Identification of substituted pyrimido[5,4-b]indoles as selective toll-like receptor 4 ligands

Article

pubs.acs.org/jmc

Identiﬁcation of Substituted Pyrimido[5,4‑b]indoles as Selective Toll-

Like Receptor 4 Ligands

Michael Chan,^†Tomoko Hayashi,^†Richard D. Mathewson,^†Afshin Nour,^†Yuki Hayashi,^†Shiyin Yao,^†

Rommel I. Tawatao,^†Brian Crain,^†Igor F. Tsigelny,^‡Valentina L. Kouznetsova,^‡,†Karen Messer,^†

Minya Pu,^†Maripat Corr,^§Dennis A. Carson,^†and Howard B. Cottam*^,†

‡

§

^†Moores Cancer Center, San Diego Supercomputer Center and Department of Neuroscience, and Department of Medicine,

University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0695, United States

ABSTRACT: A cell-based high-throughput screen to identify small molecular weight stimulators of

the innate immune system revealed substituted pyrimido[5,4-b]indoles as potent NFκB activators.

The most potent hit compound selectively stimulated Toll-like receptor 4 (TLR4) in human and

mouse cells. Synthetic modiﬁcations of the pyrimido[5,4-b]indole scaﬀold at the carboxamide, N-3,

and N-5 positions revealed diﬀerential TLR4 dependent production of NFκB and type I interferon

associated cytokines, IL-6 and interferon γ-induced protein 10 (IP-10) respectively. Speciﬁcally, a

subset of compounds bearing phenyl and substituted phenyl carboxamides induced lower IL-6 release while maintaining higher

IP-10 production, skewing toward the type I interferon pathway. Substitution at N-5 with short alkyl substituents reduced the

cytotoxicity of the leading hit compound. Computational studies supported that active compounds appeared to bind primarily to

MD-2 in the TLR4/MD-2 complex. These small molecules, which stimulate innate immune cells with minimal toxicity, could

potentially be used as adjuvants or immune modulators.

ness due to age (infants and the elderly) or disease (diabetes,

renal failure), to facilitate the use of smaller doses of antigen,

and to permit immunization with fewer doses of vaccine.⁹

Macromolecular activators of the innate immune system, such

as monophosphoryl lipid A (MPLA), a TLR4 ligand, are

utilized as adjuvants,¹⁰but development of small molecular

weight nonlipid ligands might have several advantages to

address diﬀerent immunological requirements for vaccines or

immune therapeutics.

As part of our studies on small molecules that can activate

TLRs, we conducted a high through-put screening (HTS)

campaign in which a commercially available library of

compounds was screened in a human cell-based NFκB

activation assay. Many innate signaling pathways converge on

the transcription factor NFκB. Hence it was used in the primary

screen as a broad indicator of small molecule agonism of innate

immunity. The speciﬁc innate receptor for the lead compounds

was determined using genetically modiﬁed cells and primary

mouse and human blood or bone marrow mononuclear cells.

Here we report our discovery and initial structure−activity

relationship (SAR) studies of the pyrimido[5,4-b]indole class of

ligands and their characterization as TLR4/MD-2 agonists.

INTRODUCTION

■

The innate immune response is the ﬁrst line of defense against

microbial pathogens such as viruses, bacteria, fungi, and

protozoa. A critical component of the innate immune response

is the NFκB family of transcription factors.^1,2The Toll-like

receptors (TLRs) are critical components of the innate immune

system that regulate NFκB activation. In general, the TLRs

recognize macromolecules that are associated with pathogens

and with cell stress. These pathogen-associated molecular

patterns (PAMPs) and their corresponding TLRs include:

lipopeptides (TLR2), double-stranded RNA (TLR3), lip-

opolysaccharide (LPS, TLR4),³bacterial ﬂagellin (TLR5),

guanine and uridine-rich single-stranded RNA (TLR7, 8), and

hypo-methylated CpG rich DNA (TLR9).⁴Innate immune

cells use pattern recognition receptors (PRRs) such as TLRs to

promote a rapid response to perceived threats before pathogen-

speciﬁc adaptive immunity can be established. Indeed, this

rapid response, involving multiple components of the innate

immune system, has been recognized to guide the type of

adaptive immune response that is most eﬀective for the speciﬁc

pathogenic threat.

The discovery that certain small molecules could serve as

speciﬁc innate immune receptor ligands has expanded the

possibility of designing drugs that may modulate the immune

response at its earliest stages. Notable examples of such small

molecules are the TLR7 agonists imiquimod,⁵isotorabine,⁶and

the more recent 8-oxo-9-benzyladenines,⁷as well as the TLR7/

8 agonist resiquimod.⁸A useful application of this concept is

the incorporation of adjuvants into vaccines. Adjuvants are

added to antigens in a vaccine setting to provide enhanced

response to poorly immunogenic antigens, to increase

seroconversion rates in populations with reduced responsive-

RESULTS AND DISCUSSION

■

High-Throughput Screen Study Design. A library of

compounds was acquired from the University of California, San

Francisco, Small Molecule Discovery Center consisting of

about 170000 compounds from eight suppliers (https://smdc.

ucsf.edu). The library was screened in three phases at a

Received: November 16, 2012

Published: May 8, 2013

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commercial HTS screening facility using the THP-1 human

monocytic leukemia cell line, which contained a β-lactamase

reporter gene under the control of the NFκB response element

that had been stably integrated into the cells. All screens were

performed in activator mode using LPS as a positive control,

achieving typical Z′ values above 0.75. The three-phase

screening process consisted of (1) a pilot screen of about

10000 compounds selected as representative of the entire

primary library, (2) the primary screen of the entire library, and

(3) a conﬁrmation screen of about 2000 hits found in the

primary screen. Compounds identiﬁed as active in two screens

were considered to be conﬁrmed hits. An analysis of the cluster

enrichment methods for hit selection has been recently

reported.¹¹

Discovery of Pyrimido[5,4-b]indoles as Activators of

NFκB. Following the cluster enrichment analysis, 225

compounds were selected for further in vitro biological

evaluation involving cytokine induction assays in primary

cells, including human peripheral blood mononuclear cells

(hPBMC), mouse splenocytes, mouse bone marrow derived

dendritic cells (mBMDC), and mouse bone marrow derived

macrophages (mBMDM). These cells were incubated in

triplicate, with each of the 225 compounds at a single

concentration (1 μM for splenocytes and 5 μM for all other

mouse cells and human cells), and the supernatants were tested

for the presence of NFκB dependent cytokines, IL-8 or IL-6,

released from the human or mouse cells, respectively. Thirty-

nine of the 225 compounds stimulated the human and mouse

cells to secrete IL-8 or IL-6 above the detectable limit. To

further conﬁrm activity, these compounds were repurchased

and retested by stimulating hPBMC and mBMDC with titrated

doses and assaying for IL-8 and IL-6.

(and all active derivatives) was assayed for LPS (endotoxin)

levels using a commercially available detection system and

found to contain less than 10 endotoxin units (EU)/μmol

compound. To further exclude contamination, compound 1

was resynthesized according to Scheme 1. Both samples of

compound 1 displayed indistinguishable physicochemical and

biological properties, indicating that the positive biological

activity was not due to LPS or another contaminant. Full

titration curves of the resynthesized compound were performed

using the SEAP assay with mouse and human TLR4 transfected

HEK293 cell lines, conﬁrming dose-dependent activation

(Figure 2B, C).

To conﬁrm that TLR4 was indeed the receptor, compound 1

was assayed for IL-6 production in mBMDCs from wild-type

and TLR4 deﬁcient mice (Figure 2D). Genetic disruption of

TLR4 completely abrogated IL-6 secretion induced by

compound 1. The binding of compound 1 to the TLR4/

MD-2/CD14 complex was further conﬁrmed using a

competitive antagonist for the TLR4 binding complex, LPS-

RS (LPS from Rhodococcus sphaeroides).¹²LPS-RS inhibited the

activation by compound 1 in a dose-dependent manner,

indicating that compound 1 bound to the TLR4 complex

(Figure 2E).

TLR4 signals through two distinct pathways, leading

respectively to NFκB dependent cytokines and type I interferon

(IFN) production. Several naturally occurring TLR4 ligands

and MPLA have been reported to require CD14 to activate the

type I IFN regulatory pathway.¹³

The hTLR4 transfected HEK293 cell line (Figure 2A) also

overexpresses MD-2 and CD14, which are TLR4 accessory

proteins.^14,15Compound 1, however, was not dependent on

CD14 for either IL-6 or type I IFN production, as

demonstrated using CD14 deﬁcient cells (Figure 2F, G). The

supernatants from mBMDCs stimulated with graded doses of

compound 1 were also tested for IP-10 as a surrogate marker of

type I IFN release. Results showed a dose-dependent response

for type I IFN (Figure 3A) production, which paralleled that of

IP-10 (Figure 3B).

The above assays were utilized to compare the derivatives of

the lead pyrimidoindole for their ability to activate mouse and

human TLR4. Mouse TLR4 activation was assessed using

primary mBMDC and IL-6 release (Figure 4) and conﬁrmed

using mTLR4 transfected HEK293 cells (Figure 5A, C). The

values shown in the Tables 1−3 for IL-6 release, and mTLR4

activation are area under the curve (AUC) values for titrated

doses of compounds from 312 nM to 10 μM. Each cytokine

induction curve was ﬁrst converted to a percent activity curve,

and then the AUC of the percent activity curve was calculated.

The process of converting to a percent activity curve allowed

subtracting background and adjusting for plate-to-plate

variation. Finally, the AUC values were normalized to the

activity of compound 1 within each experiment, set at 100.

Human TLR4 activation is shown for stimulation of PBMC

(IL-8) and hTLR4 HEK293 transfectomas (Figure 5B,D) at 10

μM, as these assays were not suﬃciently sensitive at lower

concentrations to make AUC comparisons. The levels of

hTLR4 activation by SAR derivatives are expressed relative to

compound 1, set at 100.

A few structurally diverse library scaﬀolds were identiﬁed in

these cytokine assays as having reproducible responses in

mouse and human stimulation assays. Among these scaﬀolds,

the pyrimido[5,4-b]indoles emerged as the most potent and

diverse class of compounds in the mouse cell assays with clear

evidence of SAR. Within this scaﬀold cluster, the leading hit

from the initial primary and secondary screens was a substituted

acetamide attached to the pyrimidoindole ring system through

a thioether linkage (Figure 1). Thus, compound 1 provided a

starting point for structure−activity studies.

Figure 1. Structure of hit compound 1.

Target Receptor Identiﬁcation. To identify the target

receptor, we used HEK293 cells stably transfected with

individual human (h) TLRs: TLR2, TLR3, TLR4/MD-2/

CD14, TLR5, TLR7, TLR8, or TLR9 expressing HEK293 cells,

along with an NFκB activation reporter producing secreted

embryonic alkaline phosphatase (SEAP). Among the tested

TLR transfected cells, only those expressing TLR4/MD-2/

CD14 responded to pyrimidoindoles, as shown in Figure 2A for

compound 1. Because TLR4/MD-2/CD14 was the receptor

complex for the active compounds in this series, it was

important to rule out the possibility that activity might have

been caused by LPS contamination. Therefore, compound 1

SAR Studies. The primary HTS library contained a family

of 452 compounds in the pyrimido[5,4-b]indole class and

therefore represented a valuable initial indication of structural

features important for activation of NFκB. Upon inspection of

the NFκB activation values in the initial HTS relative to the

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Figure 2. Target identiﬁcation of compound 1 using human TLR HEK293 reporter cell lines and genetically deﬁcient cells. (A) Human TLR2,

TLR3, TLR4/MD-2/CD14, TLR5, TLR7, TLR8, and TLR9 HEK 293 Blue cells or NF-κB/SEAPorter cells were incubated with compound 1 (10

μM) for 20−24 h, and activation was evaluated by SEAP secretion in the culture supernatants by colorimetric assay at OD₄₀₅. Data shown are mean

SEM of triplicates and representative of two to three independent experiments showing similar results. p < 0.05 was considered signiﬁcant

compared to the vehicle control using Student’s t test. (B,C) Mouse (B) or human (C) TLR4 HEK transfectomas were incubated with graded

concentrations of compound 1. TLR4 mediated NFκB activation was measured by SEAP secretion in the culture supernatant. (D) WT and Tlr4^−/−

mBMDC were incubated with compound 1 (10 μM) for18 h. IL-6 in the culture supernatants was measured by ELISA. (E) Mouse TLR4

transfectoma cells were incubated with 2.5 μM compound 1 in the presence or absence of TLR4 antagonist LPS-RS (12, 111, 1000 ng/mL).

Activation of the TLR4/NFκB pathway was evaluated by SEAP secretion in the culture supernatants. * denotes p < 0.05 considered as signiﬁcant

compared to vehicle using one way ANOVA with Dunnett’s post hoc testing. (F,G) WT and Cd14^−/−mBMDC were incubated with compound 1

(3.7 μM) overnight. IL-6 in the culture supernatants was measured by ELISA (F) and type I IFN (IFN-β) measured by luciferase release in from an

ISRE reporter cell line (G). p < 0.05 was considered as signiﬁcant compared to vehicle using Student’s t test. NS denotes “not signiﬁcant”. Data

shown are mean SEM of triplicates and representative of two independent experiments showing similar results.

various structural features, several trends were apparent. There

was a requirement for a hydrophobic moiety in the region of

the cyclohexyl group of compound 1. In this same region, the

carboxamide function was essential. Furthermore, a hydro-

phobic group at N-3, preferably a phenyl group, was also

required for activity. Substitutions on the N-3 phenyl, other

than ﬂuorine atoms, resulted in loss of activity. Removal of the

benzo ring of the indole portion of the scaﬀold also resulted in

loss of activity. When the C-4 oxo was replaced by NH to form

a triazine ring, loss of activity was observed. Finally, exchange of

the entire acetamide moiety on the C-2 thiol with the N-3

phenyl group, such that the acetamide was attached at N-3 and

the phenyl was attached at the C-2 thiol, resulted in loss of

activity. With these SAR features as an initial guide, we elected

to investigate modiﬁcations of hit compound 1 at three regions

while maintaining the core pyrimido[5,4-b]indole ring system:

the N-substitution of the S-acetamide, the N-3 substituent, and

the N-5 substituent (Figure 6).

N-Substitutions at the carboxamide moiety were ﬁrst

undertaken to probe the limitations of the hydrophobic

group requirement at this position with respect to optimization

of cytokine induction (Table 1). While keeping all other

structural features of hit compound 1 constant, we prepared a

series of carboxamides substituted with various alkyl, cycloalkyl,

aromatic, and heteroaromatic groups. The synthesis began with

construction of the appropriately substituted pyrimido[5,4-

b]indole ring system as shown in Scheme 1. 2-Amino-

benzonitrile (2) was reacted with ethyl bromoacetate to yield

ethyl 2-((2-cyanophenyl)amino)acetate (3) followed by base

catalyzed ring closure to the aminoindole^16,17(4). At this point

in the synthesis, a variety of substituents, represented by R¹in

the scheme, may be introduced that will determine the

respective N-3 substituent following annulation of the

pyrimidine ring. Thus, reaction of compound 4 with an

isothiocyanate, such as phenylisothiocyanate, provided the

substituted thioureidoindole^16,18(5), where R¹was phenyl in

this example. Ring closure of 5 using polyphosphoric acid

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a

Scheme 1. General Synthetic Route

a

Reagents and conditions: (a) BrCH₂COOEt, NaHCO₃, EtOH, reﬂux; (b) tert-BuOK, THF, <30 °C; (c) R¹-NCS, EtOH, reﬂux; (d) PPA, 110 °C;

(e) ClCH₂COOH, KOH/EtOH, reﬂux; (f) R²-NH₂(for R²substitutions refer to Table 1), HATU, DMF, room temp.

as R²in Scheme 1. Compounds 1, 13, 14, 15, 16, 17, as well as

the N-3 derivatives 37 and 38, discussed later, were registered

with Chemical Abstracts Service, but no literature references

were found for any of these compounds. The hit compound 1

was resynthesized according to Scheme 1, as discussed above.

