Structure-activity relationships in human toll-like receptor 7-active imidazoquinoline analogues

4450 J. Med. Chem. 2010, 53, 4450–4465

DOI: 10.1021/jm100358c

Structure-Activity Relationships in Human Toll-Like Receptor 7-Active Imidazoquinoline Analogues

Nikunj M. Shukla, Subbalakshmi S. Malladi, Cole A. Mutz, Rajalakshmi Balakrishna, and Sunil A. David*

Department of Medicinal Chemistry, University of Kansas, Multidisciplinary Research Building, Room 320D, 2030 Becker Drive, Lawrence

Kansas 66047

Received March 20, 2010

Engagement of toll-like receptors serve to link innate immune responses with adaptive immunity and

can be exploited as powerful vaccine adjuvants for eliciting both primary and anamnestic immune

responses. TLR7 agonists are highly immunostimulatory without inducing dominant proinflammatory

cytokine responses. A structure-activity study was conducted on the TLR7-agonistic imidazoquino-

lines, starting with 1-(4-amino-2-((ethylamino)methyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpro-

pan-2-ol as a lead. Modifications of the secondary amine of the C2 ethylaminomethylene side chain are

poorly tolerated. The 4-amino group must be retained for activity. Replacement of the imidazole ring

of the scaffold with triazole or cyclic urea led to complete loss of activity. A systematic exploration of

N¹-benzyl-C2-alkyl substituents showed a very distinct relationship between alkyl length and TLR7-

agonistic potency with the optimal compound bearing a C2-n-butyl group. Transposition of the N¹and

C2 substituents led to the identification of an extremely active TLR7-agonistic compound with an EC₅₀

value of 8.6 nM. The relative potencies in human TLR7-based primary reporter gene assays were

paralleled by interferon-R induction activities in whole human blood models.

Introduction

antigen-presenting cells as well as activating natural killer

(NK) cells. The consequence of activation of the innate

immune system mobilizes and amplifies specific adaptive

immune responses involving both T- and B-cell effector func-

tions.^5-7Thus, TLR stimuli serve to link innate and adap-

tive immunity⁵and can therefore be exploited as powerful

adjuvants in eliciting both primary and anamnestic immune

responses.

We have recently examined representative members of

virtually the entire compendium of known TLR agonists in

a series of hierarchical assays including primary TLR-reporter

assays, secondary indices of immune activation such as cyto-

kine induction and activation of lymphocytic subsets in whole

human blood, and tertiary screens characterizing transcripto-

mal activation patterns with a view to identifying optimal

immunostimulatory chemotypes.⁸Of all the innate immune

stimuli examined, we found that TLR7 agonists were extra-

ordinarily immunostimulatory, stimulating virtually all

subsets of lymphocytes, and yet without inducing dominant

proinflammatory cytokine responses (unlike TLR4, -5 or -8

agonists, which were proinflammatory and therefore may exert

systemic toxicity).⁸We therefore became especially desirous

of evaluating TLR7-active compounds as potential vaccine

adjuvants.

Toll-like receptors (TLRs^a) are pattern recognition recep-

tors that recognize specific molecular patterns present in

molecules that are broadly shared by pathogens but are

structurally distinct from host molecules.^1,2There are 10 TLRs

in the human genome.²The ligands for these receptors are

highly conserved microbial molecules such as lipopolysaccha-

rides (LPS) (recognized by TLR4), lipopeptides (TLR2 in

combination with TLR1 or TLR6), flagellin (TLR5), single-

stranded RNA (TLR7 and TLR8), double-stranded RNA

(TLR3), CpG motif-containing DNA (recognized by TLR9),

and profilin present on uropathogenic bacteria (TLR11).^3,4

TLR1, -2, -4, -5, and -6 respond to extracellular stimuli, while

TLR3, -7, -8, and -9 respond to intracytoplasmic PAMPs,

being associated with the endolysosomal compartment.²

The activation of TLRs by their cognate ligands leads to

activation of innate immune effector mechanisms, includ-

ing the production of pro-inflammatory cytokines and up-

regulation of MHC molecules and costimulatory signals in

*To whom correspondence should be addressed. Phone: 785-864-

1610. Fax: 785-864-1961. E-mail: sdavid@ku.edu.

^aAbbreviations: AP-1, activator protein-1; CD, cluster of differentia-

tion; DC, dendritic cells; DMAP, 4-dimethylaminopyridine; DMF, N,

N-dimethylformamide; EC₅₀, half-maximal effective concentration;

Long before endosomal TLR7 was discovered to serve as

the primary sensor for short, single-stranded, GU-rich RNA

sequences (ssRNA), mainly of viral origin,^9-11a number of

small molecules were synthesized and evaluated in the 1970s

and 1980s for antiviral activities owing to their pronounced

type I interferon (IFN-R and -β) inducing properties.^12-16

Although the mechanisms of innate immune stimulation of

several of these compounds (such as tilorone¹⁴and bromo-

pirone¹⁶) remain yet tobe formally elucidated, members of the

EDCI HCl, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-

3

chloride; ELISA, enzyme linked immunosorbent assay; ESI-TOF,

electrospray ionization-time-of-flight; HATU, 2-(1H-7-azabenzo-tria-

zol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate; HBTU, O-

benzotriazole-N,N,N⁰,N⁰-tetramethyl uronium hexafluorophosphate;

HEK, human embryonic kidney; IFN, interferon; LPS, lipopolysac-

charide; MHC, major histocompatibility complex; NF-κB, nuclear

factor-kappa B; NK, natural killer; PAMP, pathogen-associated mole-

cular patterns; sAP, secreted alkaline phophatase; SAR, structure-

activity relationship; ssRNA-single stranded RNA; Th1, helper T-type

1; THF, tetrahydrofuran; TLR, Toll-like receptor.

r

pubs.acs.org/jmc

Published on Web 05/18/2010

2010 American Chemical Society

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4451

1H-imidazo[4,5-c]quinolines were found to be good Type I

IFN inducers in human cell-derived assays,¹⁷and FDA

approval was obtained in 1997 for Imiquimod (1) for the

treatment of basal cell carcinoma and actinic keratosis.¹⁸It

was not until 2002, however, that the mechanistic basis of IFN

induction by the imidazoquinolines was found to be a con-

sequence of TLR7 engagement and activation.¹⁹Other than

the original landmark studies performed by investigators at

3M Pharmaceuticals,¹⁷structure-activity relationships of the

imidazoquinoline chemotype remains poorly explored, per-

haps attributable in part to recent interest in the 8-hydroxy-

adenine compounds as alternate TLR7-agonists,^20-23which

appear to lack emetic side effects observed in ferrets upon oral

administration.²²

Figure 1. Structures of Imiquimod and 2.

cytopathic effect) which predated the discovery and develop-

ment of TLR7-specific assays. In the studies reported below,

all compounds have been evaluated in human TLR7 and

TLR8 reporter gene assays; highly active compounds were

further examined for IFN-R induction in whole human blood

using analyte-specific immunoassays.

An aspect of our recent work in this area^8,24,25focuses on

developing adjuvants for transcutaneousvaccines (needle-free

vaccine patches).^26-28Given that Imiquimod is already ap-

proved for topical use, it was of particular interest not only to

explore chemical space around the imidazoquinolines but also

to carefully “immunophenotype” such compounds in human

TLR7-based assays to verify that they did not have any

TLR8-driven proinflammatory properties. One of our goals

was also to learn from such structure-activity studies optimal

positions on the scaffold that would tolerate the introduction

of electrophilic or photoactivable labels for purposes of deve-

loping self-adjuvanting vaccine constructs^29,30by covalently

modifying protein antigens. We report here the synthesis

and evaluation of a focused library of variously substitu-

ted 1H-imidazo[4,5-c]quinolines, and the identification of

a novel TLR7 agonist whose potency is 250 times that of

Imiquimod.

Recent studies show that the TLR7 agonistic imidazoquino-

lines are indeed potently adjuvantic in vaccine constructs;^38,39

however, the route of administration appears to be crucial

because intradermal but not subcutaneous administration of

the vaccine adjuvanted with 2 is immunogenic and protective.³⁸

Epidermal Langerhans cells as well as dermal dendritic cells

(myeloid and plasmacytoid subsets)⁴⁰function as specialized

professional antigen presenting cells in the skin.^41,42The plasma-

cytoid dendritic cell subset appears to be the target cell-type for

TLR7 agonists.⁴³We hypothesized that the C2 ethylamino-

methylene substituent of 2 renders the molecule sufficiently

polar as to hinder transcutaneous penetration to effectively

access and stimulate plasmacytoid dendritic cells. It was there-

fore desirable to evaluate more hydrophobic analogues of 2 as

potential candidate transcutaneous vaccine adjuvants.

