao − School of Pharmaceutical Sciences, Key

Laboratory of Bioorganic Phosphorus Chemistry & Chemical

7529

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J. Med. Chem. 2021, 64, 7507−7532

Journal of Medicinal Chemistry

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Article

Biology (Ministry of Education), Beijing Advanced

Innovation Center for Human Brain Protection and Joint

Graduate Program of Peking-Tsinghua-NIBS, School of Life

Sciences, Tsinghua University, Beijing 100084, China;

orcid.org/0000-0002-0290-894X; Phone: (+86)6279-

8433; Email: liaoxuebin@mail.tsinghua.edu.cn;

Fax: (+86)6279-8433

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00179

Author Contributions

^∥Z.W., Y.G., and L.H. contributed equally to this work.

Notes

The authors declare no competing ﬁnancial interest.

Authors

ACKNOWLEDGMENTS

Zhisong Wang − School of Pharmaceutical Sciences, Key

Laboratory of Bioorganic Phosphorus Chemistry & Chemical

Biology (Ministry of Education), Beijing Advanced

Innovation Center for Human Brain Protection and Joint

Graduate Program of Peking-Tsinghua-NIBS, School of Life

Sciences, Tsinghua University, Beijing 100084, China

Yan Gao − School of Pharmaceutical Sciences, Key Laboratory

of Bioorganic Phosphorus Chemistry & Chemical Biology

(Ministry of Education), Beijing Advanced Innovation Center

for Human Brain Protection, Tsinghua University, Beijing

100084, China; Central Laboratory, Beijing Obstetrics and

Gynecology Hospital, Capital Medical University, Beijing

100026, China

Lei He − School of Pharmaceutical Sciences, Key Laboratory of

Bioorganic Phosphorus Chemistry & Chemical Biology

(Ministry of Education), Beijing Advanced Innovation Center

for Human Brain Protection, Tsinghua University, Beijing

100084, China

Shuhao Sun − School of Pharmaceutical Sciences, Key

Laboratory of Bioorganic Phosphorus Chemistry & Chemical

Biology (Ministry of Education), Beijing Advanced

Innovation Center for Human Brain Protection, Tsinghua

University, Beijing 100084, China

Tingting Xia − School of Pharmaceutical Sciences, Key

Laboratory of Bioorganic Phosphorus Chemistry & Chemical

Biology (Ministry of Education), Beijing Advanced

Innovation Center for Human Brain Protection, Tsinghua

University, Beijing 100084, China

Lu Hu − School of Pharmaceutical Sciences, Key Laboratory of

Bioorganic Phosphorus Chemistry & Chemical Biology

(Ministry of Education), Beijing Advanced Innovation Center

for Human Brain Protection, Tsinghua University, Beijing

100084, China

Licheng Yao − School of Pharmaceutical Sciences, Key

Laboratory of Bioorganic Phosphorus Chemistry & Chemical

Biology (Ministry of Education), Beijing Advanced

Innovation Center for Human Brain Protection, Tsinghua

University, Beijing 100084, China

Liangliang Wang − School of Pharmaceutical Sciences, Key

Laboratory of Bioorganic Phosphorus Chemistry & Chemical

Biology (Ministry of Education), Beijing Advanced

Innovation Center for Human Brain Protection, Tsinghua

University, Beijing 100084, China

■

This work was supported by the Collaborative Innovation

Center for Diagnosis and Treatment of Infectious Diseases,

Tsinghua-Peking Center for Life Sciences, the Roche

Postdoctoral Fellowship Program and “1000 Talents Recruit-

ment Program”.

ABBREVIATIONS

■

br s, broad signal; DMF, N,N-dimethylformamide; DMSO,

dimethyl sulfoxide; DIEA, diisopropylethylamine; G-CSF,

granulocyte colony stimulating factor; hERG, human ether-a-

go-go-related gene; hypermutated/MSI-CRC, hypermutated/

microsatellite-instable colorectal cancer; IL, interleukin; INF-γ,

interferon γ; IP-10, interferon-inducible protein 10; IMHB,

intramolecular hydrogen bond; MyD88, myeloid diﬀer-

entiation protein 88; mDCs, myeloid dendritic cells;

mDDCs, monocyte-derived dendritic cells; MIP-1α, macro-

phage inﬂammatory protein 1 α; MCP-1, monocyte chemo-

tactic protein 1; NBS, N-bromosuccinimide; NK, natural killer;

NOD1, NOD-like receptor 1; pDCs, plasmacytoid dendritic

cells; rt, room temperature; THF, tetrahydrofuran; TNF-α,

tumor necrosis factor α; VEGF, vascular endothelial growth

factor

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Structure-Based Design of Highly Potent Toll-like Receptor 7/8 Dual Agonists for Cancer Immunotherapy

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