Thus, by this synthetic method, compounds shown in Table 1,

with R¹held constant as a phenyl group, were prepared and

evaluated in a cytokine induction assay for IL-6 in mBMDC

and compared to compound 1. Examples of the data obtained

from typical cytokine induction assays and NFκB activation

Figure 3. Type I IFN induction by compound 1. Wild-type mBMDCs

assays are shown in Figures 4 and 5, wherein compounds 1, 11,

were incubated with graded concentrations of compound 1 overnight.

12, 36, and 39 are compared for IL-6 production by mBMDC

DMSO 0.5% served as the vehicle control. The levels of type I IFN

and hPBMC.

were determined using an L929-ISRE luciferase reporter cell line and a

mIFNβ standard (A). IP-10 levels were determined by ELISA (B).

Data shown are mean SEM of triplicates and representative of two

Inspection of the IL-6 AUC values relative to the

hydrophobic group R²revealed that in general, compounds

bearing the larger cycloalkyl groups, such as cyclooctyl (9),

cycloheptyl (10), and cyclohexyl (1), were the most active,

followed by branched alkyls and then straight chain alkyls and

aromatic and heteroaromatic groups. A notable exception

would be the p-ﬂuorophenyl (14) and o-ﬂuorophenyl (15)

compounds. For the R²group, there appeared to be a strict

“hydrophobic volume” requirement for activity. Hence, the 3,3-

dimethylbutyl compound (28) was among the most active of

the alkyls, with isopentyl (27), butyl (23), and isobutyl (26)

independent experiments showing similar results. * denotes p < 0.05

compared to vehicle using one way ANOVA with Dunnett’s post hoc

testing.

yielded the pyrimido[5,4-b]indole (6) bearing the N-3

substituent R¹. Alkylation of the 2-thioxo function with

chloroacetic acid provided the versatile intermediate 7, which

was then used to prepare the ﬁnal test compounds (8) bearing

a variety of N-substitutions at the acetamide moiety, designated

Figure 4. Representative data of SAR compound screening using mouse primary dendritic cells. Biological screening of SAR compounds was

conducted using primary mBMDC. The cells were incubated with graded concentrations of the indicated compounds 1, 36, and 39 (A) or 11 and 12

(B) for 18 h. DMSO 0.5% served as the vehicle control. IL-6 levels in culture supernatants were measured by ELISA. Data shown are mean SEM

of triplicate.

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Figure 5. Representative data of SAR compound screening using TLR4 transfectomas. Mouse TLR4 (A,C), and hTLR4 (B,D) HEK transfectomas

were incubated with graded concentrations of the indicated compounds 1, 36, and 39 (A) or 11 and 12 (B) for 18 h. DMSO 0.5% served as the

vehicle control. The speciﬁc activation of the reporter cell lines was measured by SEAP activity in the supernatant by absorption at 630 nm. Data

shown are mean SEM of triplicate data.

had modiﬁcations at this position to guide the SAR. We elected

to prepare a few simple N-5 alkyl derivatives of compound 1.

The N-5 indole-like nitrogen can be alkylated if the proton is

ﬁrst removed by a strong base, such as sodium hydride. Starting

from compound 1, Scheme 2 shows the preparation of the N-5

methyl derivative (42) that also produced a dimethyl side

Figure 6. SAR regions of modiﬁcation of hit compound 1.

product (43) wherein the second methylation occurred at the

carboxamide function, yielding the N,N-disubstituted derivative.

The N-5 methyl butyrate ester (44) of compound 1 was also

prepared by this method. Table 3 compares the IL-6 inducing

activity of the N-5 derivatives relative to compound 1.

Interestingly, simple methylation at N-5 did not abrogate the

immune stimulatory activity of compound 1. Moreover,

methylation at N-5 decreased the toxicity of compound 1

(Figure 7) as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) viability assay. Dimethy-

lation, on the other hand, resulted in total loss of IL-6 activity

(compound 43). To further probe the N-5 position, we elected

to study a few N-5 alkylated derivatives of compound 1 while

avoiding dialkylation in the process. Accordingly, we prepared

the t-butyl ester of 7a (compound 45) and then alkylated the

N-5 position with several primary alkyl halides, as depicted in

Scheme 3. The resulting t-butyl esters (46a−d) were easily

hydrolyzed to the corresponding free carboxylic acids (47a−d)

and then converted to the N-cyclohexylcarboxamides (48−51)

by the same method as described for compound 1. Thus, using

this strategy, the N-5 n-propyl, n-pentyl, n-dodecyl, and

cyanomethyl derivatives were prepared. Finally, the cyano-

methyl derivative (51) was converted to the N-5 acetamide

derivative (52). All N-5 alkyl derivatives were found to be less

active than the N-5 methyl (42), with the n-propyl (48) being

the next most active of the series. The trend in the toxicity

proﬁle for these N-5 alkyl derivatives was conﬁrmed, at least for

the shorter chain alkyls, in that the N-5 methyl (42),

cyanomethyl (51), and n-propyl (48) derivatives did not

reduce cell viability at concentrations up to 10 μM (Figure 7,

shown for 42, data not shown for 48 and 51).

being somewhat less active. Interestingly, when the alkyl chain

length of R²was extended to more than ﬁve carbons or reduced

to three or fewer carbons, whether branched or not, signiﬁcant

loss of activity was observed (Table 1).

Encouraged by the SAR trends at the carboxamide moiety,

we then addressed the N-3 position (Table 2). As mentioned

above, data from the NFκB primary screen indicated that a

hydrophobic group at N-3, preferably an unsubstituted phenyl,

was required for activity. However, to draw that conclusion

with conﬁdence, more examples of compounds bearing similar

hydrophobic groups than were represented in the primary

compound collection were needed. We therefore elected to

prepare and evaluate a few additional derivatives of hit

compound 1 with variation only at N-3. Thus, the R¹group

in compound 5 was varied by reaction of 4 with the appropriate

isothiocyanate, and the remaining three steps for each

derivative were completed as outlined in Scheme 1 while

maintaining the R²group as cyclohexyl in each case. Table 2

compares the IL-6 inducing activity of compound 1 with the N-

3 derivatives. As the IL-6 data indicate, replacing the N-3

phenyl in compound 1 with a cyclohexyl group (36) caused

some loss of activity, while substitution on the N-3 phenyl (37

and 38) resulted in greater loss of activity. Replacing N-3

phenyl with a larger aromatic, such as naphthyl (39), or

extending the phenyl with an alkyl chain, as in phenethyl (40),

yielded total loss of activity. The combined substitution of R¹

with cyclohexyl and R²with cyclopentyl (41) resulted in partial

loss of activity.

The third area of interest for modiﬁcation was the N-5

position. None of the compounds in the original HTS library

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Table 1. N-Substituted Carboxamide Derivatives

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Table 1. continued

a

AUC values normalized to compound 1. Stimulation with LPS (10 ng/mL), and 5 μM of 1 released an average of 20.6 4.8 and 10.5 1.3 ng/mL

b

of IL-6, respectively. All compounds tested at 5 μM, values normalized to 1. Stimulation with LPS 10 ng/mL and 5 μM of 1 released averages of

234.8 8.6 and 590 11 pg/mL of IP-10, respectively. AUC values normalized to compound 1. TLR4 cell activation stimulated with 10 ng/mL

LPS and 5 μM compound 1 resulted in OD₆₃₀of 1.69 0.08 and 1.52 0.03, respectively. All compounds tested at 10 μM, values normalized to 1.

Stimulation with LPS 10 ng/mL and 10 μM of 1 released averages of 14.5 0.6 and 7.8 0.8 ng/mL of IL-8, respectively. All compounds tested at

c

d

e

10 μM, values normalized to 1. TLR4 cell activation stimulated with 10 ng/mL LPS and 10 μM compound 1 resulted in OD₆₃₀of 1.90 0.04 and

0.88 0.02, respectively.

Table 2. N-3 Derivatives

a

AUC values normalized to compound 1. Stimulation with LPS (10 ng/mL) and 5 μM of 1 released an average of 20.6 4.8 and 10.5 1.3 ng/mL

b

of IL-6, respectively. All compounds tested at 5 μM, values normalized to 1. Stimulation with LPS 10 ng/mL and 5 μM of 1 released averages of

234.8 8.6 and 590 11 pg/mL of IP-10, respectively. AUC values normalized to compound 1. TLR4 cell activation stimulated with 10 ng/mL

LPS and 5 μM compound 1 resulted in OD₆₃₀of 1.69 0.08 and 1.52 0.03, respectively. All compounds tested at 10 μM, values normalized to 1.

Stimulation with LPS 10 ng/mL and 10 μM of 1 released averages of 14.5 0.6 and 7.8 0.8 ng/mL of IL-8, respectively. All compounds tested at

c

d

e

10 μM, values normalized to 1. TLR4 cell activation stimulated with 10 ng/mL LPS and 10 μM compound 1 resulted in OD₆₃₀of 1.90 0.04 and

0.88 0.02, respectively.

The TLR4 transfectoma lines transmit their reporter signal

after activation of the proinﬂammatory transcription factor,

NFκB. As one might expect, the IL-6 secretion induced by the

compounds in primary mBMDCs correlated well with their

respective activities in mouse TLR4 transfectoma cell lines.

With few exceptions, the most active compounds in the IL-6

assays were also those that showed the highest activity in the

mTLR4 transfectoma line (Tables 1−3). In addition to

stimulating the production of inﬂammatory cytokines (e.g.,

IL-6) by NFκB activation, TLR4 signaling can induce the

production of type I IFN via the TRIF pathway.^19,20This

pathway results in the activation of interferon regulatory factors

(IRF), which are nuclear transcription factors that promote the

transcription of IFNs. Type I IFN is important for the

activation of antigen presenting dendritic cells, leading to good

adjuvant activity, and also promotes cellular defenses against a

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a

Scheme 2. N-5 Derivatives

a

(a) NaH, DMF, room temp, then CH₃I; (b) NaH, DMF, then Br(CH₂)₃COOCH₃

Table 3. N-5 Derivatives

a

AUC values normalized to compound 1. Stimulation with LPS (10 ng/mL) and 5 μM of 1 released an average of 20.6 4.8 and 10.5 1.3 ng/mL

b

of IL-6, respectively. All compounds tested at 5 μM, values normalized to 1. Stimulation with LPS 10 ng/mL and 5 μM of 1 released averages of

234.8 8.6 and 590 11 pg/mL of IP-10, respectively. AUC values normalized to compound 1. TLR4 cell activation stimulated with 10 ng/mL

LPS and 5 μM compound 1 resulted in OD₆₃₀of 1.69 0.08 and 1.52 0.03, respectively. All compounds tested at 10 μM, values normalized to 1.

Stimulation with LPS 10 ng/mL and 10 μM of 1 released averages of 14.5 0.6 and 7.8 0.8 ng/mL of IL-8, respectively. All compounds tested at

c

d

e

10 μM, values normalized to 1. TLR4 cell activation stimulated with 10 ng/mL LPS and 10 μM compound 1 resulted in OD₆₃₀of 1.90 0.04 and

0.88 0.02, respectively.

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Figure 7. Assessment of cytotoxicity of compounds 1 and 42. Cytotoxicity of the compounds was evaluated by MTT assay as a measure of viability.

mBMDC (10⁵/well) were incubated with graded concentrations of the compounds for 18 h and compared with vehicle (0.5% DMSO) and MPLA

(1 μg/mL). (A) IL-6 levels in the culture supernatants were determined by ELISA. (B) The cells were lysed after the overnight incubation with

MTT reagents, and absorbance at 570 nm was measured, subtracting the reference absorbance at 650 nm. Data shown are mean SEM of triplicates

and are representative of two independent experiments showing similar results.

a

Scheme 3. N-5 Alkyl Derivatives

a

(a) ClCH₂COO t-Bu, KOH, DMA,H₂O; (b) NaH, DMF, then R³X (iodopropane or iodopentane or bromododecane or bromoacetonitrile); (c)

TFA, DCM or CH₃CN; (d) cyclohexyl-NH₂, HATU, DMF, room temp; (e) compound 51, H₂SO₄, H₂O.

variety of pathogens, particularly RNA viruses. This host

defense is aided by circulating interferon inducible factors such

as IP-10. The TLR4 transfectoma reporter lines in this study

did not utilize an interferon reporter system; hence, the IP-10

data does not correlate as closely as the IL-6 data.

mean was 37 33. The cluster in the dotted circle includes

those compounds whose values were at least one SD above the

mean for both cytokines. The other cluster includes those

compounds whose values were below the mean for IL-6 and

were above the mean for IP-10. It was notable that when the

carboxamide substituent was phenyl or substituted phenyl (13,

15, 16, 17), with the exception of p-ﬂuorophenyl (14), IL-6

release was reduced while IP-10 production was maintained at a

relatively higher level. A few compounds bearing branched

aliphatic carboxamide substituents showed a similar trend, as

noted above for 29, 30, and 33.

In general, the active compounds exhibited greater activity in

the murine cell systems than in human cells. Although the

innate immune system is highly conserved among species, there

are diﬀerences between human and mouse TLR4.²¹⁻²³Similar

to the active compounds reported here nonsynthetic ligands

have been noted to have species speciﬁc behavior. Notably,

tetraacylated lipid IVa, a synthetic lipid A precursor, has been

reported to act as a weak agonist to mouse TLR4/MD-2 but as

an antagonist to human TLR4/MD-2.^24,25Recently mouse and

human TLR4/MD2 crystal structures with this ligand have

Interestingly, several of the compounds, namely 12, 13, 15,

16, 17, 29, 30, and 33, induced diminished IL-6 release

compared to compound 1 but had nearly equivalent induction

of IP-10 (Tables 1 and 2). These results suggest that there may

be a possibility of separating the two activities on the basis of

structural features. The correlation of IL-6 and IP-10

production of the selected compounds is shown graphically

in Figure 8. Two distinct groups of compounds are observed,

shown as circled regions of the plot. Thus, the group at the

upper right area (dotted circle) comprises compounds that

induce high IL-6 and high IP-10 production, including

compounds 1, 9, 10, 28, and 42. The other group is composed

of compounds that induce low IL-6 but relatively higher IP-10

production. These two clusters were selected based on mean

SD of the normalized IL-6 and IP-10 values of all SAR

compounds. For IL-6, the mean was 32 37. For IP-10, the

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Figure 9. Predicted binding mode of compound 28 to mouse TLR4/

MD-2 complex.