Unlike TLR2, TLR3, and TLR4 for which crystal struc-

tures are available as complexes with their cognate ligands,⁴⁴a

detailed structural characterization of the mode of binding of

the imidazoquinolines toTLR7isnot yet available. Therefore,

our strategy in examining structure-activity relationships

was focused rather on exploring chemical space systematically

around the validated lead compound 2 toward realizing the

two major objectives mentioned earlier. We began with deri-

vatization of the secondary amine of the C2 ethylamino-

methylene side chain in an effort to examine whether this

could be a potential site for introducing photoactivable or

electrophilic functionalities. We found that modifications on

this substituent are poorly tolerated, with only the N-ethyl

analogue 3 (Scheme 1) retaining partial activity and acyl (4, 5)

and guanidine⁴⁵(6) derivatives being bereft of activity (Table 1).

The loss of activity appeared not to be a consequence of altered

basicity of the amine; surmising that it may, in part, be due

either to the hydrophobicity of the substituent, or the confor-

mal constraint imposed by the amide or guanidine, we asked if

a polar spacer group such as triethyleneglycol (11) may miti-

gate loss of activity, but we were proven wrong because 11 was

also found to be completely inactive. Concurrent with the

syntheses of the above compounds, we also synthesized the

N-propyleneisothiocyanate derivative (14) via the N-propylene-

amino (13) and N-ethylenenitrile (12) precursors. Compound

14 was not tested in reporter gene assays because we antici-

pated spurious results on account of its reactivity; the reac-

tion of 14 with the spermine derivative (used as a model

amine) afforded the expected thiourea adduct (16) which,

disappointingly, was also inactive (Scheme 2). These results

conclusively showed that the secondary amine on the C2

substituent was not amenable to modifications. Replacement

of the terminal ethyl group on the C2 substituent with an

Results and Discussion

As alluded to earlier, long before the fact that imidazoqui-

nolines specifically engaged and activated TLR7 was known,

it was recognized that these compounds were potently

adjuvantic^31-34in addition to displaying antiviral effects via

the induction of IFN-R/β.^35,36In human blood stimulated ex

vivo with TLR7 agonists, upregulation of the surface expres-

sion of costimulatory molecules such as CD40, CD80, and

CD86 occurred in both CD11c^-plasmacytoid DCs and

CD11c^þmyeloid DCs; furthermore, the Th1 stimulatory abi-

lity of both DC subsets was enhanced in response to TLR7

ligands.³⁷Because TLR7 agonists transmit a T-helper-like

signal to antibody-producing B cells, it is a highly effective

adjuvant even for synthetic peptides that lack T-cell epitopes,

possibly even replacing the function of T-helper cells, and pro-

viding a potential T-cell-independent vaccination strategy.³³

Our initial interest in exploring TLR7 agonists as vaccine

adjuvants has been greatly reinforced by our recent observa-

tions that pure TLR7 agonists, unlike other TLR ligands, are

potently immunostimulatory without activating inflamma-

tory programs in human whole blood model systems.⁸We

elected to choose 1-(4-amino-2-((ethylamino)methyl)-1H-

imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol, 2, as our

point of departure in examining structure-activity relation-

ships in the imidazoquinolines rather than Imiquimod be-

cause we had earlier established that this analogue was more

active than the parent compound and was also verified to be a

pure TLR7 agonist in human primary cells (Figure 1).^8,24We

have recently reported the synthesis of 2.²⁴Furthermore, the

only report on a detailed examination of SAR on the imidazo-

quinolines by the 3M group¹⁷had used indirect bioassays

(indirect assay for IFN-R using inhibition of virus-induced

4452 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11

Shukla et al.

Scheme 1. Syntheses of 2-Imidazolyl Side Chain Modified Analogues of 2 ^a

^aReagents for 2-6: (i) CH₃CHO, NaCNBH₃, MeOH; (ii) CH₃COCl, Et₃N, THF; (iii) C₁₅H₃₁COCl, Et₃N, THF; (iv) EDCI, Et₃N, DMF. Reagents

for 7-11: (i) (a) TsCl, Et₃N, DMAP, CH₂Cl₂, 0 °C, (b) NaN₃, DMF, 70 °C; (ii) (Boc)₂O, Pd(OH)₂/C, H₂, MeOH; (iii) Dess-Martin periodinane,

CH₂Cl₂; (iv) (a) 10, NaCNBH₃, MeOH (b) CF₃CO₂H.

n-undecanyl group in 22 resulted in complete loss of activity

(Table 1), signaling that the length of the C2 substituent was

crucial; this was to become dramatically apparent when 32,

with a pentyl substituent on C2 was evaluated later (see

below).

Bioisosteric replacements of the C4-NH₂group with -NMe,

-OH, and -SMe groups on the Imiquimod scaffold have been

explored earlier,¹⁷and it was of interest to examine if the previ-

ously described SAR would hold true in our analogues and also

to test whether the introduction of phenyl, -NHNH₂, and

-NHOH groups would alter TLR7 activity (Scheme 4). The

4-Cl precursor (44) andthedes-amino analogue 50, aswellasthe

4-OH (46) and 4-Ph (47) analogues, were completely inactive,

while the 4-NHOH (48) and 4-NHNH₂(49) compounds were

substantially weaker (Table 1) than their 4-NH₂counterparts

(31, 32). These results indicate the importance of the preserva-

tion of the NH₂group on C4 for maximal activity.

Up to this point in our SAR studies, the most active and

chemically distinct species were compound 2 and the N¹-benzyl-

C2-n-butyl analogue 31. We were curious to examine whether

transposing the N¹and C2 substituents on these molecules would

result in enhanced activity. Fortuitously, both the “hybrids” (52,

54; Scheme 5) were very active. 54 was found to be extraordi-

narily potent, with an EC₅₀of 8.6 nM (Figure 4, Table 1).

Finally, we attempted to modify the imidazole ring of the

scaffold. The triazole compounds 55 and 56, as well as the

cyclic urea 57, were completely inactive, which emphasizes the

role of the recognition of the imidazole ring system by TLR7

(Scheme 6). Mention must also be made that these studies have

been useful in delineating possible sites for introducing label-

ing functionalities. Preliminary studies (data not shown) show

that the N¹-benzyl substituent may be appropriate for intro-

ducing arylazido functional groups, while the quinoline ring

may be amenable to introducing electrophilic groups. These

results will be communicated in a future publication.

Desiring more lipophilic analogues which are expected to

permeate the dermal barrier better, we sought to examine N¹-

benzyl analogues (Scheme 3), noting that similar substitutions

have resulted in augmented activity in the 8-hydroxy-adenine

series.^20-23Compound 32 was the first to be synthesized in

this series which, to our pleasant surprise, showed an activity

20-fold greater than 2. Coupled with our observation that 22 was

completely inactive, we sought to systematically explore SAR of

the N¹-benzyl C2 alkyl substituents. As depicted in Figure 2, a

very distinct relationship between alkyl length and TLR7-

agonistic potency was observed, with the optimal compound

being 31 (C2-n-butyl), with an EC₅₀of 59 nM (Table 1).

n-Butylene (38) and n-butylyne (43) analogues showed decrease

in potency. It is noteworthy that a C2-n-butyl, N¹-phenyl ana-

logue synthesized by the 3M group¹⁷was not the most active; our

results indicate that an N¹-benzyl substituent is preferred.

In recognition that bioactivity readouts using cell-culture

systems may not always reflect with fidelity in vivo behavior

owing to differential plasma protein binding (that we our-

selves have observed and characterized),⁴⁶it was important to

verify that the activity profiles observed in TLR7-specific

reporter gene assays (Table 1) was also seen in whole blood

with its full complement of plasma proteins. It was gratifying

that we observed the expected 31 . 2 ∼ 34 activity profile in

whole blood IFN-R induction experiments (Figure 3).

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4453

Table 1. EC₅₀Values ( μM) of Compounds in Human TLR7-Specific Reporter Gene Assay^a

4454 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11

Table 1. Continued

Shukla et al.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4455

Table 1. Continued

^aND denotes no activity detected at 100 μM.

All of the C2-alkyl compounds reported herein were tested

for TLR8 activity using the thiazoloquinolineCL075 (3M002)

as a reference TLR8 agonist,^47,48and none had any appreci-

able activity in these assays. It should also be pointed out that

while potencies of compounds are conventionally represented

as EC₅₀(or IC₅₀) values, that metric alone appears to be

inadequate in rank-ordering the activities of the active com-

pounds tested. An inspection of Figure 4 indicates, for

instance, that while the EC₅₀values of both Imiquimod and

2 are comparable (∼2 μM), compound 2 induces a more

robust response than Imiquimod. Similarly, the area-under-

curve estimates of 54 (analogous to combined “avidity” and

affinity measures of antigen-antibody interactions)^49,50in-

dicate that consideration of the EC₅₀values alone may under-

estimate its potency.

compartment activity, rather than governing specific mole-

cular interactions with TLR7. A comparison of observed

retention times on C₁₈-reverse-phase mass-chromatograms

(a measure of hydrophobicity)^51,52with the potencies of the

active compounds indicate a distinct deviation from the trend

of 54, suggesting specific and idiosyncratic effects rather than

bulk effects (Figure 5).