Figure 8. Correlation plot of IL-6 versus IP-10 induced by SAR

derivatives. IL-6 (AUC) and IP-10 values in Tables 1−3 were plotted.

Two distinct groups, compounds in the dotted circled (upper right)

and in solid (left) circled areas, were selected based on the induction

of IL-6 and IP-10. Numbers in the legends indicate compound ID.

These clusters were selected based on mean SD of the normalized

IL-6 and IP-10 values of all SAR compounds. For IL-6. the mean SD

was 32 37. For IP-10, the mean SD was 37 33. The group at the

upper right area comprises compounds that induce high IL-6 and high

IP-10 production, including compounds 1, 9, 10, 28, and 42, whose

values were at least one SD above the mean for both cytokines. The

other cluster is composed of compounds (13, 16, 17, 29, 30, and 33)

that induce low IL-6 but relatively higher IP-10 production, whose

values were below the mean for IL-6 and were above the mean for IP-

10.

that may keep the compound in the MD-2 pocket bound to

both TLR4 and MD-2 is depicted in Figure 10. There is a set of

suggested that the diﬀerent charge distributions of mouse and

human TLR4/MD-2 aﬀect the positions of the phosphate

groups of lipid IVa, orienting them in a manner that would limit

receptor dimerization by human TLR4.^22,26

Computational Studies. TLR4, in association with MD-2,

is responsible for the physiological recognition of LPS.^27,28The

structural basis of receptor speciﬁcity and of the mechanism of

activation by LPS have recently been elucidated by determining

the crystal structure of the TLR4/MD-2-LPS complex at 3.1 A

resolution.²⁹Binding of agonistic ligands such as LPS causes

dimerization of the extracellular domains to form a TLR4/MD-

2-LPS macromolecular complex. Like the extracellular domains

of other TLRs, TLR4 contains leucine-rich repeats and adopts a

characteristic horseshoe-like shape. MD-2 is noncovalently

bound to the side of the horseshoe ring and also directly

interfaces with the ligand. MD-2 has a β-cup fold structure

composed of two antiparallel β-sheets forming a large

hydrophobic pocket for ligand binding. LPS binds to this

pocket and directly mediates dimerization of the two TLR4/

MD-2 complexes. TLR4 can be activated by structurally diverse

LPS molecules, which have been predicted to occupy this

pocket in MD-2.

Small molecule hit compounds discovered in the present

study were also thought to bind to the TLR4/MD-2 complex in

such a way as to facilitate dimerization. Accordingly, we

examined the predicted binding mode(s) of one of the most

active compounds to the murine TLR4/MD-2 complex by

conducting molecular docking of compound 28 to the crystal

structure of the mouse complex (PDB 2Z64) using the

programs HEX³⁰and AMPAC.³¹We selected the best

conﬁgurations of 28 bound to this complex based on molecular

surface shape complementarity and the most favorable

intermolecular energy of interactions. It is noteworthy that

the best docking position for 28 was within the LPS pocket.²⁹

Figure 9 shows the predicted binding mode of compound 28 in

the TLR4/MD-2 model, while the set of binding interactions

Figure 10. Predicted binding interactions of compound 28 with mouse

TLR4/MD-2 complex (PDB 2Z64).

favorable electrostatic interactions resulting in possible hydro-

gen bonds formed by the residues Glu439 of TLR4 and Arg90

of MD-2 with compound 28, although the former would

approach a hydrogen bond interaction due to the ﬂexibility of

the Glu439 side chain. There are also multiple potential

hydrophobic interactions with MD-2 and TLR4. Such

interactions of the compound with two proteins can improve

the free energy of complex formation by approximately 8−10

kcal/mol.

Interestingly, the predicted binding model for the pyrimi-

doindoles is similar to that proposed for tricyclic antidepres-

sant, amitriptyline.³²This molecule also has some TLR4/MD-2

binding and has a similar three-ring scaﬀold. Other small

molecules such as paclitaxel,³³opioids,³⁴and a peptide³⁵have

also been reported to bind to the MD-2 binding pocket.

CONCLUSION

In the course of an HTS designed to identify activators of

innate immunity, a series of substituted pyrimido[5,4-b]indoles

■

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General Procedure A for the Synthesis of Compound 5. To a

solution of compound 4 (1 equiv) in warm EtOH was added the

appropriate isothiocyanate (1.1 equiv) dropwise with stirring. The

reaction was reﬂuxed for 6 h and cooled overnight. Solids were ﬁltered,

washed with EtOH, and dried overnight in vacuo to give compound 5.

General Procedure B for the Synthesis of Compound 6b−d.

Compound 5 was dissolved in polyphosphoric acid and stirred at 110

°C for 3.5 h. Solution was added to ice-cold water and extracted with

EtOAc and dried over MgSO₄. The solid was then dried in vacuo

overnight to give compound 6.

General Procedure C for the Synthesis of Compound 7. In a

ﬂame-dried ﬂask, compound 6a (1 equiv) and KOH (2 equiv) were

dissolved in anhydrous EtOH with heat. In a separate ﬂame-dried ﬂask,

chloroacetic acid (1 equiv) was added to anhydrous EtOH.

Chloroacetic acid solution was then added to the reaction mixture

and reﬂuxed for 6 h. The reaction was concentrated by half and

acidiﬁed with 3 M HCl to pH 4. The solids were collected, washed

with water, and dried in vacuo to give compound 7.

General Procedure D for the Synthesis of Compound 8.

Compound 7 (1 equiv), triethylamine (2 equiv), and the appropriate

amine (1.1 equiv) were dissolved in anhydrous DMF. To this solution,

HATU (1.1 equiv) dissolved in DMF was added and stirred until

complete and concentrated in vacuo. The crude material was then

recrystallized in MeOH to give compound 8.

General Procedure E for the Synthesis of Compound 46. NaH

(1.1 equiv) was added to a solution of compound 45 (1 equiv) in

DMF. The reaction mixture was stirred for 5 min, and then the

appropriate alkyl halide (1.1 equiv) was added and stirred until

complete. The crude product was extracted with EtOAc and dried over

MgSO₄. Crude material was ﬁnally recrystallized with MeOH to give

compound 46.

were discovered as selective TLR4 ligands. Small molecules of

this class are unique among TLR4 activators in that they are

“non-lipid-like”. Structure−activity evaluation in both mouse

and human cells revealed that, to maintain activity, the

carboxamide region of this scaﬀold must contain a hydrophobic

moiety of signiﬁcant “volume”. Interestingly, a subset of the

compounds bearing phenyl and substituted phenyl carbox-

amides induced lower NFκB-dependent inﬂammatory cytokine

release while maintaining interferon-dependent IP-10 produc-

tion. Varying the substituents at N-3 indicated an even greater

restriction for a hydrophobic moiety at this position, with a

phenyl group being preferred for activity. Finally, N-5

substitution revealed that short alkyl substituents at this

position attenuate cell toxicity relative to the corresponding

nonsubstituted derivative while maintaining TLR4 activity.

Both the inﬂammatory cytokine and type I IFN inducing

activities of these compounds were CD14-independent.

Computational studies with one of the active compounds

predicted binding primarily to MD-2 in the murine TLR4/MD-

2 complex. Lead optimization studies to improve the activity of

the compounds using computational methods are currently

underway in our laboratories. To date, no single adjuvant can

elicit the optimal immune response to all pathogens in vaccines.

Here we have described a panel of small molecules that

stimulate immune cells to produce distinct proﬁles of NFκB

and interferon associated cytokines. This panel of molecules

may allow for diﬀerential analysis of the relative pathway

induction needed for adequate immunoprotection and

immunotherapy for diverse pathogens.

General Procedure F for the Synthesis of Compound 47.

Compound 46 was dissolved in 1:1 acetonitrile/triﬂouroacetic acid

and stirred at room temperature overnight. Depending on the N-alkyl

substitution, crude material either precipitated as pure product or was

puriﬁed by chromatography to give compound 47.

EXPERIMENTAL SECTION

■

Chemistry. Materials. Reagents were purchased as at least reagent

grade from Sigma-Aldrich (St. Louis, MO) unless otherwise speciﬁed

and used without further puriﬁcation. Solvents were purchased from

Fischer Scientiﬁc (Pittsburgh, PA) and were either used as purchased

or redistilled with an appropriate drying agent. HTS compound library

was obtained from UCSF Small Molecule Discovery Center (San

Francisco, CA). Commercial HTS service was provided by Invitrogen

(Grand Island, NY). Compounds 13, 14, 15, 16, 17, 37, and 38 were

purchased from Life Chemicals (Burlington, ON, Canada). All

synthesized compounds, intermediates, and purchased compounds

from Life Chemicals were determined to be >95% pure by HPLC

utilizing an Agilent 1100 LC/MSD. Endotoxin levels of active

compounds were measured with Endosafe-PTS (Charles River,

Wilmington, MA) and found to have less than 10 EU/μmol. Reaction

room temperature was maintained between 22 and 24 °C.

N-Cyclohexyl-2-((4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-

b]indol-2-yl)thio)acetamide (1). Compound 7a (50 mg, 0.14 mmol),

triethylamine (40 μL, 0.28 mmol), and cyclohexylamine (18 μL, 0.16

mmol) were dissolved in anhydrous DMF (1 mL). HATU (59.5 mg,

0.16 mmol) dissolved in 0.2 mL of DMF was added to the reaction

mixture and stirred for 20 min and then concentrated in vacuo. The

crude material was recrystallized with MeOH to give 61 mg in near

1

quantitative yield. H NMR (300 MHz, DMSO-d₆) δ ppm 0.97−1.33

(m, 4 H), 1.51 (d, J = 8.53 Hz, 1 H), 1.56−1.80 (m, 4 H), 3.48 (m, 1

H), 3.87 (s, 2 H), 7.24 (t, J = 7.29 Hz, 1 H), 7.38−7.67 (m, 6 H), 8.05

(d, J = 7.98 Hz, 1 H), 8.15 (d, J = 7.70 Hz, 1 H), 12.09 (s, 1 H).

HRMS calcd for C₂₄H₂₄N₄O₂SNa (M + Na)⁺, 455.1512; found,

455.1511.

Ethyl 2-((2-Cyanophenyl)amino)acetate (3). Anthranilonitrile

(30.29 g, 256 mmol), ethyl bromoacetate (29.46 mL, 267 mmol),

and sodium bicarbonate (25.6 g, 300 mmol) were combined in

anhydrous EtOH (90 mL) and reﬂuxed for 42 h. After cooling slightly,

solution was decanted from precipitate into a prewarmed ﬂask. Further

cooling of the decantate yielded crystals, which were then ﬁltered and

washed with cold water to give 23.6 g of white crystalline product. ¹H

NMR (500 MHz, DMSO-d₆) δ ppm 1.13−1.27 (m, 3 H), 4.04 (d, J =

6.41 Hz, 2 H), 4.12 (q, J = 7.22 Hz, 2 H), 6.36 (t, J = 6.25 Hz, 1 H),

6.64 (d, J = 8.54 Hz, 1 H), 6.69 (t, J = 7.47 Hz, 1 H), 7.41 (t, J = 7.78

Hz, 1 H), 7.49 (dd, J = 7.93, 1.53 Hz, 1 H). HRMS calcd for

C₁₁H₁₂N₂O₂Na (M + Na)⁺, 227.0791; found, 227.0794.

Ethyl 3-Amino-1H-indole-2-carboxylate (4). In a ﬂame-dried ﬂask,

a suspension of potassium t-butoxide (7.124 g, 63.5 mmol) in

anhydrous THF was stirred and maintained below 30 °C under argon.

To this solution was added a solution of compound 3 (16 g, 63.5

mmol) in anhydrous THF over 45 min and stirred for an additional 2

h. The reaction was then poured into ice water, extracted with EtOAc,

and dried over MgSO₄. The solid was then dissolved in minimal

EtOH, and water was added dropwise until just cloudy and allowed to

precipitate at room temperature. Precipitate was ﬁltered and washed

Instrumentation. Analytical TLC was performed using precoated

TLC silica gel 60 F₂₅₄aluminum sheets purchased from EMD

(Gibbstown, NJ) and visualized using UV light. Flash chromatography

was carried out on EMD silica gel 60 (40−63 μm) system or with a

Biotage Isolera One (Charlotte, NC) using the speciﬁed solvent.

Reactions were monitored using an Agilent 1100 LC/MSD (Santa

Clara, CA) with either a Supelco Discovery HS C18 column (Sigma-

Aldrich) or an Onyx Monolithic C18 (Phenomenex, Torrance, CA)

with purity above 98% by percent area. All synthesized compounds

and intermediates were analyzed by high-resolution MS using an

Agilent 6230 ESI-TOFMS (Santa Clara, CA). Selected compounds

were analyzed by IR (KBr window method) to conﬁrm presence of

functional groups having useful diagnostic frequencies using a Perkin-

Elmer 1600 series. ¹H NMR spectra were obtained on a Varian

Mercury 300 or 400 (Varian, Inc., Palo Alto, CA). ¹³C spectra were

obtained on Varian 500 with Xsens probe. The chemical shifts are

expressed in parts per million (ppm) using suitable deuterated NMR

solvents in reference to TMS at 0 ppm. The 3D structures were

prepared and optimized using the AMPAC semiempirical quantum

chemistry program (Accelrys, San Diego, CA).

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1

with cold EtOH to give 6.6 g of compound 4 in 51% yield. H NMR

(500 MHz, DMSO-d₆) δ ppm 1.33 (t, J = 7.02 Hz, 3 H), 4.29 (q, J =

7.12 Hz, 2 H), 5.68 (s, 2 H), 6.88 (ddd, J = 8.08, 5.80, 1.98 Hz, 1 H),

7.15−7.25 (m, 2 H), 7.74 (d, J = 7.93 Hz, 1 H), 10.34 (s, 1 H). HRMS

calcd for C₁₁H₁₂N₂O₂(M + H)⁺, 205.0972; found, 205.0973.

polyphosphoric acid was reacted according to general procedure B.

Crude product was puriﬁed by automated ﬂash chromatography

(Biotage) (DCM/MeOH = 99:1) to give 27.1 mg of compound 6d in

61.1% yield. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.95 (t, J = 7.33

Hz, 2 H), 3.67 (t, J = 6.20 Hz, 2 H), 6.96−7.63 (m, 8 H), 7.91 (d, J =

7.70 Hz, 1 H), 8.31 (d, J = 4.77 Hz, 1 H), 11.50−11.89 (m, 1 H).

HRMS calcd for C₁₈H₁₅N₃OS (M + H)⁺, 321.0936; found, 321.0939.

2-((4-Oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-yl)-

thio)acetic Acid (7a). Compound 6a (3.77 g, 12.9 mmol), KOH (1.44

g, 25.8 mmol), chloroacetic acid (1.25 g, 12.9 mmol, 6.5 mL of

anhydrous EtOH), and 115 mL of anhydrous EtOH were reacted

according to general procedure C to give 3.66 g of compound 7a in

81% yield. IR: 3251 (OH), 1702 (CO carboxyl), 1660 (CO amide)

Ethyl 3-(3-Phenylthioureido)-1H-indole-2-carboxylate (5a). Com-

pound 4 (5.21 g, 25.5 mmol) was reacted with phenyl isothiocyanate

(3.36 mL, 28.1 mmol) in warm EtOH (75 mL) according to general

1

procedure A to give 6.732 g compound 5a in 75% yield. H NMR

(400 MHz, DMSO-d₆) δ ppm 1.32 (t, J = 7.07 Hz, 3 H), 4.32 (q, J =

7.31 Hz, 2 H), 7.09 (dt, J = 14.75, 7.50 Hz, 2 H), 7.22−7.38 (m, 3 H),

7.43 (d, J = 8.29 Hz, 1 H), 7.51 (d, J = 7.80 Hz, 2 H), 7.56 (d, J = 8.29

Hz, 1 H), 9.40 (s, 1 H), 9.69 (br s, 1 H), 11.79 (s, 1 H). HRMS calcd

for C₁₈H₁₇N₃O₂SNa (M + Na)⁺, 362.0934; found, 362.0935.

Ethyl 3-(3-Cyclohexylthioureido)-1H-indole-2-carboxylate (5b).

Compound 4 (100 mg, 0.49 mmol) was reacted with cyclohexyl

isothiocyanate (169.15 mg, 0.54 mmol) in warm EtOH (750 μL)

according to general procedure A to give 104 mg of compound 5b in

61.8% yield. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.01−1.42 (m, 9

H), 1.49−1.62 (m, 1 H), 1.62−1.74 (m, 2 H), 1.77−1.96 (m, 2 H),

4.13 (br s, 1 H), 4.29 (q, J = 7.09 Hz, 2 H), 7.05 (t, J = 7.33 Hz, 1 H),

7.25 (t, J = 8.07 Hz, 1 H), 7.41 (d, J = 8.07 Hz, 1 H), 7.49 (d, J = 8.07

Hz, 1 H), 8.91 (s, 1 H), 11.71 (br s, 1 H). HRMS calcd for

C₁₈H₂₃N₃O₂S (M + H)⁺, 345.1511; found, 345.15142.

Ethyl 3-(3-(Naphthalen-1-yl)thioureido)-1H-indole-2-carboxylate

(5c). Compound 4 (50 mg, 0.28 mmol) was reacted with 1-naphthyl

isothiocyanate (49.9 mg, 0.27 mmol) in warm EtOH (750 μL)

according to general procedure A to give 58.6 mg of compound 5c in

61.5% yield. HRMS calcd for C₂₂H₁₉N₃O₂SNa (M + Na)⁺, 412.1090;

found, 412.1091.

Ethyl 3-(3-Benzylthioureido)-1H-indole-2-carboxylate (5d). Com-

pound 4 (100 mg, 0.49 mmol) was reacted with phenethyl

isothiocyanate (80.4 μL, 0.54 mmol) in warm EtOH (1.5 mL)

according to general procedure A to give 147 mg of compound 5d in

81.8% yield. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.31 (t, J = 6.97

Hz, 3 H), 4.27 (q, J = 7.21 Hz, 2 H), 7.06 (t, J = 7.70 Hz, 1 H), 7.09−

7.33 (m, 7 H), 7.43 (dd, J = 8.07, 4.03 Hz, 2 H), 7.56 (br s, 1 H), 9.12

(br s, 1 H), 11.79 (br s, 1 H). HRMS calcd for C₂₀H₂₂N₃O2S (M +

H)⁺, 368.1427; found, 368.1431

1

cm⁻¹. H NMR (400 MHz, DMSO-d₆) δ ppm 3.94 (s, 2 H), 7.14−

7.36 (m, 1 H), 7.38−7.84 (m, 7 H), 7.93 (d, J = 7.70 Hz, 1 H), 12.09

(br s, 1 H). HRMS calcd for C₁₈H₁₃N₃O₃SNa (M + Na)⁺, 374.0570;

found, 374.0572.

2-((3-Cyclohexyl-4-oxo-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-

yl)thio)acetic Acid (7b). Compound 6b (165.8 mg, 0.55 mmol), KOH

(62 mg, 1.1 mmol), chloroacetic acid (52.3 mg, 0.55 mmol, 3 mL of

anhydrous EtOH), and 62 mL of anhydrous EtOH were reacted

according to general procedure C to give 35.6 mg of compound 7b in

17.9% yield. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.05−1.52 (m, 4

H), 1.59−2.05 (m, 6 H), 3.94 (s, 2 H), 4.20−4.46 (m, 1 H), 7.15 (t, J

= 7.33 Hz, 1 H), 7.30−7.59 (m, 2 H), 7.88 (d, J = 8.07 Hz, 1 H), 11.79

(br s, 1 H). HRMS calcd for C₁₈H₁₉N₃O₃SNa (M + Na)⁺, 380.1039;

found, 380.1042.

2-((3-(Naphthalen-2-yl)-4-oxo-4,5-dihydro-3H-pyrimido[5,4-b]-

indol-2-yl)thio)acetic Acid (7c). Compound 6c (120 mg, 0.35 mmol),

KOH (39.1 mg, 70 mmol), chloroacetic acid (33 mg, 0.35 mmol, 3 mL

of anhydrous EtOH), and 62 mL of anhydrous EtOH were reacted

according to general procedure C to give 67.1 mg of compound 7c in

1

47.8% yield. H NMR (400 MHz, DMSO-d₆) δ ppm 3.90 (s, 2 H),

7.26 (t, J = 7.40 Hz, 1 H), 7.33−7.66 (m, 5 H), 7.67−7.80 (m, 2 H),

7.98 (d, J = 7.70 Hz, 1 H), 8.09 (d, J = 8.43 Hz, 1 H), 8.17 (t, J = 4.77

Hz, 1 H), 12.14 (br s, 1 H). HRMS calcd for C₂₂H₁₅N₃O₃SNa (M +

Na)⁺, 424.0726; found, 424.0730.

2-((4-Oxo-3-phenethyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-

yl)thio)acetic Acid (7d). Compound 6d (271 mg, 0.84 mmol), KOH

(94.5 mg, 1.69 mmol), chloroacetic acid (79.8 mg, 0.84 mmol, 3 mL of

anhydrous EtOH), and 62 mL of anhydrous EtOH were reacted

according to general procedure C to give 160 mg of compound 7d in

3-Phenyl-2-thioxo-2,3-dihydro-1H-pyrimido[5,4-b]indol-4(5H)-

one (6a). To a ﬂame-dried ﬂask with cold anhydrous EtOH (75 mL)

was added cold acetyl chloride (7 mL, 98.5 mmol) under argon with

stirring. In a separate ﬂame-dried ﬂask charged with argon, compound

5a (6.5 g, 19 mmol) was dissolved in anhydrous EtOH (25 mL) and

added to the acetyl chloride solution. The reaction was reﬂuxed for 12

h and cooled upon completion. Precipitate was ﬁltered and

recrystallized with EtOH to give 3.77 g of compound 6a in 75%

1

50% yield. H NMR (400 MHz, DMSO-d₆) δ ppm 3.02 (t, J = 7.80

Hz, 2 H), 4.02 (s, 2 H), 4.35 (t, J = 7.31 Hz, 2 H), 6.87−7.83 (m, 8

H), 7.95 (d, J = 7.31 Hz, 1 H), 11.92 (br s, 1 H). HRMS calcd for

C₂₀H₁₇N₃O₃SNa (M + Na)⁺, 402.0883; found, 402.0885.

N-Cycloheptyl-2-((4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-

b]indol-2-yl)thio)acetamide (9). Compound 7a (50 mg, 0.14 mmol),

cycloheptylamine (39.86 μL, 0.16 mmol), triethylamine (39.63 μL,

0.28 mmol), and HATU (59.52 mg, 0.16 mmol) were reacted

according to general procedure D to give 12.46 mg in 18.5% yield. ¹H

NMR (300 MHz, DMSO-d₆) δ ppm 1.18−1.65 (m, 10 H), 1.66−1.88

(m, 2 H), 3.59−3.77 (m, 1 H), 3.87 (s, 2 H), 7.25 (t, J = 7.01 Hz, 1

H), 7.40−7.55 (m, 4 H), 7.56−7.68 (m, 3 H), 8.06 (d, J = 7.98 Hz, 1

H), 8.20 (d, J = 7.70 Hz, 1 H), 12.10 (s, 1 H). ¹³C NMR (126 MHz,

DMSO-d₆) δ ppm 24.12, 28.28, 34.69, 37.17, 50.51, 113.31, 119.7,

120.48, 120.78, 120.81, 127.77, 130.00, 130.01, 130.32, 136.51, 137.68,

139.35, 153.01, 155.39, 165.92. HRMS calcd for C₂₅H₂₆N₄O₂SNa (M

+ Na)⁺, 469.1669; found, 469.1670.

1

yield. H NMR (400 MHz, DMSO-d₆) δ ppm 7.09−7.31 (m, 3 H),

7.31−7.61 (m, 6 H), 8.20 (d, J = 8.07 Hz, 1 H), 12.17 (br s, 1 H).

HRMS calcd for C₁₆H₁₂N₃OS (M + H)⁺, 294.0696; found, 294.0698.

3-Cyclohexyl-2-thioxo-2,3-dihydro-1H-pyrimido[5,4-b]indol-

4(5H)-one (6b). Compound 5b (70 mg, 0.2 mmol) and 1 mL of

polyphosphoric acid was reacted according to general procedure B to

1

give 60.6 mg of compound 6b in quantitative yield. H NMR (400

MHz, DMSO-d₆) δ ppm 1.07−1.49 (m, 5 H), 1.53−1.70 (m, 1 H),

1.70−1.84 (m, 2 H), 2.01 (s, 2 H), 3.86−4.09 (m, 1 H), 7.11 (t, J =

8.07 Hz, 1 H), 7.29−7.54 (m, 2 H), 7.87 (d, J = 8.07 Hz, 1 H), 8.06

(d, J = 6.97 Hz, 1 H), 11.60 (s, 1 H). HRMS calcd for C₁₆H₁₈N₃OS

(M + H)⁺, 300.1165; found, 300.1169.

3-(Naphthalen-1-yl)-2-thioxo-2,3-dihydro-1H-pyrimido[5,4-b]-

indol-4(5H)-one (6c). Compound 5c (150 mg, 0.38 mmol) and 1 mL

of polyphosphoric acid was reacted according to general procedure B

N-Cyclooctyl-2-((4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]-

indol-2-yl)thio)acetamide (10). Compound 7a (25 mg, 0.07 mmol),

cyclooctylamine (24.15 μL, 0.08 mmol), triethylamine (19.82 μL, 0.14

mmol), and HATU (29.76 mg, 0.08 mmol) were reacted according to

1

to give 132 mg of compound 6b in quantitative yield. H NMR (400

MHz, DMSO-d₆) δ ppm 7.02−7.18 (m, 1 H), 7.33−7.48 (m, 2 H),

7.48−7.67 (m, 3 H), 7.74 (d, J = 8.07 Hz, 1 H), 7.87 (d, J = 8.07 Hz, 1

H), 7.84 (d, J = 7.70 Hz, 1 H), 7.94−8.03 (m, 1 H), 8.03−8.18 (m, 1

H), 11.83 (s, 1 H). HRMS calcd for C₂₀H₁₄N₃OS (M + H)⁺,

344.0852; found, 344.0855.

1

general procedure D to give 30.77 mg in quantitative yield. H NMR

(400 MHz, DMSO-d₆) δ ppm 1.23−1.89 (m, 14 H), 3.68−3.82 (m, 1

H), 3.87 (s, 2 H), 7.24 (t, J = 6.97 Hz, 1 H), 7.37−7.75 (m, 7 H), 8.06

(d, J = 8.07 Hz, 1 H), 8.18 (d, J = 7.70 Hz, 1 H), 12.10 (br s, 1 H). ¹³C

NMR (126 MHz, DMSO-d₆) δ ppm 23.75, 25.47, 27.25, 31.87, 37.16,

49.49, 113.32, 119.7, 120.48, 120.78, 120.81, 127.78, 130.00, 130.01,

3-Phenethyl-2-thioxo-2,3-dihydro-1H-pyrimido[5,4-b]indol-

4(5H)-one (6d). Compound 5d (50.7 mg, 0.138 mmol) and 1 mL of

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130.32, 136.51, 137.68, 139.36, 153.02, 155.39, 165.90. HRMS calcd

for C₂₆H₂₉N₄O₂S (M + H)⁺, 461.2006; found, 461.2010.

mmol), 2-aminothiazole (31.35 mg, 0.16 mmol), triethylamine (39.63

μL, 0.28 mmol), and HATU (59.52 mg, 0.16 mmol) were reacted

according to general procedure D to give 12.82 mg in 21.1% yield. ¹H

NMR (400 MHz, DMSO-d₆) δ ppm 4.17 (s, 2 H), 6.90−7.27 (m, 2

H), 7.30−7.78 (m, 8 H), 7.83 (d, J = 7.70 Hz, 1 H), 12.03−12.16 (m,

1 H). ¹³C NMR (126 MHz, DMSO-d₆) δ ppm 20.55, 28.58, 36.97,

46.95, 113.30, 119.71, 120.54, 120.77, 120.80, 127.76, 130.01, 130.32,

136.49, 137.68, 139.35, 152.91, 155.40, 167.27. HRMS calcd for

C₂₁H₁₅N₅O₂S₂Na (M + Na)⁺, 456.0559; found, 456.0562.

N-Cyclopentyl-2-((4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-

b]indol-2-yl)thio)acetamide (11). Compound 7a (50 mg, 0.14

mmol), cyclopentylamine (31 μL, 0.16 mmol), triethylamine (39.63

μL, 0.28 mmol), and HATU (59.52 mg, 0.16 mmol) were reacted

1

according to general procedure D to give 48.9 mg in 76% yield. H

NMR (300 MHz, DMSO-d₆) δ ppm 1.32−1.55 (m, 4 H), 1.55−1.70

(m, 2 H), 1.71−1.86 (m, 2 H), 3.87 (s, 2 H), 3.97 (dd, J = 13.07, 6.46

Hz, 1 H), 7.25 (t, J = 7.29 Hz, 1 H), 7.35−7.72 (m, 7 H), 8.05 (d, J =

7.98 Hz, 1 H), 8.26 (d, J = 6.88 Hz, 1 H), 12.10 (s, 1 H). ¹³C NMR

(126 MHz, DMSO-d₆) δ ppm 23.95, 32.74, 37.13, 51.12, 113.32,

119.69, 120.5, 120.76, 120.79, 127.77, 129.99, 130.01, 130.32, 136.50,

137.67, 139.35, 153.02, 155.39, 166.64. HRMS calcd for

C₂₅H₂₆N₄O₂SNa (M + Na)⁺, 441.1356; found, 441.1367.

N-Ethyl-2-((4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-

2-yl)thio)acetamide (22). Compound 7a (50 mg, 0.14 mmol),

ethylamine (17.73 μL, 0.16 mmol), triethylamine (39.63 μL, 0.28

mmol), and HATU (59.52 mg, 0.16 mmol) were reacted according to

1

general procedure D to give 22.9 mg in 43.3% yield. H NMR (400

MHz, DMSO-d₆) δ ppm 1.02 (t, J = 7.15 Hz, 3 H), 3.04−3.16 (m, 2

H), 3.88 (s, 2 H), 7.26 (t, J = 7.33 Hz, 1 H), 7.39−7.54 (m, 3 H),

7.56−7.69 (m, 3 H), 8.05 (d, J = 8.07 Hz, 1 H), 8.26 (m, 1 H), 12.11

(s, 1 H). ¹³C NMR (126 MHz, DMSO-d₆) δ ppm 15.12, 34.34, 37.01,

113.30, 119.70, 120.59, 120.74, 120.81, 127.76, 130.01, 130.32, 136.49,

137.68, 139.35, 152.88, 155.41, 167.02. HRMS calcd for

C₂₀H₁₈N₄O₂SNa (M + Na)⁺, 401.1043; found, 401.1046.