These structure-activity studies have been instructive and

rewarding in that they not only complement the seminal work at

3M but also have led to the discovery of highly potent, lipophilic,

human TLR7 agonist, 54. We note, not without irony, that

this highly potent compound differs from its parent molecule by

only a replacement of a secondary amine by a methylene unit.

The oral bioavailability of 54 is predicted to be good, and it is

possible that the systemic IFN-Rinducing effects of this imidazo-

quinoline may find additional uses in the treatment of conditions

such as hepatitis⁵³and chronic myelogenous⁵⁴and hairy cell

leukemias.⁵⁵However, for purposes of developing dermal vac-

cine adjuvants, 31 may be more efficacious for reasons discussed

earlier. Compounds 54 and 31 are being exhaustively immuno-

profiled and characterized in a variety of assays.

Given that TLR7 is sequestered in an acidic endolysosomal

compartment, and these compounds are weak bases and are

therefore expected to accumulate in that acidic organelle, we

wondered if the observed SAR could simply be a consequence

of differential hydrophobicity of the compounds, modulat-

ing uptake into the cell and entry into the endolysosomal

4456 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11

Shukla et al.

Scheme 2. Syntheses of 2-Imidazolyl Side Chain Modified Analogues of 2^a

^aReagents for 2-16: (i) CH₂dCHCN, Et₃N, MeOH; (ii) Pd(OH)₂/C, H₂, CH₃CO₂H; (iii) CS₂, Et₃N, (Boc)₂O, EtOH; (iv) CH₂Cl₂. Reagents for

17-22: (i) C₁₀H₂₁CHO, NaCNBH₃, MeOH; (ii) (a) (Boc)₂O, Et₃N, MeOH, (b) LiOH, THF/MeOH; (iii) (a) 19, HATU, Et₃N, DMAP, DMF,

(b) NaOH, EtOH, reflux; (iv) (a) 3-chloroperoxybenzoic acid, CH₂Cl₂, CHCl₃, MeOH, (b) benzoyl isocyanate, CH₂Cl₂(c) NaOCH₃, MeOH,

(d) CF₃CO₂H.

Scheme 3. Syntheses of 1-Benzyl-2-(alkyl)-1H-imidazo[4,5-c]quinolin-4-amines^a

^aReagents for 23-38: (i) HNO₃, 75 °C; (ii) PhPOCl₂, 135 °C; (iii) benzylamine, Et₃N, CH₂Cl₂; (iv) Pt/C, H₂, Na₂SO₄, EtOAc; (v) (a) RCOCl, Et₃N,

THF, (b) NH₃/MeOH, 150 °C. Reagents for 39-43: (i) benzylamine, Et₃N, CH₂Cl₂; (ii) Pt/C, H₂, Na₂SO₄, EtOAc; (iii) (a) 4-pentynoic acid, HBTU,

Et₃N, DMAP, DMF, 90 °C; (iv) (a) 3-chloroperoxybenzoic acid, CH₂Cl₂, CHCl₃, MeOH, (b) benzoyl isocyanate, CH₂Cl₂, (c) NaOCH₃, MeOH.

Experimental Section

unless otherwise mentioned, while thin-layer chromatogra-

phy was carried out on silica gel CCM precoated aluminum

sheets. Purity for all final compounds was confirmed to be

greater than 97% by LC-MS using a Zorbax Eclipse Plus

4.6 mm ꢀ 150 mm, 5 μm analytical reverse phase C₁₈column

with H₂O-2-propanol or H₂O-CH₃CN gradients and an

Agilent ESI-TOF mass spectrometer (mass accuracy of 3 ppm)

operating in the positive ion (or negative ion, as appropriate)

acquisition mode.

Chemistry. All of the solvents and reagents used were

obtained commercially and used as such unless noted other-

wise. Moisture- or air-sensitive reactions were conducted

under nitrogen atmosphere in oven-dried (120 °C) glass appa-

ratus. The solvents were removed under reduced pressure

using standard rotary evaporators. Flash column chromato-

graphy was carried out using RediSep Rf “Gold” high per-

formance silica columns on CombiFlash Rf instrument

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4457

Figure 2. Alkyl chain length dependence of TLR7-agonistic acti-

vity profiles of the N¹-benzyl-C2-alkyl compounds (right panel). The

activity of 2 (reference compound) is shown on the left. Error bars

represent standard deviations obtained on quadruplicate samples.

Figure 3. Dose-dependent induction of IFN-R in whole human

blood by 2, 31, and 34. Plasma IFN-R was assayed in triplicate by

ELISA.

0.15 mmol) and palmitoyl chloride (16 mg, 0.06 mmol). After

30 min, the solvent was removed under vacuum, and residue was

dissolved in EtOAc and washed with water. The EtOAc fraction

was then dried under vacuum, and the residue was purified

using column chromatography (5% MeOH/dichloromethane)

to obtain the compound 5 (10 mg, 30%). ¹H NMR (400 MHz,

CDCl₃) δ 8.12 (d, J = 8.1 Hz, 1H), 7.88 (d, J = 8.3 Hz, 1H), 7.54

(t, J = 7.6 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 4.75 (s, 2H), 3.56 (q,

J = 6.8 Hz, 2H), 2.45-2.34 (m, 2H), 1.76-1.57 (m, 2H),

1.58-1.11 (m, 35H), 0.90 (t, J = 6.8 Hz, 3H). ¹³C NMR (101

MHz, CDCl₃) δ 192.82, 173.95, 152.03, 150.36, 135.45, 130.90,

128.29, 124.10, 123.24, 120.62, 114.81, 71.96, 55.58, 42.81,

42.30, 33.06, 31.92, 29.68, 29.65, 29.52, 29.47, 29.42, 29.35,

25.37, 22.69, 14.12, 13.70, 5.61. MS (ESI) calcd for C₃₃H₅₃N₅-

O₂, m/z 551.42, found 552.41 (M þ H)^þ.

Synthesis of Compound 3: 1-(4-Amino-2-((diethylamino)methyl)-

1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol. To a solution

of trifluoroacetate salt of 2 (75 mg, 0.14 mmol) in anhydrous

MeOH were added acetaldehyde (6.6 mg, 0.15 mmol), 12-14

drops of acetic acid, and sodium cyanoborohydride (10 mg, 0.15

mmol). After 4 h, the solvent was removed using vacuum, and the

residue was purified using column chromatography (7% MeOH/

1

dichloromethane) to obtain the compound 3 (16 mg, 34%). H

NMR (500 MHz, MeOD) δ8.36 (d, J= 7.8 Hz, 1H), 7.73 (dd, J =

8.4, 1.0 Hz, 1H), 7.69-7.64 (m, 1H), 7.52 (ddd, J= 8.4, 7.2, 1.2 Hz,

1H), 4.83 (s, 2H), 4.75-4.63 (m, 2H), 3.42 (dd, J = 14.2, 7.0 Hz,

4H), 1.39-1.29 (m, 6H), 1.21 (dd, J= 13.5, 6.2 Hz, 6H). ¹³CNMR

(126 MHz, MeOD) δ 150.85, 150.16, 138.41, 136.09, 131.52,

126.42, 126.18, 123.69, 120.11, 114.65, 72.54, 56.68, 49.70, 49.56,

27.65, 9.45. MS (ESI) calcd for C₁₉H₂₇N₅O, m/z 341.22, found

342.24 (M þ H)^þ.

Synthesis of Compound 6: 1-((4-Amino-1-(2-hydroxy-2-methyl-

propyl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)-3-(3-(dimethyl-

amino)propyl)-1,2-diethylguanidine. To a solution of trifluoro-

acetate salt of 2 (50 mg, 0.092 mmol) in anhydrous DMF were

Synthesis of Compound 4: N-((4-Amino-1-(2-hydroxy-2-methy-

lpropyl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)-N-ethylacetamide.

To a solution of trifluoroacetate salt of 2 (40 mg, 0.074 mmol) in

anhydrous THF were added triethlyamine (23 mg, 0.22 mmol) and

acetyl chloride (6 mg, 0.08 mmol). After 30 min, the solvent was

removed under vacuum. The residue was dissolved in EtOAc,

washed with water, and dried over Na₂SO₄. The EtOAc fraction

was then concentrated under vacuum, and the residue was purified

using column chromatography (6% MeOH/dichloromethane) to

obtain the compound 4 (22 mg, 85%). ¹H NMR (400 MHz,

MeOD) δ 8.34 (d, J = 8.3 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H),

7.53 (t, J = 7.7 Hz, 1H), 7.38 (t, J = 7.7 Hz, 1H), 5.23 (s, 2H), 4.81

(s, 2H), 3.63 (q, J = 7.1 Hz, 2H), 2.23 (s, 3H), 1.34-1.13 (m, 9H).