N-Cyclobutyl-2-((4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-

b]indol-2-yl)thio)acetamide (12). Compound 7a (50 mg, 0.14

mmol), cyclobutylamine (26.73 μL, 0.16 mmol), triethylamine

(39.63 μL, 0.28 mmol), and HATU (59.52 mg, 0.16 mmol) were

reacted according to general procedure D to give 41.6 mg in 73.5%

1

yield. H NMR (300 MHz, DMSO-d₆) δ ppm 1.48−1.72 (m, 2 H),

1.93 (q, J = 9.70 Hz, 2 H), 2.05−2.26 (m, 2 H), 3.85 (s, 2 H), 4.08−

4.26 (m, 1 H), 7.26 (t, J = 7.29 Hz, 1 H), 7.37−7.70 (m, 6 H), 8.07 (d,

J = 7.98 Hz, 1 H), 8.56 (d, J = 7.70 Hz, 1 H), 12.10 (s, 1 H). ¹³C NMR

(126 MHz, DMSO-d₆) δ ppm 15.11, 30.72, 37.03, 44.72, 113.32,

119.70, 120.52, 120.81, 127.78, 130.00, 130.01, 130.33, 136.49, 137.69,

139.35, 152.95, 155.4, 166.21. HRMS calcd for C₂₂H₂₀N₄O₂SNa (M +

Na)⁺, 427.1199; found, 427.1200.

N-(Naphthalen-2-yl)-2-((4-oxo-3-phenyl-4,5-dihydro-3H-

pyrimido[5,4-b]indol-2-yl)thio)acetamide (18). Compound 7a (50

mg, 0.14 mmol), 2-aminonaphthalene (44.82 mg, 0.16 mmol),

triethylamine (39.63 μL, 0.28 mmol), and HATU (59.52 mg, 0.16

mmol) were reacted according to general procedure D to give 40.8 mg

in 61% yield. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 4.16 (br s, 2 H),

7.11 (t, J = 7.70 Hz, 1 H), 7.26−8.07 (m, 14 H), 8.31 (br s, 1 H),

10.65 (br s, 1 H), 12.10 (br s, 1 H). ¹³C NMR (126 MHz, DMSO-d₆)

δ ppm 37.99, 113.27, 115.48, 119.71, 120.21, 120.49, 120.68, 120.72,

125.07, 126.92, 127.71, 127.74, 127.90, 128.89, 130.01, 130.06, 130.18,

130.41, 133.84, 136.49, 137.16, 137.63, 139.30, 152.89, 155.37, 166.96.

HRMS calcd for C₂₈H₂₀N₄O₂SNa (M + Na)⁺, 499.1199; found,

499.1201.

N-(Furan-2-ylmethyl)-2-((4-oxo-3-phenyl-4,5-dihydro-3H-

pyrimido[5,4-b]indol-2-yl)thio)acetamide (19). Compound 7a (50

mg, 0.14 mmol), furfurylamine (27.66 μL, 0.16 mmol), triethylamine

(39.63 μL, 0.28 mmol), and HATU (59.52 mg, 0.16 mmol) were

reacted according to general procedure D to give 50.37 mg in 83.6%

yield. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.95 (s, 2 H), 4.29 (d, J

= 5.50 Hz, 2 H), 6.25 (d, J = 3.30 Hz, 1 H), 6.32 (t, J = 2.57 Hz, 1 H),

7.24 (t, J = 6.97 Hz, 1 H), 7.35−7.72 (m, 7 H), 7.97 (d, J = 7.70 Hz, 1

H), 8.73 (t, J = 5.32 Hz, 1 H), 12.10 (br s, 1 H). ¹³C NMR (126 MHz,

DMSO-d₆) δ ppm 36.46, 36.76, 107.46, 107.48, 107.49, 110.86,

110.87, 110.89, 113.23, 119.71, 120.60, 120.80, 120.88, 127.76, 130.01,

130.02, 130.34, 136.50, 137.69, 139.34, 142.64, 152.32, 152.81, 155.41,

167.44. HRMS calcd for C₂₃H₁₈N₄O₃SNa (M + Na)⁺, 453.0992;

found, 453.0995.

N-(1H-Indazol-6-yl)-2-((4-oxo-3-phenyl-4,5-dihydro-3H-

pyrimido[5,4-b]indol-2-yl)thio)acetamide (20). Compound 7a (50

mg, 0.14 mmol), 2-aminoindazole (41.68 mg, 0.16 mmol), triethyl-

amine (39.63 μL, 0.28 mmol), and HATU (59.52 mg, 0.16 mmol)

were reacted according to general procedure D to give 32.7 mg in 50%

yield. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 4.11 (s, 2 H), 7.09 (t, J

= 7.33 Hz, 1 H), 7.17 (d, J = 8.43 Hz, 1 H), 7.32−7.77 (m, 9 H),

7.87−8.01 (m, 2 H), 8.11 (s, 1 H), 10.55 (br s, 1 H), 12.07 (br s, 1 H).

¹³C NMR (126 MHz, DMSO-d₆) δ ppm 38.00, 99.22, 113.29, 114.43,

N-Butyl-2-((4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-

2-yl)thio)acetamide (23). Compound 7a (50 mg, 0.14 mmol),

butylamine (30.94 μL, 0.16 mmol), triethylamine (39.63 μL, 0.28

mmol), and HATU (59.52 mg, 0.16 mmol) were reacted according to

1

general procedure D to give 18.6 mg in 44.7% yield. H NMR (400

MHz, DMSO-d₆) δ ppm 0.78 (t, J = 7.31 Hz, 3 H), 1.17−1.30 (m, 2

H), 1.32−1.46 (m, 2 H), 2.98−3.13 (m, 2 H), 3.89 (s, 2 H), 7.25 (t, J

= 6.83 Hz, 1 H), 7.39−7.55 (m, 4 H), 7.60 (m, J = 5.40, 5.40 Hz, 3 H),

8.04 (d, J = 7.80 Hz, 1 H), 8.17−8.25 (m, 1 H), 12.10 (s, 1 H). ¹³C

NMR (126 MHz, DMSO-d₆) δ ppm 13.61, 19.53, 31.2, 36.52, 38.7,

112.84, 119.26, 120.08, 120.32, 120.38, 127.3, 129.54, 129.55, 129.86,

136.05, 137.25, 138.93, 152.43, 154.96, 166.7. HRMS calcd for

C₂₂H₂₂N₄O₂SNa (M + Na)⁺, 429.1356; found, 429.1359.

2-((4-Oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-yl)-

thio)-N-pentylacetamide (24). Compound 7a (25 mg, 0.07 mmol), n-

amylamine (20 μL, 0.16 mmol), triethylamine (19.82 μL, 0.14 mmol),

and HATU (29.76 mg, 0.08 mmol) were reacted according to general

1

procedure D to give 19.5 mg in 66% yield. H NMR (300 MHz,

DMSO-d₆) δ ppm 0.74 (t, J = 7.15 Hz, 3 H), 1.07−1.25 (m, 4 H),

1.31−1.45 (m, 2 H), 2.98−3.15 (m, 2 H), 3.88 (s, 2 H), 7.24 (t, J =

6.88 Hz, 1 H), 7.40−7.54 (m, 3 H), 7.56−7.68 (m, 3 H), 8.04 (d, J =

7.98 Hz, 1 H), 8.20 (t, J = 5.36 Hz, 1 H), 12.10 (s, 1 H). ¹³C NMR

(126 MHz, DMSO-d₆) δ ppm 14.26, 22.29, 29.03, 29.25, 36.92,

113.27, 119.69, 120.52, 120.78, 120.81, 127.76, 130.00, 130.32, 136.48,

137.69, 139.35, 152.85, 155.40, 167.16. HRMS calcd for C₂₃H₂₅N₄O₂S

(M + H)⁺, 421.1693; found, 421.1689.

N-Hexyl-2-((4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]-

indol-2-yl)thio)acetamide (25). Compound 7a (25 mg, 0.07 mmol),

hexylamine (21 μL, 0.16 mmol), triethylamine (19.82 μL, 0.14 mmol),

and HATU (29.76 mg, 0.08 mmol) were reacted according to general

procedure D to give 20 mg in 65% yield. ¹H NMR (300 MHz, DMSO-

d₆) δ ppm 0.76 (t, J = 6.46 Hz, 3 H), 1.01−1.49 (m, 8 H), 3.06 (q, J =

6.60 Hz, 2 H), 3.87 (s, 2 H), 7.24 (t, J = 6.80 Hz, 1 H), 7.41−7.55 (m,

3 H), 7.55−7.66 (m, 3 H), 8.05 (d, J = 7.70 Hz, 1 H), 8.19 (t, J = 5.23

Hz, 1 H), 12.10 (s, 1 H). ¹³C NMR (126 MHz, DMSO-d₆) δ ppm

14.29, 22.40, 26.49, 29.51, 31.40, 36.82, 113.28, 119.63, 120.55,

120.79, 120.81, 127.82, 129.94, 130.04, 130.36, 136.41, 137.72, 139.36,

152.80, 155.41, 167.33. HRMS calcd for C₂₄H₂₇N₄O₂S (M + H)⁺,

435.1849; found, 435.1846.

N-Isobutyl-2-((4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]-

indol-2-yl)thio)acetamide (26). Compound 7a (50 mg, 0.14 mmol),

isobutylamine (31.11 μL, 0.16 mmol), triethylamine (39.63 μL, 0.28

mmol), and HATU (59.52 mg, 0.16 mmol) were reacted according to

1

general procedure D to give 17.71 mg in 31% yield. H NMR (400

119.60, 119.65, 120.55, 120.69, 121.03, 121.24, 127.81, 129.95, 130.10,

130.45, 133.80, 136.42, 137.66, 137.73, 139.31, 140.74, 148.09, 152.88,

155.37, 162.89, 167.00. HRMS calcd for C₂₅H₁₈N₆O₂SNa (M + Na)⁺,

489.1104; found, 489.1106.

2-((4-Oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-yl)-

thio)-N-(thiazol-2-yl)acetamide (21). Compound 7a (50 mg, 0.14

MHz, DMSO-d₆) δ ppm 0.72−0.92 (m, 6 H), 1.68 (dt, J = 13.47, 6.64

Hz, 1 H), 2.91 (t, J = 6.60 Hz, 2 H), 3.93 (s, 2 H), 7.25 (t, J = 6.97 Hz,

1 H), 7.40−7.56 (m, 3 H), 7.61 (d, J = 6.23 Hz, 3 H), 8.04 (d, J = 7.70

Hz, 1 H), 8.23 (t, J = 6.23 Hz, 1 H), 12.11 (br s, 1 H). ¹³C NMR (126

MHz, DMSO-d₆) δ ppm 20.55, 28.58, 36.97, 46.95, 113.30, 119.71,

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120.54, 120.77, 120.80, 127.76, 130.01, 130.32, 136.49, 137.68, 139.35,

152.91, 155.40, 167.27. HRMS calcd for C₂₂H₂₂N₄O₂SNa (M + Na)⁺,

429.1356; found, 429.1359.

167.06. HRMS calcd for C₂₃H₂₅N₄O₂S (M + H)⁺, 421.1693; found,

421.1695.

N-(tert-Butyl)-2-((4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-

b]indol-2-yl)thio)acetamide (32). Compound 7a (50 mg, 0.14

mmol), t-butylamine (32.9 μL, 0.16 mmol), triethylamine (39.63 μL,

0.28 mmol), and HATU (59.52 mg, 0.16 mmol) were reacted

N-Isopentyl-2-((4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]-

indol-2-yl)thio)acetamide (27). Compound 7a (25 mg, 0.07 mmol),

isopentylamine (20 μL, 0.16 mmol), triethylamine (19.82 μL, 0.14

mmol), and HATU (29.76 mg, 0.08 mmol) were reacted according to

general procedure D to give 20 mg in 67% yield. ¹H NMR (300 MHz,

DMSO-d₆) δ ppm 0.78 (d, J = 6.60 Hz, 6 H), 1.29 (q, J = 6.88 Hz, 2

H), 1.54 (tt, J = 13.38, 6.84 Hz, 1 H), 3.08 (s, 2 H), 3.88 (s, 2 H), 7.24

(t, J = 7.01 Hz, 1 H), 7.36−7.68 (m, 6 H), 8.04 (d, J = 7.98 Hz, 1 H),

8.17 (t, J = 5.50 Hz, 1 H), 12.10 (s, 1 H). ¹³C NMR (126 MHz,

DMSO-d₆) δ ppm 22.71, 25.45, 36.86, 37.68, 38.43, 113.29, 119.64,

120.55, 120.77, 120.80, 127.83, 129.95, 130.03, 130.35, 136.42, 137.71,

139.34, 152.82, 155.40, 167.23. HRMS calcd for C₂₃H₂₅N₄O₂S (M +

H)⁺, 421.1693; found, 421.1689.

1

according to general procedure D to give 22.6 mg in 39.7% yield. H

NMR (400 MHz, DMSO-d₆) δ ppm 1.15−1.37 (m, 9 H), 3.85 (s, 2

H), 7.26 (t, J = 6.83 Hz, 1 H), 7.41−7.56 (m, 4 H), 7.57−7.71 (m, 3

H), 7.93 (s, 1 H), 8.07 (d, J = 7.80 Hz, 1 H), 12.11 (s, 1 H). ¹³C NMR

(126 MHz, DMSO-d₆) δ ppm 28.86, 37.86, 50.89, 113.34, 119.68,

120.54, 120.74, 120.78, 127.76, 129.99, 130.00, 130.31, 136.54, 137.68,

139.37, 153.13, 155.40, 166.60. HRMS calcd for C₂₂H₂₂N₄O₂SNa (M

+ Na)⁺, 429.1356; found, 429.1359.

2-((4-Oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-yl)-

thio)-N-((tetrahydrofuran-2-yl)methyl)acetamide (33). Compound

7a (50 mg, 0.14 mmol), tetrahydrofurfurylamine (32.31 μL, 0.16

mmol), triethylamine (39.63 μL, 0.28 mmol) and HATU (59.52 mg,

0.16 mmol) were reacted according to general procedure D to give 35

mg in 57.5% yield. IR: 3372, 3193 (NH), 1651 (CO) cm⁻¹. ¹H NMR

(400 MHz, DMSO-d₆) δ ppm 1.32−1.61 (m, 1 H), 1.63−2.06 (m, 3

H), 2.96−3.26 (m, 2 H), 3.52−3.64 (m, 1 H), 3.64−3.75 (m, 1 H),

3.76−3.86 (m, 1 H), 3.92 (s, 2 H), 7.25 (t, J = 6.97 Hz, 1 H), 7.35−

7.71 (m, 6 H), 8.07 (d, J = 8.06 Hz, 1 H), 8.30 (t, J = 5.50 Hz, 1 H),

12.10 (br s, 1 H). ¹³C NMR (126 MHz, DMSO-d₆) δ ppm 25.12, 28.4,

36.38, 43.03, 67.15, 77.11, 112.81, 119.25, 120.11, 120.39, 127.32,

129.55, 129.57, 129.88, 136.05, 137.25, 138.92, 152.4, 154.95, 167.14.

HRMS calcd for C₂₃H₂₂N₄O₃SNa (M + Na)⁺, 457.1305; found,

457.1308.

2-((4-Oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-yl)-

thio)-N-(piperidin-4-yl)acetamide (34). Compound 7a (25 mg, 0.07

mmol), 4-aminopiperidine (16.6 uL, 0.16 mmol), triethylamine (19.82

μL, 0.14 mmol), and HATU (29.76 mg, 0.08 mmol) were reacted

according to general procedure D to give 17 mg in 65% yield. IR:

3372, 3193 (NH), 1651 (CO) cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆)

δ ppm 1.27 (d, J = 11.36 Hz, 1 H), 1.46 (d, J = 9.90 Hz, 1 H), 1.77−

2.05 (m, 2 H), 2.64−2.85 (m, 1 H), 4.00−4.43 (m, 3 H), 7.14−7.38

(m, 2 H), 7.41−7.80 (m, 6 H), 8.04 (d, J = 7.70 Hz, 1 H). ¹³C NMR

(126 MHz, DMSO-d₆) δ ppm 33.41, 35.16, 36.00, 36.46, 40.91, 44.69,

45.37, 48.25, 113.30, 119.70, 120.67, 120.75, 120.79, 127.76, 130.00,

130.03, 130.33, 136.51, 137.63, 137.68, 139.36, 152.93, 152.97, 155.40,

165.44, 166.23. HRMS calcd for C₂₃H₂₄N₅O₂S (M + H)⁺, 434.1645;

found, 434.1648.

N-(3,3-Dimethylbutyl)-2-((4-oxo-3-phenyl-4,5-dihydro-3H-

pyrimido[5,4-b]indol-2-yl)thio)acetamide (28). Compound 7a (25

mg, 0.07 mmol)), 3,3-dimethylbutylamine (20 μL, 0.16 mmol),

triethylamine (19.82 μL, 0.14 mmol), and HATU (29.76 mg, 0.08

mmol) were reacted according to general procedure D and puriﬁed by

reverse-phase chromatography (C18, 10:90 to 90:10 water:methanol

gradient) to give 18 mg in 60% yield. ¹H NMR (300 MHz, DMSO-d₆)

δ ppm 0.83 (s, 9 H), 1.26−1.47 (m, 2 H), 3.00−3.16 (m, 2 H), 3.86 (s,

2 H), 7.24 (t, J = 7.29 Hz, 1 H), 7.39−7.55 (m, 3 H), 7.56−7.70 (m, 3

H), 8.04 (d, J = 7.98 Hz, 1 H), 8.17 (t, J = 5.50 Hz, 1 H), 12.10 (s, 1

H). ¹³C NMR (126 MHz, DMSO-d₆) δ ppm 24.12, 28.28, 34.68,

37.17, 50.51, 113.31, 119.70, 120.48, 120.78, 120.81, 127.77, 130.00,

130.01, 130.32, 136.51, 137.68, 139.35, 153.01, 155.39, 165.92. HRMS

calcd for C₂₄H₂₇N₄O₂S (M + H)⁺, 435.1849; found, 435.1846.

N-(3-Methylpentyl)-2-((4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido-

[5,4-b]indol-2-yl)thio)acetamide (29). Compound 7a (25 mg, 0.07

mmol), 3-methylpentylamine (20 μL, 0.16 mmol), triethylamine

(19.82 μL, 0.14 mmol) and HATU (29.76 mg, 0.08 mmol) according

togeneral procedure D and puriﬁed by reverse-phase chromatography

(C18, 10:90 to 90:10 water:methanol gradient) to give 17 mg in 55%

1

yield. H NMR (400 MHz, DMSO-d₆) δ ppm 0.67−0.85 (m, 6 H),

0.96−1.11 (m, 1 H), 1.13−1.26 (m, 2 H), 1.31 (dd, J = 12.65, 6.42 Hz,

1 H), 1.37−1.51 (m, 1 H), 2.99−3.17 (m, 2 H), 3.87 (s, 2 H), 7.24 (t,

J = 7.52 Hz, 1 H), 7.41−7.55 (m, 4 H), 7.56−7.69 (m, 3 H), 8.04 (d, J

= 8.07 Hz, 1 H), 8.17 (t, J = 5.50 Hz, 1 H), 12.10 (s, 1 H). ¹³C NMR

(126 MHz, DMSO-d₆) δ ppm 11.42, 19.22, 29.22, 31.70, 36.16, 37.49,

113.27, 119.69, 120.50, 120.81, 127.77, 130.00, 130.01, 130.33, 136.48,

137.69, 139.36, 152.84, 155.40, 167.11. HRMS calcd for

C₂₄H₂₆N₄O₂SNa (M + Na)⁺, 457.1669; found, 457.1670.

(Z)-3-((Hydroxy(2-(2-((4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido-

[5,4-b]indol-2-yl)thio)acetamido)ethoxy)phosphoryl)oxy)propane-

1,2-diyl Dioleate (35). Compound 7a (50 mg, 0.14 mmol), DOPE

(32.9 μL, 0.16 mmol), triethylamine (39.63 μL, 0.28 mmol), and

HATU (59.52 mg, 0.16 mmol) were reacted according to general

2-((4-Oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-yl)-

thio)-N-(pentan-2-yl)acetamide (30). Compound 7a (25 mg, 0.07

mmol), 2-aminopentane (18.54 μL, 0.16 mmol), triethylamine (19.82

μL, 0.14 mmol), and HATU (29.76 mg, 0.08 mmol) according to

1

procedure D to give 16.4 mg in 10.9% yield. H NMR (400 MHz,

DMSO-d₆) δ ppm 0.84 (t, J = 6.83 Hz, 6 H), 1.05−1.75 (m, 50 H),

1.96 (br s, 8 H), 2.23 (t, J = 7.31 Hz, 4 H), 3.02 (s, 3 H), 3.14−3.26

(m, 2 H), 3.59−3.83 (m, 3 H), 3.90 (s, 2 H), 4.02−4.17 (m, 1 H), 4.26

(d, J = 9.26 Hz, 1 H), 5.06 (br s, 1 H), 5.30 (br s, 3 H), 7.24 (t, J =

7.31 Hz, 1 H), 7.38−7.75 (m, 6 H), 8.09 (d, J = 7.80 Hz, 1 H), 8.87

(br s, 1 H), 10.12 (br s, 1 H), 12.08 (br s, 1 H). ¹³C NMR (126 MHz,

DMSO-d₆) δ ppm 8.84, 14.38, 14.39, 22.58, 24.85, 24.91, 27.02, 27.05,

28.88, 28.91, 28.98, 29.00, 29.07, 29.12, 29.19, 29.33, 29.43, 29.45,

29.57, 31.77, 33.82, 34.00, 37.11, 45.62, 62.78, 63.05, 70.84, 70.89,

113.19, 119.66, 120.61, 120.86, 120.89, 127.62, 129.96, 130.00, 130.27,

136.50, 137.72, 139.36, 152.72, 155.40, 167.22, 172.67, 172.91. HRMS

calcd for C₅₉H₈₉N₄O₁₀PSNa (M + Na)⁺, 1099.5929; found,

1099.5930.

1

general procedure D to give 19.1 mg in 65% yield. H NMR (400

MHz, DMSO-d₆) δ ppm 0.74 (t, J = 6.97 Hz, 3 H), 1.01 (d, J = 6.60

Hz, 3 H), 1.13−1.62 (m, 4 H), 3.64−3.81 (m, 1 H), 3.89 (s, 2 H), 7.24

(t, J = 7.33 Hz, 1 H), 7.41−7.72 (m, 6 H), 8.05 (t, J = 6.60 Hz, 2 H),

12.09 (br s, 1 H). ¹³C NMR (126 MHz, DMSO-d₆) δ ppm 14.25,

19.32, 21.23, 37.11, 38.84, 44.94, 113.28, 119.68, 120.47, 120.77,

120.84, 127.75, 130.01, 130.32, 136.51, 137.68, 139.35, 152.97, 155.39,

166.36. HRMS calcd for C₂₃H₂₄N₄O₂SNa (M + Na)⁺, 443.1512;

found, 443.1514.

2-((4-Oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-yl)-

thio)-N-(pentan-3-yl)acetamide (31). Compound 7a (25 mg, 0.07

mmol), 3-aminopentane (18.24 μL, 0.16 mmol), triethylamine (19.82

μL, 0.14 mmol), and HATU (29.76 mg, 0.08 mmol) were reacted

N-Cyclohexyl-2-((3-cyclohexyl-4-oxo-4,5-dihydro-3H-pyrimido-

[5,4-b]indol-2-yl)thio)acetamide (36). Compound 7b (17.7 mg, 0.05

mmol), triethylamine (13.8 μL, 0.1 mmol), and cyclohexylamine (6.2

μL, 0.06 mmol) were dissolved in anhydrous DMF (0.5 mL). HATU

(20.74 mg, 0.06 mmol) dissolved in 0.2 mL of DMF was added to the

reaction mixture and stirred for 20 min and concentrated in vacuo.

The crude material was recrystallized in MeOH to give 9.1 mg in 41%

1

according to general procedure D to give 19.44 mg in 65% yield. H

NMR (400 MHz, DMSO-d₆) δ ppm 0.77 (d, J = 6.83 Hz, 6 H), 1.21−

1.62 (m, 4 H), 3.47−3.58 (m, 1 H), 3.93 (s, 1 H), 7.24 (t, J = 6.83 Hz,

1 H), 7.37−7.76 (m, 6 H), 7.93 (d, J = 7.31 Hz, 1 H), 8.05 (d, J = 7.80

Hz, 1 H), 12.10 (br s, 1 H). ¹³C NMR (126 MHz, DMSO-d₆) δ ppm

10.87, 27.40, 37.00, 52.36, 113.29, 119.64, 120.50, 120.73, 120.86,

127.82, 129.95, 130.05, 130.36, 136.43, 137.71, 139.34, 152.98, 155.40,

1

yield. H NMR (400 MHz, DMSO-d₆) δ ppm 0.99−2.17 (m, 16 H),

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2.60−2.88 (m, 4 H), 3.99 (br s, 2 H), 4.17−4.45 (m, 1 H), 7.20 (t, J =

7.33 Hz, 1 H), 7.34−7.65 (m, 2 H), 8.01 (d, J = 8.07 Hz, 1 H), 8.22

(d, J = 6.97 Hz, 1 H), 11.86 (br s, 1 H). ¹³C NMR (126 MHz, DMSO-

d₆) δ ppm 19.02, 24.92, 25.28, 25.63, 26.36, 28.79, 32.87, 37.21, 48.49,

56.47, 61.95, 113.22, 120.27, 120.56, 120.72, 120.82, 127.59, 136.47,

139.26, 151.90, 155.96, 166.22. HRMS calcd for C₂₄H₃₀N₄O₂SNa (M

+ Na)⁺, 461.1982; found, 461.1983.

129.99, 130.34, 136.35, 137.47, 140.40, 153.27, 155.75, 166.14. HRMS

calcd for C₂₅H₂₇N₄O₂S (M + H)⁺, 447.1849; found, 447.1852. 43: ¹H

NMR (400 MHz, DMSO-d₆) δ ppm 0.76−1.98 (m, 10 H), 2.10 (s, 3

H), 3.97 (s, 2 H), 4.23 (s, 3 H), 7.16 (t, J = 7.52 Hz, 1 H), 7.26−7.85

(m, 8 H), 8.07 (d, J = 7.70 Hz, 1 H). ¹³C NMR (126 MHz, DMSO-d₆)

δ ppm 24.85, 25.60, 31.43, 32.28, 38.02, 50.24, 62.03, 111.44, 119.63,

120.34, 120.85, 121.08, 128.20, 129.74, 130.25, 135.43, 136.73, 139.34,

155.99, 156.50, 169.99. HRMS calcd for C₂₆H₂₉N₄O₂S (M + H)⁺,

461.2006; found, 461.2007.

N-Cyclohexyl-2-((3-(naphthalen-2-yl)-4-oxo-4,5-dihydro-3H-

pyrimido[5,4-b]indol-2-yl)thio)acetamide (39). Compound 7c (26.1

mg, 0.065 mmol), triethylamine (18.1 μL, 0.13 mmol), and

cyclohexylamine (8.2 μL, 0.071 mmol) were dissolved in anhydrous

DMF (0.1 mL). HATU (27.2 mg, 0.071 mmol) dissolved in 0.2 mL of

DMF was added to the reaction mixture and stirred for 20 min and

concentrated in vacuo. The crude material was recrystallized in MeOH

Methyl 4-(2-((2-(Cyclohexylamino)-2-oxoethyl)thio)-4-oxo-3-

phenyl-3H-pyrimido[5,4-b]indol-5(4H)-yl)butanoate (44). To a

ﬂame-dried ﬂask, bromobutyric acid (50 μL, 0.43 mmol) was dissolved

in anhydrous MeOH (3 mL) followed by the addition of TMSCl

(272.45 μL, 2.15 mmol). The solution was stirred overnight at room

temperature and concentrated in vacuo to remove all trace of MeOH

and HCl. In a separate ﬂame-dried ﬂask, compound 1 (25 mg, 0.06

mmol) was dissolved in DMF (1 mL). NaH 60% dispersion (2.31 mg,

0.06 mmol) was added to the solution. After stirring at room

temperature for 10 min, methyl iodide (3.6 μL, 0.06 mmol) was added

and stirred for an additional 12 h. The reaction mixture was

concentrated in vacuo and puriﬁed by preparative thin-layer

chromatography to give 1.3 mg in 4% yield. IR: 3293 (NH), 1732

(CO ester), 1683 (CO amide) cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆)

δ ppm 0.99−1.32 (m, 5 H), 1.52 (d, J = 12.19 Hz, 1 H), 1.57−1.80

(m, 4 H), 1.91−2.10 (m, 2 H), 2.29 (t, J = 7.31 Hz, 2 H), 3.47 (s, 3

H), 3.47 (br s, 1 H), 3.89 (s, 2 H), 4.62 (t, J = 6.83 Hz, 2 H), 7.30 (t, J

= 7.56 Hz, 1 H), 7.39−7.51 (m, 2 H), 7.52−7.68 (m, 4 H), 7.74 (s, 1

H), 8.11 (d, J = 7.80 Hz, 1 H), 8.17 (d, J = 7.80 Hz, 1 H). ¹³C NMR

(126 MHz, DMSO-d₆) δ ppm 24.88, 25.59, 26.00, 30.71, 32.79, 37.02,

51.71, 111.34, 118.74, 120.19, 120.80, 121.09, 128.18, 129.93, 130.02,

130.37, 136.29, 137.81, 139.77, 153.45, 155.47, 166.28, 173.19. HRMS

calcd for C₂₉H₃₂N₄O₄SNa (M + Na)⁺, 555.2036; found, 555.2036.

tert-Butyl 2-((4-Oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]-

indol-2-yl)thio)acetate (45). Compound 6a (100 mg, 0.34 mmol)

and KOH (38.2 g, 0.68 mmol) were suspended in 2 mL of

dimethylacetamide in a ﬂame-dried ﬂask with stirring. H₂O was added

dropwise until KOH was completely dissolved. t-Butyl chloroacetate

(49 μL, 0.34 mmol) was immediately added to the reaction mixture

and stirred at room temperature monitoring with thin-layer

chromatography (1:99 MeOH:DCM). Upon completion, reaction

mixture was extracted with ethyl acetate (20 mL) and water (40 mL),

dried over MgSO₄, and concentrated in vacuo. EtOH (5 mL) was

added to the resulting viscous liquid, and pure product was ﬁltered to

give 55 mg in 40% yield. IR: 3192 (NH), 1732 (CO ester), 1674 (CO

1

to give 27 mg in 86% yield. H NMR (400 MHz, DMSO-d₆) δ ppm

0.76−1.38 (m, 5 H), 1.42−1.95 (m, 5 H), 3.43−3.55 (m, 1 H), 3.74−

4.00 (m, 2 H), 7.29 (t, J = 6.97 Hz, 1 H), 7.41 (d, J = 8.43 Hz, 1 H),

7.47−7.66 (m, 4 H), 7.68−7.81 (m, 2 H), 8.01−8.36 (m, 4 H), 12.17

(s, 1 H). ¹³C NMR (126 MHz, DMSO-d₆) δ ppm 24.82, 25.57, 31.23,

32.72, 32.75, 36.27, 36.97, 48.36, 113.39, 119.55, 120.69, 120.81,

120.84, 122.13, 126.31, 127.24, 127.99, 128.25, 128.88, 129.03, 130.07,

130.92, 132.83, 134.46, 138.05, 139.48, 153.40, 155.47, 162.87, 166.19.