¹³C NMR (101 MHz, MeOD) δ 172.18, 154.62, 136.88, 134.07,

129.68, 129.48, 124.60, 122.64, 122.31, 118.21, 113.67, 70.85, 65.49,

55.12, 43.70, 41.79, 19.68, 14.03, 12.30. MS (ESI) calcd for

C₁₉H₂₅N₅O₂, m/z 355.20, found 356.21 (M þ H)^þ.

added triethylamine (51 mg, 0.51 mmol), EDCI HCl (53 mg,

3

0.28 mmol), and a catalytic amount of DMAP. The reaction was

stirred for 24-30 h. The solvent was then removed under

vacuum, and the residue was purified using C₁₈reverse-phase

column chromatography to obtain the compound 6 (9 mg,

1

24%). H NMR (400 MHz, MeOD) δ 8.48 (d, J = 8.4 Hz,

1H), 7.84 (d, J = 7.4 Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H), 7.63 (t,

J = 7.2 Hz, 1H), 5.18 (s, 2H), 4.85-4.71 (m, 2H), 3.58 (q, J =

7.3 Hz, 2H), 3.54-3.41 (m, 4H), 3.27-3.18 (m, 2H), 2.90 (d, J =

3.7 Hz, 6H), 2.27-2.00 (m, 2H), 1.30 (dt, J = 14.4, 7.2 Hz, 12H).

¹³C NMR (101 MHz, MeOD) δ 160.10, 153.79, 137.08, 134.45,

129.86, 124.93, 122.19, 118.63, 113.38, 71.07, 55.30, 54.69, 46.25,

45.16, 42.06, 41.41, 39.78, 24.50, 13.79, 11.77. MS (ESI) calcd

for C₂₅H₄₀N₈O, m/z 468.33, found 469.33 (M þ H)^þand 235.17

(M þ 2H)^2þ

.

Synthesis of Compound 5: N-((4-Amino-1-(2-hydroxy-2-methyl-

propyl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)-N-ethylpalmita-

mide. To a solution of trifluoroacetate salt of 2 (20 mg, 0.05

mmol) in anhydrous THF were added triethylamine (15 mg,

Synthesis of Compound 9: tert-Butyl 2-(2-(2-hydroxyethoxy)-

ethoxy)ethylcarbamate. To a solution of 7 (500 mg, 2.1 mmol) in

anhydrous dichloromethane, cooled to 0 °C, were added triethyl-

amine (420 mg, 4.2 mmol), tosyl chloride (609 mg, 3.54 mmol),

4458 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11

Shukla et al.

Scheme 4. Syntheses of 4-Substituted-1-benzyl-2-(alkyl)-1H-imidazo[4,5-c]quinolines^a

^aReagents: (i) (a) RCOCl, Et₃N, THF, (b) CaO, MeOH, 110 °C; (ii) 6N aq. HCl, 110 °C; (iii) polystyrene-bound PPh₃-Pd, phenyl boronic acid,

K₂CO₃, THF/H₂O; (iv) NH₂OH HCl, Et₃N, MeOH, 50 °C; (v) (a) t-butyl carbazate, 80 °C, MeOH, (b) CF₃CO₂H; (vi) (a) RCOCl, Et₃N, THF, (b)

3

CaO, MeOH, 110 °C.

Scheme 5. Syntheses of C-2/N-1 Substituent-Swapped 31/2 Hybrids^a

^aReagents: (i) HBTU, 2-(tert-butoxycarbonyl(ethyl)amino)acetic acid, Et₃N, DMAP, DMF, 70 °C; (ii) (a) 3-chloroperoxybenzoic acid, CH₂Cl₂,

CHCl₃, MeOH, (b) benzoyl isocyanate, CH₂Cl₂, (c) NaOCH₃, MeOH, (d) CF₃CO₂H; (iii) (a) C₄H₉COCl, Et₃N, THF, (b) CaO, MeOH, 110 °C; (iv) (a)

3-chloroperoxybenzoic acid, CH₂Cl₂, CHCl₃, MeOH, (b) benzoyl isocyanate, CH₂Cl₂(c) NaOCH₃, MeOH.

and a catalytic amount of DMAP. The reaction was stirred for

16 h, and then the mixture was washed with water and dried over

Na₂SO₄. The solvent was then removed under vacuum. This

residue O-tosylated compound was dissolved in anhydrous

DMF, to which sodium azide (273 mg, 4.2 mmol) was added

and the reaction was heated at 70 °C for 1 h. The solvent was

then removed under vacuum, and the residue was dissolved in

EtOAc, washed with water, and dried over Na₂SO₄. The

EtOAc fraction was concentrated under vacuum to obtain

the compound 8 (600 mg). This 8 was then dissolved in MeOH,

and di-tert-butyl dicarbonate (687 mg, 3.5 mmol) was added to

it. The solution was subjected to catalytic hydrogenolysis using

Pd(OH)₂/C at 60 psi hydrogen pressure for 4 h. The solution

was then filtered through celite, and filtrate was evaporated

under reduced pressure to afford the residue, which was

purified using column chromatography (3% MeOH/dichloro-

methane) to obtain the compound 9 (320 mg, 62%). ¹H

NMR (400 MHz, CDCl₃) δ 5.13 (s, 1H), 3.77 (d, J = 3.5 Hz,

2H), 3.70-3.61 (m, 6H), 3.58 (t, J = 5.2 Hz, 2H), 3.34 (d,

J = 4.6 Hz, 2H), 2.50 (s, 1H), 1.46 (s, 9H). ¹³C NMR (101

MHz, CDCl₃) δ 159.62, 79.34, 72.55, 70.43, 70.29, 61.78, 40.34,

28.41. MS (ESI) calcd for C₁₁H₂₃NO₅, m/z 249.16, found

272.15 (M þ Na^þ).

Synthesis of Compound 11: 1-(4-Amino-2-(((2-(2-(2-amino-

ethoxy)ethoxy)ethyl) (ethyl)amino)methyl)-1H-imidazo[4,5-c]qui-

nolin-1-yl)-2-methylpropan-2-ol. To a solution of 9 (200 mg, 0.8

mmol) in 4 mL of 0.3 M Dess-Martin periodinane solution in

dichloromethane, 10-12 drops of acetic acid were added and the

reaction was stirred for 4-5 h. The solvent was then removed under

vacuum, and the residue was purified using column chromatogra-

phy (40-50% EtOAc/dichloromethane) to obtain the intermedi-

ate aldehyde derivative 10 (71 mg). To a solution of trifluoroacetate

salt of 2 (150 mg, 0.29 mmol) in anhydrous MeOH were added 10

(71 mg, 0.29 mmol), 6-8 drops of acetic acid, and sodium

cyanoborohydride (22 mg, 0.35 mmol). The solution was stirred

for 14-15 h, and the solvent was removed under vacuum to obtain

the residue, which was purified using column chromatography

(10% MeOH/dichloromethane) to obtain the intermediate N-Boc

derivative. This was dissolved in 5 mL of trifluoroacetic acid and

stirred for 30 min, followed by removal of the solvent by purging

nitrogen and drying under vacuum to obtain the trifluoroacetate

1

salt of the compound 11 (110 mg, 60%). H NMR (400 MHz,

MeOD) δ 8.49 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 7.4 Hz, 1H), 7.76

(t, J = 7.3 Hz, 1H), 7.63 (t, J = 7.2 Hz, 1H), 4.82 (s, 4H), 3.88 (t,

J = 5.1 Hz, 2H), 3.70 (d, J =6.5 Hz, 6H), 3.50 (s, 2H), 3.38 (s, 2H),

3.17-3.09 (m, 2H), 1.55-1.10 (m, 9H). ¹³C NMR (101 MHz,

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4459

MeOD) δ 149.53, 148.21, 136.86, 134.61, 130.12, 122.23, 118.64,

113.15, 71.15, 69.99, 69.93, 66.50, 64.83, 55.36, 52.60, 49.74, 48.69,

46.46, 39.10, 26.24, 7.96. MS (ESI) calcd for C₂₃H₃₆N₆O₃, m/z

Synthesis of Compound 13: 1-(4-Amino-2-(((3-aminopropyl)-

(ethyl)amino)methyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methyl-

propan-2-ol. To a solution of 12 (160 mg, 0.44 mmol) in glacial

acetic acid, a catalytic amount of Pd(OH)₂/C was added and the

reaction mixture was subjected to hydrogenation at 55 psi

hydrogen pressure for 4 h. The solution was then filtered. The

filtrate was concentrated to obtain the acetate salt of the

compound 13 (55 mg). This residue (20 mg, 0.05 mmol) was

then dissolved in MeOH and di-tert-butyl dicarbonate (18 mg,

0.08 mmol) was added. After 30 min, the solvent was removed

under vacuum and the residue was purified using column

chromatography (15% MeOH/dichloromethane) to obtain

the intermediate N-Boc derivative. This was then dissolved in

2 mL of HCl solution in dioxane and stirred for 2 h. The solvent

was then removed under vacuum to obtain the hydrochloride

444.29, found 467.27 (M þ Na^þ) and 223.15 (M þ 2H)^2þ

.

Synthesis of Compound 12: 3-(((4-Amino-1-(2-hydroxy-2-

methylpropyl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)(ethyl)amino)-

propanenitrile. To a solution of 2 (250 mg, 0.46 mmol) in anhydrous

MeOH were added triethylamine (117 mg, 1.16 mmol) and acrylo-

nitrile (61 mg, 1.16 mmol). The reaction was stirred for 30 h,

followed by removal of the solvent under vacuum. The residue

was dissolved in EtOAc, washed with water, and dried over Na₂SO₄.

The EtOAc fraction was evaporated under vacuum, and the residue

was purified using column chromatography (11% MeOH/dichloro-

methane) to obtain the compound 12 (145 mg, 86%). ¹H NMR

(400 MHz, CDCl₃) δ 8.06 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 8.3 Hz,

1H), 7.55 (t, J = 7.6 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 6.04 (s, 2H),

4.88 (s, 2H), 4.12 (s, 2H), 2.94 (t, J = 6.7 Hz, 2H), 2.75 (q, J = 7.0

Hz, 2H), 2.54 (t, J = 6.7 Hz, 2H), 1.35 (s, 6H), 1.19 (t, J = 7.2 Hz,

3H). ¹³C NMR (101 MHz, CDCl₃) δ150.98, 150.41, 135.44, 127.81,

126.16, 122.48, 122.40, 122.18, 120.20, 118.64, 115.30, 71.49, 56.48,

50.85, 47.92, 47.64, 27.99, 15.33, 11.12. MS (ESI) calcd for

1

salt of the compound 13 (15 mg, 74%). H NMR (400 MHz,

MeOD) δ 8.51 (d, J = 8.2 Hz, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.78

(t, J = 7.9 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 5.09 (s, 2H),

3.74-3.50 (m, 6H), 3.12 (d, J = 7.0 Hz, 2H), 2.32 (s, 2H), 1.51

(t, J = 6.9 Hz, 3H), 1.35 (s, 6H). ¹³C NMR (126 MHz, MeOD)

δ 156.18, 151.90, 140.30, 137.56, 129.79, 126.47, 124.63, 123.13,

123.00, 72.46, 56.49, 53.02, 52.27, 48.77, 40.46, 27.84, 24.89,

C₂₀H₂₆N₆O, m/z 366.22, found 367.22 (M þ H)^þ

.

Figure 4. TLR7-agonistic activities of active compounds in a hu-

man TLR7-specific reporter gene assay. Error bars represent stan-

dard deviations obtained on quadruplicate samples.

Figure 5. Correlation of TLR7-agonistic potency and hydropho-

bicity (measured as retention time on C₁₈reverse-phase chromato-

graphy).

Scheme 6. Syntheses of Imidazole-Modified Analogues^a

^aReagents: (i) NaNO₂, CH₃CO₂H, H₂O, 0 °C; (ii) NH₃/MeOH, 150 °C; (iii) n-propyl isocyanate, Et₃N, 100 °C, THF.

4460 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11

Shukla et al.

11.74. MS (ESI) calcd for C₂₀H₃₀N₆O, m/z 370.25, found 371.26

(M þ H)^þ.

solution was refluxed at 45-50 °C for 40 min. The solvent was

then removed and the residue was purified using column chro-

matography (7% MeOH/dichloromethane) to obtain the N-

oxide derivative (75 mg). This was then dissolved in anhydrous

dichloromethane, followed by the addition of benzoyl isocyanate

(37 mg, 0.25 mmol) and heated at 45 °C for 15 min. The solvent

was thenremoved under vacuum, andthe residuewas dissolvedin

anhydrous MeOH, followed by the addition of excess sodium

methoxide and heating at 80 °C for 1 h. The solvent was then

removed under vacuum, and the residue was purified using

column chromatography (8% MeOH/dichloromethane) to ob-

tain the intermediate N-Boc derivative (27 mg). This was dis-

solved in 5 mL of trifluoroacetic acid and stirred for 30 min,

followed by removal of the solvent by purging nitrogen and

drying under vacuum to obtain the trifluoroacetate salt of the

compound22(33mg, 12%). ¹H NMR (400 MHz, CDCl₃) δ14.65

(s, 1H), 10.11 (s, 2H), 8.06 (s, 1H), 7.99 (d, J = 8.3 Hz, 1H), 7.44

(t, J = 7.6 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.20 (d, J = 8.6 Hz,

1H), 4.76(s, 2H), 4.61 (s, 2H), 3.23 (s, 2H), 1.93 (s, 2H), 1.50-1.11

(m, 22H), 0.91 (t, J = 6.7 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃)

δ 149.04, 148.91, 136.14, 134.18, 129.98, 124.99, 124.25, 120.76,

119.24, 112.18, 72.07, 49.07, 44.01, 31.89, 29.55, 29.49, 29.34,

29.31, 28.98, 26.58, 25.46, 22.67, 14.10, 5.61. MS (ESI) calcd for

C₂₆H₄₁N₅O, m/z 439.33, found 440.33 (M þ H)^þand 220.67

Synthesis of Compound 16: 1-(3-(((4-amino-1-(2-hydroxy-2-

methylpropyl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)(ethyl)amino)-

propyl)-3-(3-(4-(3-aminopropylamino)butylamino)propyl) thiourea.

To a solution of 13 (35 mg, 0.07 mmol) in anhydrous ethanol were

added triethylamine (22 mg, 0.21 mmol) and CS₂(54 mg, 0.71

mmol). The reaction mixture was stirred for 30 min and then cooled

to 0 °C. Di-tert-butyl dicarbonate (15 mg, 0.07 mmol) and a catalytic

amount of DMAP dissolved in ethanol were added to the reaction

mixture. After 30 min, the solvent was removed under vacuum, and

the residue was purified using column chromatography to obtain the

compound 15. To a solution of 15 (10 mg, 0.02 mmol) in anhydrous

dichloromethane N¹,N⁵,N¹⁰-tris boc spermine (37 mg, 0.073 mmol)

was added. The reaction mixture was stirred for 3 h. The solvent was

then removed under vacuum, and the residue was purified using

column chromatography (6% MeOH/dichloromethane) to obtain

the intermediate N-Boc derivative. This was dissolved in 5 mL of

trifluoroacetic acid and stirred for 30 min, followed by removal of

the solvent by purging nitrogen and drying under vacuum to obtain

the trifluoroacetate salt of the compound 16 (5 mg, 20%). ¹H NMR

(500 MHz, MeOD) δ 8.45 (d, J = 8.5 Hz, 1H), 7.83 (d, J = 8.4 Hz,

1H), 7.76 (t, J = 7.8 Hz, 1H), 7.67-7.56 (m, 1H), 4.96 (d, J = 6.9

Hz, 2H), 4.81-4.70 (m, 2H), 3.77-3.38 (m, 8H), 3.13 (dd, J= 18.0,

10.2 Hz, 2H), 3.10-2.96 (m, 8H), 2.16-2.04(m,4H),1.97-1.88(m,

(M þ 2H)^2þ

.

2H), 1.84-1.74 (m, 4H), 1.44 (t, J = 7.2 Hz, 3H), 1.29 (s, 6H). ¹³

C

Synthesis of Compound 24: 3-Nitroquinoline-2,4-diol. Com-

pound 23 (5 g, 31 mmol) was dissolved in 30 mL of nitric acid

and stirred at room temperature for 10 min, followed by heating

the reaction mixture at 75 °C for another 15 min. The reaction

mixture was then allowed to cool down to room temperature

and was added to ice-water mixture to precipitate the product.

The solid, yellow precipitate was filtered and dried to obtain the

NMR (126 MHz, MeOD) δ 150.81, 138.54, 138.41, 135.99, 131.60,

126.50, 123.71, 120.06, 114.65, 72.55, 56.76, 52.24, 50.71, 49.86,

49.68, 48.26, 48.06, 46.25, 45.87, 37.80, 27.48, 25.39, 24.29, 9.47. MS

(ESI) calcd for C₃₁H₅₄N₁₀OS, m/z 614.42, found 615.43 (M þ H)^þ

and 308.22 (M þ 2H)^2þ

.

Synthesis of Compound 19: 2-(tert-Butoxycarbonyl(undecyl)-

amino)acetic acid.²⁴To a solution of 17 (200 mg, 1.6 mmol) in

anhydrous MeOH were added undecanal (218 mg, 1.28 mmol),

5-6 drops of acetic acid, and sodium cyanoborohydride (111 mg,

1.76 mmol). The reaction was stirred for 3 h, and then the solvent

was removed under vacuum. The residue was dissolved in

dichloromethane and washed with water, brine, and dried over

Na₂SO₄. The dichloromethane fraction was then concentrated

to obtain the residue 18, which was dissolved in MeOH,

followed by the addition of triethylamine (202 mg, 2 mmol)

and di-tert-butyl dicarbonate (436 mg, 2 mmol). The reaction

was stirred for 4 h, and the solvent was removed under vacuum.