HRMS calcd for C₂₈H₂₆N₄O₂SNa (M + Na)⁺, 505.1669; found,

505.1671.

N-Cyclohexyl-2-((4-oxo-3-phenethyl-4,5-dihydro-3H-pyrimido-

[5,4-b]indol-2-yl)thio)acetamide (40). Compound 7d (25 mg, 0.066

mmol), triethylamine (19.5 μL, 0.13 mmol), and cyclohexylamine (8.2

μL, 0.073 mmol) were dissolved in anhydrous DMF (0.1 mL). HATU

(27.2 mg, 0.073 mmol) dissolved in 0.2 mL of DMF was added to the

reaction mixture and stirred for 20 min and concentrated in vacuo.

The crude material was recrystallized in MeOH to give 18.28 mg in

1

60% yield. H NMR (400 MHz, DMSO-d₆) δ ppm 0.83−1.42 (m, 5

H), 1.44−2.08 (m, 5 H), 2.87−3.17 (m, 2 H), 3.95−4.24 (m, 2 H),

4.25−4.62 (m, 2 H), 7.33 (br s, 6 H), 7.87−8.48 (m, 3 H), 11.99 (s, 2

H). ¹³C NMR (126 MHz, DMSO-d₆) δ ppm 24.91, 25.63, 32.83,

34.09, 36.63, 45.66, 48.51, 113.26, 119.40, 120.37, 120.67, 120.84,

127.16, 127.76, 129.10, 137.30, 138.26, 139.36, 151.68, 154.92, 166.19.

HRMS calcd for C₂₆H₂₈N₄O₂SNa (M + Na)⁺, 483.1825; found,

483.1827.

2-((3-Cyclohexyl-4-oxo-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-

yl)thio)-N-cyclopentylacetamide (41). Compound 7b (25 mg, 0.07

mmol), triethylamine (19.5 μL, 0.14 mmol), and cyclopentylamine

(8.82 μL, 0.08 mmol) were dissolved in anhydrous DMF (0.5 mL).

HATU (29.3 mg, 0.08 mmol) dissolved in 0.2 mL of DMF was added

to the reaction mixture and stirred for 20 min and concentrated in

vacuo. The crude material was recrystallized in MeOH to give 9.3 mg

in 30.3% yield. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.97−2.09 (m,

18 H), 2.61−2.83 (m, 4 H), 4.02 (br s, 1 H), 4.17−4.37 (m, 1 H), 7.20

(t, J = 7.30 Hz, 1 H), 7.36−7.56 (m, 2 H), 8.00 (d, J = 8.06 Hz, 1 H),

8.32 (d, J = 7.33 Hz, 1 H), 11.86 (br s, 1 H). ¹³C NMR (126 MHz,

DMSO-d₆) δ ppm 23.97, 25.25, 26.34, 28.78, 32.73, 37.02, 51.21,

61.99, 113.23, 120.31, 120.54, 120.67, 120.79, 127.66, 136.48, 139.24,

151.93, 155.94, 166.85. HRMS calcd for C₂₃H₂₈N₄O₂SNa (M + Na)⁺,

447.1825; found, 447.1827.

1

amide) cm⁻¹. H NMR (400 MHz, DMSO-d₆) δ ppm 1.32−1.48 (m,

9 H), 3.91 (s, 2 H), 7.26 (t, J = 7.31 Hz, 1 H), 7.42−7.55 (m, 4 H),

7.56−7.69 (m, 3 H), 7.96 (d, J = 8.29 Hz, 1 H), 12.13 (s, 1 H). HRMS

calcd for C₂₂H₂₁N₃O₃SNa (M + Na)⁺, 430.1196; found, 430.1197.

tert-Butyl 2-((4-Oxo-3-phenyl-5-propyl-4,5-dihydro-3H-pyrimido-

[5,4-b]indol-2-yl)thio)acetate (46a). Compound 45 (50 mg, 0.12

mmol), NaH 60% dispersion in mineral oil (3.3 mg, 0.14 mmol),

iodopropane (13.1 μL, 0.14 mmol), and DMF (1 mL) were reacted

according to general procedure E and puriﬁed by silica gel

chromatography (10:90 ethyl acetate:hexane) to give 32.8 mg in

1

61% yield. H NMR (400 MHz, DMSO-d₆) δ ppm 0.82 (t, J = 7.31

N-Cyclohexyl-2-((5-methyl-4-oxo-3-phenyl-4,5-dihydro-3H-

pyrimido[5,4-b]indol-2-yl)thio)acetamide (42) and N-Cyclohexyl-N-

methyl-2-((5-methyl-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-

b]indol-2-yl)thio)acetamide (43). To a solution of compound 1 (50

mg, 0.12 mmol) in DMF (1 mL) in a ﬂame-dried ﬂask was added NaH

60% dispersion in mineral oil (9.24 mg, 0.24 mmol) and stirred at

room temperature for 10 min. Methyl iodide (14.3 μL, 0.24 mmol)

was then added to the solution and stirred overnight. Reaction mixture

was concentrated and puriﬁed by preparative thin-layer chromatog-

raphy (40:60 ethyl acetate:hexane) to give 18.18 mg in 33.9% yield of

Hz, 3 H), 1.40 (s, 9 H), 1.66−1.95 (m, 2 H), 3.91 (s, 2 H), 4.56 (t, J =

6.58 Hz, 2 H), 7.30 (br s, 1 H), 7.39−7.68 (m, 6 H), 7.75 (d, J = 8.29

Hz, 1 H), 7.99 (d, J = 7.80 Hz, 1 H). HRMS calcd for C₂₅H₂₇N₃O₃SNa

(M + Na)⁺, 472.1665; found, 472.1668.

tert-Butyl 2-((4-Oxo-5-pentyl-3-phenyl-4,5-dihydro-3H-pyrimido-

[5,4-b]indol-2-yl)thio)acetate (46b). Compound 45 (50 mg, 0.12

mmol), NaH 60% dispersion in mineral oil (3.3 mg, 0.14 mmol),

iodopentane (17.64 μL, 0.14 mmol), and DMF (1 mL) were reacted

according to general procedure E and puriﬁed by silica gel

chromatography (10:90 ethyl acetate:hexane) to give 33.8 mg in

1

compound 42 and 5.3 mg in 9.6% yield of compound 43. 42: H

1

NMR (400 MHz, DMSO-d₆) δ ppm 0.93−1.37 (m, 5 H), 1.40−1.90

(m, 5 H), 3.44−3.58 (m, 1 H), 3.88 (s, 2 H), 4.15 (s, 3 H), 7.31 (t, J =

7.33 Hz, 1 H), 7.39−7.53 (m, 2 H), 7.53−7.78 (m, 5 H), 8.05−8.27

(m, 2 H). ¹³C NMR (126 MHz, DMSO-d₆) δ ppm 24.90, 25.62, 31.47,

32.82, 37.14, 48.41, 111.31, 119.12, 120.09, 120.74, 120.92, 128.03,

59% yield. H NMR (400 MHz, DMSO-d₆) δ ppm 0.80 (t, J = 6.83

Hz, 3 H), 1.25 (d, J = 6.83 Hz, 4 H), 1.32−1.55 (m, 9 H), 1.64−1.83

(m, 2 H), 3.91 (s, 2 H), 4.59 (t, J = 7.31 Hz, 2 H), 7.30 (t, J = 7.56 Hz,

1 H), 7.46 (dd, J = 7.56, 1.71 Hz, 2 H), 7.54 (t, J = 7.31 Hz, 1 H),

7.57−7.67 (m, 3 H), 7.72 (d, J = 8.78 Hz, 1 H), 7.99 (d, J = 7.80 Hz, 1

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H). HRMS calcd for C₂₇H₃₁N₃O₃SNa (M + Na)⁺, 500.1978; found,

500.1975.

Hz, 1 H), 8.17 (d, J = 8.07 Hz, 1 H). ¹³C NMR (126 MHz, DMSO-d₆)

δ ppm 11.35, 24.15, 24.89, 25.61, 32.83, 37.12, 45.80, 48.40, 111.49,

118.75, 120.09, 120.61, 120.99, 127.98, 129.96, 129.98, 130.29, 136.38,

137.68, 139.83, 153.35, 155.43, 166.14. HRMS calcd for C₂₇H₃₁N₄O₂S

(M + H)⁺, 475.2162; found, 475.2164.

tert-Butyl 2-((5-Dodecyl-4-oxo-3-phenyl-4,5-dihydro-3H-

pyrimido[5,4-b]indol-2-yl)thio)acetate (46c). Compound 45 (50

mg, 0.12 mmol), NaH 60% dispersion in mineral oil (3.3 mg, 0.14

mmol), bromododecane (32.4 μL, 0.14 mmol), and DMF (1 mL)

were reacted according to general procedure E and puriﬁed by silica

gel chromatography (10:90 ethyl acetate:hexane) to give 37.4 mg in

N-Cyclohexyl-2-((4-oxo-5-pentyl-3-phenyl-4,5-dihydro-3H-

pyrimido[5,4-b]indol-2-yl)thio)acetamide (49). Compound 47b (34

mg, 0.08 mmol), HATU (34 mg, 0.088 mmol), triethylamine (22.5 μL,

0.16 mmol), cyclohexylamine (10.2 μL, 0.088 mmol), and DMF (1

mL) were reacted according to general procedure D to give 21 mg in

1

54% yield. H NMR (400 MHz, DMSO-d₆) δ ppm 0.84 (t, J = 6.83

Hz, 3 H), 1.21 (br s, 18 H), 1.40 (s, 9 H), 1.64−1.86 (m, 3 H), 3.91 (s,

2 H), 4.59 (t, J = 7.07 Hz, 2 H), 7.29 (t, J = 7.80 Hz, 1 H), 7.45 (dd, J

= 7.31, 1.95 Hz, 2 H), 7.54 (t, J = 7.80 Hz, 1 H), 7.58−7.67 (m, 3 H),

7.72 (d, J = 8.29 Hz, 1 H), 7.98 (d, J = 7.80 Hz, 1 H). HRMS calcd for

C₃₄H₄₅N₃O₃SNa (M + Na)⁺, 598.3074; found, 598.3070.

tert-Butyl 2-((5-(Cyanomethyl)-4-oxo-3-phenyl-4,5-dihydro-3H-

pyrimido[5,4-b]indol-2-yl)thio)acetate (46d). Compound 45 (318

mg, 0.78 mmol), NaH 60% dispersion (38.16 mg, 0.86 mmol),

bromoacetonitrile (65.5 μL, 0.86 mmol), and DMF (20 mL) were

reacted according to general procedure E to give 247.8 mg in 71%

yield. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.41 (s, 9 H), 3.94 (s, 2

H), 5.87 (s, 2 H), 7.43 (t, J = 7.31 Hz, 1 H), 7.47−7.55 (m, 2 H),

7.58−7.72 (m, 4 H), 7.88 (d, J = 8.78 Hz, 1 H), 8.03 (d, J = 8.29 Hz, 1

H). HRMS calcd for C₂₄H₂₂N₄O₃SNa (M + Na)⁺, 469.1305; found,

469.1303.

1

52% yield. H NMR (400 MHz, DMSO-d₆) δ ppm 0.80 (t, J = 6.97

Hz, 3 H), 1.00−1.40 (m, 9 H), 1.43−1.92 (m, 7 H), 3.44−3.58 (m, 1

H), 3.88 (s, 2 H), 4.58 (t, J = 7.15 Hz, 2 H), 7.29 (t, J = 7.33 Hz, 1 H),

7.40−7.68 (m, 6 H), 7.72 (d, J = 8.43 Hz, 1 H), 8.03−8.32 (m, 2 H).

¹³C NMR (126 MHz, DMSO-d₆) δ ppm 14.35, 22.29, 24.91, 25.62,

28.70, 30.56, 32.84, 37.11, 44.38, 48.42, 111.45, 118.70, 120.13,

120.64, 121.02, 128.02, 129.99, 130.31, 136.39, 137.70, 139.74, 153.36,

155.44, 166.16. HRMS calcd for C₂₉H₃₄N₄O₂S (M + H)⁺, 502.24025;

found, 502.24018.

N-Cyclohexyl-2-((5-dodecyl-4-oxo-3-phenyl-4,5-dihydro-3H-

pyrimido[5,4-b]indol-2-yl)thio)acetamide (50). Compound 47c

(28.6 mg, 0.055 mmol), HATU (23 mg, 0.061 mmol), triethylamine

(15.33 μL, 0.110 mmol), cyclohexylamine (7 μL, 0.061 mmol), and

DMF (1 mL) were reacted according to general procedure D and

puriﬁed by silica gel chromatography (20:80 ethyl acetate:hexane) to

2-((4-Oxo-3-phenyl-5-propyl-4,5-dihydro-3H-pyrimido[5,4-b]-

indol-2-yl)thio)acetic Acid (47a). Compound 46a (29.74 mg, 0.07

mmol), acetonitrile (1 mL), and triﬂuoroacetic acid (1 mL) were

reacted according to general procedure F to give 26 mg in quantitative

1

give 22 mg in 67% yield. H NMR (400 MHz, DMSO-d₆) δ ppm

0.73−0.92 (m, 4 H), 1.20 (d, J = 6.97 Hz, 22 H), 1.41−1.89 (m, 7 H),

3.45−3.61 (m, 1 H), 3.88 (s, 2 H), 4.58 (t, J = 6.60 Hz, 2 H), 7.28 (t, J

= 7.52 Hz, 1 H), 7.36−7.80 (m, 7 H), 8.05−8.26 (m, 2 H). ¹³C NMR

(126 MHz, DMSO-d₆) δ ppm 11.85, 14.50, 22.63, 24.97, 25.70, 26.59,

29.19, 29.44, 30.88, 31.82, 32.90, 37.20, 44.48, 48.42, 111.54, 118.70,

120.22, 120.69, 121.09, 128.06, 130.05, 130.39, 136.46, 137.79, 139.83,

153.41, 155.50, 166.17. HRMS calcd for C₃₆H₄₉N₄O₂S (M + H)⁺,

601.3571; found, 601.3570.