The residue was then dissolved in a solvent mixture of THF:

MeOH (3:1) and lithium hydroxide that was dissolved in water

was added until the pH went above 12. The reaction was stirred

for 24 h, followed by removal of the solvent under vacuum. The

residue was dissolved in water, and the solution was acidified

using 10% HCl until pH was less than 2. The water fraction was

then extracted with EtOAc, followed by washing the EtOAc

fraction with water and drying over Na₂SO₄. The EtOAc

fraction was then concentrated under vacuum to obtain the

compound 19 (320 mg, 61%). MS (ESI) calcd for C₁₈H₃₅NO₄,

m/z 329.26, found 328.25 (M - H)^-.

1

compound 24 (5.9 g, 92%). H NMR (400 MHz, DMSO) δ

12.00 (s, 1H), 8.04 (d, J = 7.9 Hz, 1H), 7.66 (t, J = 7.5 Hz, 1H),

7.34 (d, J = 8.2 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1H). ¹³C NMR (101

MHz, DMSO) δ 156.69, 156.23, 138.56, 133.61, 127.71, 124.92,

122.81, 116.32, 114.47. MS (ESI) calcd for C₉H₆N₂O₄, m/z

206.03, found 205.04 (M - H)^-.

Synthesis of Compound 25: 2,4-Dichloro-3-nitroquinoline.

Compound 24 (2 g, 9.7 mmol) was dissolved in 20 mL of

phenylphosphonyl dichloride and heated at 135 °C for 3 h.

The reaction mixture was poured into ice-water and stirred

vigorously to obtain the precipitate, which was filtered and dried

to afford the compound 25 (2.24 g, 74%). ¹H NMR (400 MHz,

CDCl₃) δ 8.30 (ddd, J = 8.4, 1.4, 0.6 Hz, 1H), 8.14 (ddd, J = 8.5,

1.2, 0.6 Hz, 1H), 7.97 (ddd, J = 8.5, 7.0, 1.4 Hz, 1H), 7.84 (ddd,

J = 8.3, 7.0, 1.2 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 146.64,

139.73, 135.62, 133.44, 129.76, 129.37, 125.22, 124.46.

Synthesis of Compound 26: N-Benzyl-2-chloro-3-nitroquinolin-

4-amine. To a solution of 25 (2.24 g, 9.26 mmol) in 30 mL of

anhydrous dichloromethane were added triethylamine (1.4 g,

13.9 mmol) and benzylamine (1.19 g, 11.1 mmol). The reaction

mixture was refluxed at 45 °C for 30 min. The solvent was then

evaporated under vacuum, and water was added to the residue

to obtain the precipitate. This was filtered, washed several times

with water, and dried to obtain the compound 26 (2.1 g, 73%).

¹H NMR (400 MHz, DMSO) δ 8.54 (dd, J = 12.9, 7.3 Hz, 2H),

7.89-7.81 (m, 2H), 7.69 (td, J = 8.6, 5.0 Hz, 1H), 7.39-7.21 (m,

5H), 4.46 (d, J = 6.3 Hz, 2H). ¹³C NMR (101 MHz, DMSO) δ

145.79, 144.82, 141.51, 138.27, 132.70, 129.08, 128.94, 127.89,

127.40, 127.15, 126.96, 123.52, 120.12, 47.24. MS (ESI) calcd for

C₁₆H₁₂ClN₃O₂, m/z 313.06, found 312.06 (M - H)^-.

Synthesis of Compound 22: 1-(4-Amino-2-((undecylamino)-

methyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol. To

a solution of 19 (171 mg, 0.52 mmol) in anhydrous DMF were

added HATU (217 mg, 0.57 mmol), triethylamine (58 mg, 0.57

mmol), a catalytic amount of DMAP, and 20 (120 mg, 0.52

mmol). The reaction mixture was stirred for 3-4 h. The solvent

was evaporated under vacuum. The residue was dissolved in

EtOAc, washed with water, and dried over Na₂SO₄to obtain the

residue. This was dissolved in 10 mL of ethanol, and a solution of

NaOH (105 mg, 2.62 mmol) dissolved in1 mL of water was added

to it. The reaction mixture was refluxed for 5-6 h, followed by

evaporation under reduced pressure. The residue was purified by

column chromatography (5% MeOH/dichloromethane) to ob-

tain the compound 21 (165 mg). To a solution of 21 in a solvent

mixture of MeOH:dichloromethane:chloroform (0.1:1:1), 3-chloro-

peroxybenzoic acid (135 mg, 0.79 mmol) was added and the

Synthesis of Compound 27: N⁴-Benzyl-2-chloroquinoline-3,4-

diamine. To a solution of 26 (2.21 g, 7.06 mmol) in 20 mL of

EtOAc were added a catalytic amount of Pt/C and Na₂SO₄. The

reaction mixture was subjected to hydrogenation at 55 psi

hydrogen pressure for 3 h. The reaction mixture was then

filtered through celite, and the filtrate was evaporated under

vacuum to obtain the compound 27 (1.8 g, 90%). ¹H NMR (400

MHz, MeOD) δ 7.90 (d, J = 8.2 Hz, 1H), 7.70 (d, J = 8.4 Hz,

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4461

1H), 7.43 (t, J = 7.6 Hz, 1H), 7.38-7.31 (m, 3H), 7.25 (t, J = 7.5

Hz, 2H), 7.20 (d, J = 7.0 Hz, 1H), 4.51 (s, 2H). ¹³C NMR

(101 MHz, MeOD) δ 141.54, 141.28, 139.93, 137.86, 128.45,

128.04, 127.44, 126.96, 126.80, 126.22, 125.31, 123.22, 121.40,

49.56. MS (ESI) calcd for C₁₆H₁₄ClN₃, m/z 283.09, found 284.10

(M þ H)^þ.

33: 1-Benzyl-2-hexyl-1H-imidazo[4,5-c]quinolin-4-amine. ¹H

NMR (400 MHz, CDCl₃) δ 7.82 (d, J = 8.4 Hz, 1H), 7.72 (d,

J = 8.2 Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H), 7.38-7.29 (m, 3H),

7.13 (t, J = 7.6 Hz, 1H), 7.07 (d, J = 7.4 Hz, 2H), 5.73 (s, 2H),

5.63 (s, 2H), 2.93-2.83 (m, 2H), 1.87-1.73 (m, 2H), 1.40 (dd,

J = 14.3, 6.7 Hz, 2H), 1.34-1.24 (m, 4H), 0.88 (t, J = 6.5 Hz,

3H). ¹³C NMR (101 MHz, CDCl₃) δ 154.01, 151.10, 144.59,

135.38, 133.98, 129.25, 128.02, 127.02, 126.87, 126.69, 125.54,

122.17, 119.70, 115.20, 48.84, 31.44, 29.05, 27.90, 27.46, 22.46,

14.01. MS (ESI) calcd for C₂₃H₂₆N₄, m/z 358.22, found 359.22

(M þ H)^þ.

General Procedure for Synthesis of C-2 alkyl imidazoquioline

compounds (28-38). Synthesis of Compound 32: 1-Benzyl-2-

pentyl-1H-imidazo[4,5-c]quinolin-4-amine. To a solution of 27

(50 mg, 0.18 mmol) in anhydrous THF were added triethyl-

amine (26 mg, 0.19 mmol) and hexanoyl chloride. The reaction

mixture was stirred at room temperature for 6 h. The solvent was

then removed under vacuum, and the residue was dissolved in

ethyl acetate and washed with water. The ethyl acetate fraction

was dried using Na₂SO₄and evaporated under vacuum to

obtain the intermediate amide compound. This was dissolved

in 0.1 mL of 2 M solution of ammonia in MeOH. The sealed

reaction vessel was heated in microwave at 150 °C for 1 h. The

solvent was then removed under vacuum, and the residue was

purified using column chromatography (7% MeOH/dichloro-

methane) to obtain the compound 32 (8 mg; unoptimized

1

34: 1-Benzyl-2-heptyl-1H-imidazo[4,5-c]quinolin-4-amine. H

NMR (500 MHz, CDCl₃) δ 7.81 (dd, J = 8.4, 0.8 Hz, 1H), 7.71

(dd, J = 8.3, 1.0 Hz, 1H), 7.44 (ddd, J = 8.4, 7.1, 1.3 Hz, 1H),

7.35-7.29 (m, 3H), 7.12 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 7.05 (d,

J = 6.9 Hz, 2H), 5.72 (s, 4H), 2.86 (dd, J = 18.2, 10.2 Hz, 2H),

1.79 (dt, J = 15.5, 7.7 Hz, 2H), 1.39 (dt, J = 14.9, 7.0 Hz, 2H),

1.34-1.18 (m, 6H), 0.86 (t, J = 7.0 Hz, 3H). ¹³C NMR (126

MHz, CDCl₃) δ 152.52, 149.22, 142.11, 133.55, 132.39, 127.57,

126.36, 125.49, 124.86, 124.69, 123.80, 120.69, 118.05, 113.31,

47.15, 29.89, 27.60, 27.19, 26.21, 25.75, 20.85, 12.34. MS (ESI)

calcd for C₂₄H₂₈N₄, m/z 372.23, found 373.24 (M þ H)^þ.

35: 1-Benzyl-2-nonyl-1H-imidazo[4,5-c]quinolin-4-amine. ¹H

NMR (500 MHz, CDCl₃) δ 7.85-7.79 (m, 1H), 7.71 (dd, J =

8.3, 0.9 Hz, 1H), 7.44 (qd, J = 6.9, 3.4 Hz, 1H), 7.38-7.29 (m,

3H), 7.13 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 7.05 (d, J = 6.9 Hz,

2H), 5.85 (s, 2H), 5.72 (s, 2H), 2.90-2.84 (m, 2H), 1.87-1.71 (m,

2H), 1.44-1.33 (m, 2H), 1.33-1.18 (m, 10H), 0.87 (t, J = 7.0

Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 152.31, 148.72, 141.13,

133.07, 132.10, 127.20, 126.00, 125.25, 124.35, 123.92, 123.40,

120.47, 117.71, 112.77, 46.79, 29.72, 27.59, 27.26, 27.24, 27.13,

27.12, 25.78, 25.35, 20.53, 12.00. MS (ESI) calcd for C₂₆H₃₂N₄,

m/z 400.26, found 401.27 (M þ H)^þ.

1

yields). H NMR (500 MHz, CDCl₃) δ 7.81 (d, J = 8.3 Hz,

1H), 7.72 (dd, J = 8.3, 1.0 Hz, 1H), 7.44 (qd, J = 7.0, 3.5 Hz,

1H), 7.38-7.29 (m, 3H), 7.18-7.10 (m, 1H), 7.06 (d, J = 6.9 Hz,

2H), 5.74 (s, 2H), 5.60 (s, 2H), 2.95-2.79 (m, 2H), 1.80 (dt,

J = 15.6, 7.6 Hz, 2H), 1.42-1.27 (m, 4H), 0.87 (t, J = 7.2 Hz,

3H). ¹³C NMR (126 MHz, CDCl₃) δ 153.50, 150.28, 134.61,

133.41, 128.61, 127.40, 126.51, 125.94, 125.85, 124.85, 121.69,

119.07, 114.42, 48.20, 30.85, 26.97, 26.77, 21.65, 13.23. MS (ESI)

calcd for C₂₂H₂₄N₄, m/z 344.20, found 345.21 (M þ H)^þ. 2D

¹H COSY and NOESY experiments (included in Suppor-

ting Information) provided unambiguous assignment of ¹H

resonances.

28: 1-Benzyl-2-methyl-1H-imidazo[4,5-c]quinolin-4-amine. ¹H

NMR (500 MHz, CDCl₃) δ 7.81 (dd, J = 8.4, 0.8 Hz, 1H), 7.75

(dd, J = 8.3, 1.0 Hz, 1H), 7.45 (ddd, J = 8.4, 7.1, 1.4 Hz, 1H),

7.38-7.28 (m, 3H), 7.15 (ddd, J = 8.3, 7.1, 1.2 Hz, 1H),

36: 1-Benzyl-2-undecyl-1H-imidazo[4,5-c]quinolin-4-amine.

¹H NMR (500 MHz, CDCl₃) δ 7.81 (d, J = 8.2 Hz, 1H), 7.71

(d, J = 7.7 Hz, 1H), 7.47-7.42 (m, 1H), 7.36-7.28 (m, 3H),

7.15-7.11 (m, 1H), 7.06 (d, J = 7.0 Hz, 2H), 5.73 (s, 2H), 5.70 (s,

2H), 3.02-2.71 (m, 2H), 1.79 (dt, J = 15.5, 7.8 Hz, 2H),

1.44-1.34 (m, 2H), 1.34-1.18 (m, 14H), 0.88 (t, J = 7.0 Hz,

3H). ¹³C NMR (126 MHz, CDCl₃) δ 152.56, 149.23, 142.11,

133.58, 132.43, 127.60, 126.39, 125.53, 124.89, 124.73, 123.83,

120.73, 118.08, 113.34, 47.19, 30.20, 27.88, 27.73, 27.67, 27.62,

27.56, 26.24, 25.78, 20.98, 12.43. MS (ESI) calcd for C₂₈H₃₆N₄,

m/z 428.29, found 429.30 (M þ H)^þ.

7.10-7.05 (m, 2H), 5.73 (s, 2H), 5.64 (s, 2H), 2.63 (s, 3H). ¹³

C

NMR (126 MHz, CDCl₃) δ 149.17, 148.91, 142.26, 133.27,

132.64, 127.74, 126.56, 125.74, 124.90, 124.82, 123.96, 120.90,

117.99, 113.37, 47.59, 12.20. MS (ESI) calcd for C₁₈H₁₆N₄, m/z

288.14, found 289.15 (M þ H)^þ.

1

29: 1-Benzyl-2-ethyl-1H-imidazo[4,5-c]quinolin-4-amine. H

NMR (500 MHz, CDCl₃) δ 7.80 (dd, J = 8.4, 0.8 Hz, 1H), 7.73

(dd, J = 8.3, 1.0 Hz, 1H), 7.44 (qd, J = 7.2, 3.7 Hz, 1H),

7.37-7.28 (m, 3H), 7.13 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H),

7.09-7.01 (m, 2H), 5.73 (s, 2H), 5.59 (s, 2H), 2.97-2.86 (m,

2H), 1.41 (t, J = 7.5 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ

153.83, 149.91, 143.24, 134.14, 133.05, 128.19, 126.96, 126.04,

125.64, 125.52, 124.43, 121.20, 118.62, 114.05, 47.69, 19.72, 10.98.

MS (ESI) calcd for C₁₉H₁₈N₄, m/z 302.15, found 303.16 (M þ H)^þ.

30: 1-Benzyl-2-propyl-1H-imidazo[4,5-c]quinolin-4-amine. ¹H

NMR (500 MHz, CDCl₃) δ 7.80 (dd, J = 8.4, 0.8 Hz, 1H), 7.71

(dd, J = 8.3, 1.0 Hz, 1H), 7.49-7.42 (m, 1H), 7.40-7.30 (m,

3H), 7.18-7.10 (m, 1H), 7.10-7.05 (m, 2H), 5.74 (s, 2H), 5.66 (s,

2H), 2.92-2.80 (m, 2H), 1.91-1.85 (m, 2H), 1.03 (t, J = 7.4 Hz,

3H). ¹³C NMR (126 MHz, CDCl₃) δ 153.50, 150.46, 143.54,

134.77, 133.58, 128.82, 127.60, 126.73, 126.12, 126.05, 125.01,

121.90, 119.30, 114.59, 48.36, 28.88, 20.92, 13.52. MS (ESI)

calcd for C₂₀H₂₀N₄, m/z 316.17, found 317.17 (M þ H)^þ.

37: 1-Benzyl-2-pentadecyl-1H-imidazo[4,5-c]quinolin-4-amine.

¹H NMR (500 MHz, CDCl₃) δ 7.81 (dd, J = 8.4, 0.8 Hz, 1H),

7.72 (dd, J = 8.3, 1.0Hz, 1H), 7.44 (ddd, J =8.4, 7.1, 1.3Hz, 1H),

7.36-7.27 (m, 3H), 7.13 (ddd, J = 8.3, 7.1, 1.2 Hz, 1H),

7.09-7.02 (m, 2H), 5.73 (s, 2H), 5.69 (s, 2H), 2.91-2.83 (m,

2H), 1.79 (dt, J = 15.5, 7.7 Hz, 2H), 1.59-1.33 (m, 2H),

1.33-1.16 (m, 22H), 0.88 (t, J = 7.0 Hz, 3H). ¹³C NMR (126

MHz, CDCl₃) δ 151.99, 148.66, 141.59, 133.01, 131.85, 127.03,

125.82, 124.96, 124.32, 124.17, 123.26, 120.16, 117.50, 112.78,

46.62, 29.66, 27.43, 27.40, 27.39, 27.37, 27.32, 27.17, 27.11, 27.10,

26.99, 25.68, 25.21, 20.43, 11.86. MS (ESI) calcd for C₃₂H₄₄N₄,

m/z 484.36, found 485.36 (M þ H)^þ.