1

yield. H NMR (400 MHz, DMSO-d₆) δ ppm 0.81 (t, J = 6.58 Hz, 3

H), 1.75 (m, 2 H), 3.96 (s, 2 H), 4.56 (t, J = 7.07 Hz, 2 H), 7.29 (t, J =

7.31 Hz, 1 H), 7.38−7.88 (m, 7 H), 7.99 (d, J = 8.29 Hz, 1 H). HRMS

calcd for C₂₁H₁₉N₃O₃SNa (M + Na)⁺, 416.1039; found, 416.1036.

2-((4-Oxo-5-pentyl-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]-

indol-2-yl)thio)acetic Acid (47b). Compound 46b (30.98 mg, 0.065

mmol), acetonitrile (1 mL), and triﬂuoroacetic acid (1 mL) were

reacted according to general procedure F to give 27 mg in quantitative

2-((5-(Cyanomethyl)-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido-

[5,4-b]indol-2-yl)thio)-N-cyclohexylacetamide (51). Compound 47d

(150 mg, 0.39 mmol), HATU (161 mg, 0.42 mmol), triethylamine (59

μL, 0.77 mmol), cyclohexylamine (49 μL, 0.42 mmol), and DMF (5

mL) were reacted according to general procedure D to give 157.3 mg

1

yield. H NMR (400 MHz, DMSO-d₆) δ ppm 0.81 (t, J = 6.58 Hz, 3

H), 0.99−1.43 (m, 4 H), 1.62−1.84 (m, 2 H), 3.96 (s, 2 H), 4.59 (t, J

= 7.07 Hz, 2 H), 7.29 (t, J = 7.31 Hz, 1 H), 7.45 (br s, 2 H), 7.51−7.68

(m, 4 H), 7.72 (d, J = 8.29 Hz, 1 H), 7.98 (d, J = 8.29 Hz, 1 H).

HRMS calcd for C₂₃H₂₃N₃O₃SNa (M + Na)⁺, 444.1352; found,

444.1351.

2-((5-Dodecyl-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]-

indol-2-yl)thio)acetic Acid (47c). Compound 46c (34.81 mg, 0.06

mmol), acetonitrile (1 mL), and triﬂuoroacetic acid (1 mL) were

reacted according to general procedure F to give 28.6 mg in 92% yield.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.82 (t, J = 6.83 Hz, 3 H),

1.19 (d, J = 7.80 Hz, 18 H), 1.71 (d, J = 6.34 Hz, 2 H), 3.94 (s, 2 H),

4.56 (t, J = 7.07 Hz, 2 H), 7.27 (t, J = 7.80 Hz, 1 H), 7.36−7.47 (m, 2

H), 7.59 (s, 4 H), 7.70 (d, J = 8.29 Hz, 1 H), 7.96 (d, J = 7.80 Hz, 1

H), 12.74 (br s, 1 H). HRMS calcd for C₃₀H₃₇N₃O₃SNa (M + Na)⁺,

542.2448; found, 542.2444.

1

in 86.7% yield. IR: 3240 (NH), 1697 (CO) cm⁻¹. H NMR (400

MHz, DMSO-d₆) δ ppm 0.97−1.33 (m, 5 H), 1.45−1.87 (m, 5 H),

3.45−3.58 (m, 1 H), 3.90 (s, 2 H), 5.87 (s, 2 H), 7.42 (t, J = 7.80 Hz, 1

H), 7.47−7.56 (m, 2 H), 7.57−7.74 (m, 4 H), 7.88 (d, J = 8.29 Hz, 1

H), 8.17 (dd, J = 13.16, 7.80 Hz, 2 H). ¹³C NMR (126 MHz, DMSO-

d₆) δ ppm 24.92, 25.62, 32.84, 33.04, 37.21, 48.45, 111.45, 116.75,

118.20, 121.01, 121.35, 122.12, 129.02, 129.90, 130.08, 130.56, 135.95,

138.98, 139.65, 155.44, 155.50, 166.02. HRMS calcd for C₂₆H₂₆N₅O₂S

(M + H)⁺, 472.1802; found, 472.1803.

2-((5-(2-Amino-2-oxoethyl)-4-oxo-3-phenyl-4,5-dihydro-3H-

pyrimido[5,4-b]indol-2-yl)thio)-N-cyclohexylacetamide (52). A

KOH (6 mg, 0.11 mmol) solution in water (50 μL) was added to a

solution of compound 51 in dimethylacetamide (200 μL) and stirred

at rt overnight. Reaction mixture was acidiﬁed with 3 M HCl, extracted

with ethyl acetate and water, dried over MgSO₄, and concentrated to

dryness in vacuo. Further recrystallization in MeOH gives 20 mg in

2-((5-(Cyanomethyl)-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido-

[5,4-b]indol-2-yl)thio)acetic Acid (47d). Compound 46a (29.74 mg,

0.07 mmol), acetonitrile (1 mL), and triﬂuoroacetic acid (1 mL) were

reacted according to general procedure F to give 26 mg in quantitative

1

37% yield. H NMR (400 MHz, DMSO-d₆) δ ppm 0.94−1.36 (m, 5

1

H), 1.43−1.89 (m, 5 H), 3.43−3.62 (m, 1 H), 3.89 (s, 2 H), 5.24 (s, 2

H), 7.12−7.22 (m, 1 H), 7.30 (t, J = 7.33 Hz, 1 H), 7.42 (m, J = 7.30,

2.20 Hz, 2 H), 7.48−7.67 (m, 5 H), 8.10 (d, J = 7.70 Hz, 1 H), 8.18

(d, J = 7.70 Hz, 1 H). ¹³C NMR (126 MHz, DMSO-d₆) δ ppm 24.90,

25.59, 32.82, 37.05, 46.85, 48.48, 111.36, 119.30, 120.36, 120.91,

120.95, 128.13, 129.87, 130.05, 130.41, 136.14, 137.82, 140.64, 153.47,

155.65, 166.26, 169.77. HRMS calcd for C₂₆H₂₇N₅O₃SNa (M + Na)⁺,

512.1727; found, 512.1726.

yield. H NMR (400 MHz, DMSO-d₆) δ ppm 3.84−4.15 (m, 2 H),

5.87 (s, 2 H), 7.42 (t, J = 7.56 Hz, 1 H), 7.47−7.56 (m, 2 H), 7.57−

7.74 (m, 4 H), 7.88 (d, J = 8.29 Hz, 1 H), 8.03 (d, J = 7.80 Hz, 1 H),

12.89 (br s, 1 H). HRMS calcd for C₂₀H₁₄N₄O₃SNa (M + Na)⁺,

413.0679; found, 413.0676.

N-Cyclohexyl-2-((4-oxo-3-phenyl-5-propyl-4,5-dihydro-3H-

pyrimido[5,4-b]indol-2-yl)thio)acetamide (48). Compound 47a

(29.4 mg, 0.075 mmol), HATU (31.3 mg, 0.082 mmol), triethylamine

(20.8 μL, 0.15 mmol), cyclohexylamine (9.43 μL, 0.082 mmol), and

DMF (1 mL) were reacted according to general procedure D to give 4

Biological Studies. Animals. Seven−nine-week-old C57BL/6

(wild-type, WT) and Cd14^−/−(C57BL/6 background) were

purchased from the Jackson Laboratories (Bar Harbor, MA).

Tlr4^−/−mice were a gift from Dr. Shizuo Akira (Osaka University,

Japan) and backcrossed for 10 generations onto the C57BL/6

background at University of California, San Diego (UCSD). All animal

1

mg in 11% yield. H NMR (400 MHz, DMSO-d₆) δ ppm 0.82 (t, J =

7.33 Hz, 3 H), 1.00−1.37 (m, 5 H), 1.42−1.99 (m, 7 H), 3.88 (s, 2 H),

4.56 (t, J = 6.97 Hz, 2 H), 7.29 (t, J = 7.70 Hz, 1 H), 7.41−7.50 (m, 1

H), 7.50−7.68 (m, 4 H), 7.74 (d, J = 8.43 Hz, 1 H), 8.10 (d, J = 8.07

4221

dx.doi.org/10.1021/jm301694x | J. Med. Chem. 2013, 56, 4206−4223

Journal of Medicinal Chemistry

Article

experiments were approved by the UCSD Institutional Animal Care

and Use Committee.

software was used to obtain p-values for comparison between groups

(p < 0.05 was considered signiﬁcant). For the in vitro studies, two-

tailed Student’s t test was used to compare two groups, and one-way

ANOVA Dunnett’s test was used to compare multiple groups.

In Vitro Cytokine Induction in Bone Marrow Derived Dendritic

Cells (BMDC). BMDC were prepared from C57BL/6 mice as

described.³⁶BMDC (10⁵cells per well) were plated in 96-well plates

in 200 μL of complete RPMI1640 supplemented with 10% fetal calf

serum (FCS; Sigma Aldrich), 100 U/mL penicillin, and 100 μg/mL

streptomycin (Invitrogen). The cells were incubated with graded

concentrations of the compounds for 18 h at 37 °C, 5% CO₂. After 18

h incubation, the cell culture supernatants were collected. LPS

(puriﬁed LPS, Invivogen, San Diego, CA) or MPLA (1 μg/mL

synthetic MPLA, Invivogen, San Diego, CA) were used as positive

controls. The levels of IL-6 in the culture supernatants were

determined by ELISA (BD Biosciences, La Jolla, CA).³⁷The AUC

was calculated from BMDC dose−response curves using Prism 5

(GraphPad, San Diego). Each cytokine induction curve was ﬁrst

converted to a percent activity curve, and then the AUC of the percent

activity curve was calculated. The process of converting to a percent

activity curve allowed subtracting background and adjusting for plate-

to-plate variation. Finally, the AUC values were normalized to the

activity of compound 1 within each experiment, set at 100. The

cultures stimulated with LPS 10 ng/mL, and 5 μM compound 1

released an average of 20.6 ng/mL 4.7 SD and 10.5 ng/mL 1.3

SD of IL-6, respectively.

In Vitro Assays Using TLR Reporter Cell Lines. Murine or

humanTLR4 HEK Blue cells (Invivogen, 2.5 × 10⁴cells per well of

a 96-well plate), or NFκB/SEAPorter HEK 293 cells (Imgenex, San

Diego, CA) for human TLR2, TLR3, TLR5, TLR7, TLR8, or TLR9 (5

× 10⁴cells per well of 96 well plate) were incubated with graded doses

of compound 1. The culture supernatants were harvested after a 20−

24 h incubation period. SEAP activity in the supernatants was

determined by a colorimetric assay, using either the SEAPorter Assay

Kit (Imgenex), with absorbance read at 405 nm, or QuantiBlue

(Invivogen), with absorbance read at 630 nm. Stimulation of the

human TLR4 cells with 10 ng/mL LPS resulted in OD₆₃₀of 1.90

relative to 0.88 of cells stimulated with 10 μM compound 1. The

relative reporter activation for the murine TLR4 cells for cells

stimulated with 10 ng/mL LPS and 5 μM compound 1 was 1.70

0.08 and 1.52 0.03, respectively.

In Vitro Activities in hPBMC. Human PBMC were isolated from

buﬀy coats obtained from the San Diego Blood Bank (San Diego, CA)

as described previously.^37,38PBMC (1 × 10⁶/mL) were incubated with

various compounds in complete RPMI for 18 h at 37 °C, 5% CO₂, and

culture supernatants were collected. The levels of IL-8 in the

supernatants were determined by ELISA (BD Biosciences, La Jolla,

CA). Human cell cultures were treated with 10 ng/mL LPS as the

positive control. Cultures treated with LPS 10 ng/mL and compound

1 10 μM released averages of 14.5 0.6 ng/mL SD and 7.8 0.8 ng/

mL SD of IL-8, respectively.

Type I IFN Assay. L929 cells stably expressing an interferon sensitive

response element (ISRE) luciferase reporter construct were kindly

provided by Dr. B. Beutler (UT Southwestern, Texas).³⁹The

bioactivity of type I IFN in mBMDC supernatants was measured by

luciferase assay using L929-ISRE cells as described previously.³⁹L929-

ISRE cells were plated at 5× 10⁴cells per well in Dulbecco’s Modiﬁed

Eagle Medium (Invitrogen) supplemented with 10% FCS, 100 U/mL

penicillin, and 100 μg/mL streptomycin (DMEM-10) in a 96-well

white-walled clear-bottom plate. Thus, 50 μL of mBMDC supernatant

was incubated with L929-ISRE cells in 50 μL of DMEM for 6 h. Mu-

IFN Beta Standard (PBL Interferon Source, Piscataway, NJ) was used

as a standard. The luciferase activities were measured by Steady-Glo

luciferase assay buﬀer (Promega, Madison, WI).

AUTHOR INFORMATION

■

Corresponding Author

*Phone: 858-534-5424. Fax: 858-246-1586. E-mail: hcottam@

ucsd.edu. Address: Sanford Consortium for Regenerative

Medicine, University of California, San Diego, La Jolla,

California 92093, United States.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We are grateful for the assistance provided by Dr. Xu of the

Department of Chemistry at University of California, San

Diego for high resolution mass spectrometry. We acknowledge

the services provided by the University of California, San

Francisco Small Molecule Discovery Center. This project was

supported by a contract from the National Institute of Allergy

and Infectious Diseases (HHSN272200900034C) from the

National Institutes of Health (to DAC). The content is solely

the responsibility of the authors and does not necessarily

represent the oﬃcial views of the National Institutes of Health.

ABBREVIATIONS USED

■

AUC, area under the curve; HTS, high throughput screen;

TLR, Toll-like receptor; IL, interleukin; TNF, tumor necrosis

factor; LPS, lipopolysaccharide; MyD88, myeloid diﬀerentia-

tion primary response gene 88; MD-2, myeloid diﬀerentiation

protein-2; MPLA, monophosphoryl lipid A; PBMC, peripheral

blood mononuclear cells; EtOH, ethanol; THF, tetrahydrofur-

an; TMSCl, trimethylsilyl chloride or chlorotrimethylsilane;

DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DMF,

N,N-dimethylformamide; HATU, O-(7-azabenzotriazol-1-yl)-

N,N,N⁰,N⁰-tetramethyluroniumhexaﬂuorophosphate; DCM, di-

chloromethane; MeOH, methanol; TEA, triethylamine;

BMDC, bone marrow derived dendritic cells; BMDM, bone

marrow derived macrophages; NFκB, nuclear factor κ B; MTT,

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;

ISRE, interferon response element; IFN, interferon; SEAP,

secreted embryonic alkaline phosphatase; ELISA, enzyme-

linked immunosorbent assay; IP-10, interferon γ-induced

protein 10; PRR, pattern-recognition receptors

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Identification of substituted pyrimido[5,4-b]indoles as selective toll-like receptor 4 ligands

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