38: 1-Benzyl-2-(but-3-enyl)-1H-imidazo[4,5-c]quinolin-4-amine.

¹H NMR (500 MHz, CDCl₃) δ7.80 (dd, J =8.4, 0.8Hz, 1H), 7.73

(dd, J = 8.3, 1.0 Hz, 1H), 7.46-7.42 (m, 1H), 7.36-7.29 (m, 3H),

7.13 (ddd, J = 8.2, 7.1, 1.2Hz, 1H), 7.08-7.05 (m, 2H), 5.96-5.84

(m, 1H), 5.74 (s, 2H), 5.58 (s, 2H), 5.11-4.97 (m, 2H), 3.03-2.93

(m, 2H), 2.64-2.54 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ

150.99, 148.87, 134.50, 133.06, 131.93, 127.16, 125.97, 125.02,

124.64, 124.58, 123.43, 120.17, 117.57, 113.92, 46.72, 29.59, 24.77.

MS (ESI) calcd for C₂₁H₂₀N₄, m/z 328.17, found 329.18 (M þ H)^þ.

Synthesis of Compound 40: N-Benzyl-3-nitroquinolin-4-amine.

To a solution of 39 (2.03 mg, 9.76 mmol) in 20 mL of anhydrous

dichloromethane were added triethylamine (1.48 g, 14.64 mmol)

and benzylamine (1.36 g, 12.69 mmol). The reaction mixture was

refluxed at 45 °C for 30 min. The solvent was then evaporated

1

31: 1-Benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine. H

NMR (500 MHz, CDCl₃) δ 7.80 (dd, J = 8.4, 0.8 Hz, 1H), 7.72

(dd, J = 8.3, 1.0 Hz, 1H), 7.43 (ddd, J = 8.4, 7.0, 1.4 Hz, 1H),

7.36-7.28 (m, 3H), 7.12 (ddd, J = 8.3, 7.1, 1.2 Hz, 1H),

7.08-7.04 (m, 2H), 5.73 (s, 2H), 5.56 (s, 2H), 2.91-2.83 (m,

2H), 1.84-1.74 (m, 2H), 1.43 (dq, J = 14.8, 7.4 Hz, 2H), 0.92 (t,

J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 152.56, 149.54,

142.97, 133.87, 132.54, 127.81, 126.58, 125.61, 125.34, 125.21,

124.06, 120.78, 118.25, 113.71, 47.38, 28.54, 25.71, 21.04, 12.28.

MS (ESI) calcd for C₂₁H₂₂N₄, m/z330.18, found 331.19 (M þ H)^þ.

4462 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11

Shukla et al.

under vacuum, and water was added to the residue to obtain the

precipitate This was filtered, washed several times with water,

and dried to obtain the compound 40 (2.6 g, 95%). H NMR

(d, J = 8.4 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.42 (t, J = 7.7 Hz,

1H), 7.38-7.30 (m, 3H), 7.05 (d, J = 7.0 Hz, 2H), 5.81 (s, 2H),

3.05-2.95 (m, 2H), 1.84 (dt, J = 15.6, 7.6 Hz, 2H), 1.48-1.24

(m, 4H), 0.88 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃)

δ 156.40, 143.96, 143.60, 135.40, 134.74, 133.74, 129.93, 129.42,

128.30, 127.56, 126.58, 125.40, 119.91, 117.29, 49.19, 31.63,

27.91, 27.70, 22.29, 13.90. MS (ESI) calcd for C₂₂H₂₂ClN₃,

m/z 363.15, found 364.15 (M þ H)^þ.

1

(400 MHz, CDCl₃) δ 9.89 (s, 1H), 9.42 (s, 1H), 8.31 (d, J = 8.6

Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.79 (t, J = 7.6 Hz, 1H),

7.56-7.35 (m, 6H), 5.13 (d, J = 5.7 Hz, 2H). ¹³C NMR (101

MHz, CDCl₃) δ 151.01, 150.64, 147.37, 136.84, 132.73, 130.51,

129.36, 128.54, 127.16, 126.84, 126.33, 125.58, 119.14, 53.06.

MS (ESI) calcd for C₁₆H₁₃N₃O₂, m/z 279.10, found 280.11

(M þ H)^þ.

Synthesis of Compound 46: 1-Benzyl-2-pentyl-1H-imidazo-

[4,5-c]quinolin-4-ol. The compound 44 (50 mg, 0.14 mmol) was

dissolved in 6 M HCl, and the solution was heated in a micro-

wave at 110 °C for 1 h. The solvent was then removed under

vacuum, and the residue was purified using column chromato-

graphy (7% MeOH/dichloromethane) to obtain the compound

46 (38 mg, 80%). ¹H NMR (400 MHz, DMSO) δ 11.59 (s, 1H),

7.74 (d, J = 8.2 Hz, 1H), 7.43 (d, J = 8.2 Hz, 1H), 7.39-7.30 (m,

J = 8.3 Hz, 3H), 7.30-7.23 (m, J = 7.2 Hz, 1H), 7.08-7.00 (m,

J = 7.5 Hz, 3H), 5.83 (s, 2H), 2.83 (t, J = 7.5 Hz, 2H), 1.79-1.65

(m, 2H), 1.40-1.16 (m, 4H), 0.82 (t, J = 7.0 Hz, 3H). ¹³C NMR

(101 MHz, DMSO) δ 157.78, 154.36, 137.04, 136.73, 134.44,

131.09, 129.43, 128.08, 127.95, 125.94, 121.98, 121.27, 116.69,

112.04, 48.44, 31.22, 27.11, 26.69, 22.30, 14.26. MS (ESI) calcd

for C₂₂H₂₃N₃, m/z 345.18, found 346.19 (M þ H)^þ.

Synthesis of Compound 41: N⁴-Benzylquinoline-3,4-diamine.

To a solution of 40 (2.6 g, 7.06 mmol) in 10 mL of EtOAc were

added a catalytic amount of Pt/C and Na₂SO₄. The reaction

mixture was subjected to hydrogenation at 55 psi hydrogen

pressure for 4 h. The reaction mixture was then filtered through

celite, and the filtrate was evaporated under vacuum to obtain

the compound 41 (2.3 g, 96%). ¹H NMR (400 MHz, CDCl₃) δ

8.53 (s, 1H), 8.00 (d, J = 8.3 Hz, 1H), 7.75 (d, J = 8.2 Hz, 1H),

7.56-7.29 (m, 7H), 4.43 (s, 2H), 4.05 (s, 1H), 3.78 (s, 2H). ¹³

C

NMR (101 MHz, CDCl₃) δ 143.59, 143.48, 141.10, 139.77,

129.89, 129.84, 128.81, 127.77, 127.60, 126.05, 125.76, 120.06,

99.98, 50.98. MS (ESI) calcd for C₁₆H₁₅N₃, m/z 249.13, found

250.13 (M þ H)^þ.

Synthesis of Compound 43: 1-Benzyl-2-(but-3-ynyl)-1H-imidazo-

[4,5-c]quinolin-4-amine. To a solution of pentynoic acid (37 mg,

0.38 mmol) in anhydrous DMF were added HBTU (142 mg, 0.38

mmol), triethylamine (52 mg, 0.51 mmol), a catalytic amount of

DMAP, and 41 (85 mg, 0.34 mmol). The reaction mixture was then

heated to 90 °C for 1 h. The solvent was then removed under

vacuum. The residue was dissolved in EtOAc, washed with water,

dried over Na₂SO₄, and then concentrated to obtain the residue.

This was purified using column chromatography (7% MeOH/

dichloromethane) to obtain the compound 42 (60 mg). To a

solution of 42 in a solvent mixture of MeOH:dichloromethane:

chloroform (0.1:1:1) was added 3-chloroperoxybenzoic acid

(83 mg, 0.48 mmol), and the solution was refluxed at 45-50 °C for

40 min. The solvent was then removed under vacuum, and the

residue was purified using column chromatography (8% MeOH/

dichloromethane) to obtain the N-oxide derivative (30 mg). This

was then dissolved in anhydrous dichloromethane, followed by the

addition of benzoyl isocyanate (20 mg, 0.14 mmol) and heated at

45 °C for 15 min. The solvent was then removed under vacuum,

and the residue was dissolved in anhydrous MeOH, followed by

the addition of excess sodium methoxide. The reaction mixture

was then heated at 80 °C for 1 h. The solvent was removed under

vacuum, and the residue was purified using column chromato-

graphy (6% MeOH/dichloromethane) to obtain the compound 43

(15 mg, 12%). ¹H NMR (500 MHz, CDCl₃) δ 7.81 (dd, J = 8.4,

0.8 Hz, 1H), 7.74 (dd, J = 8.3, 1.0 Hz, 1H), 7.46 (ddd, J = 8.4, 5.7,

1.3 Hz, 1H), 7.37-7.30 (m, 3H), 7.15 (ddd, J = 8.3, 7.1, 1.2 Hz,

1H), 7.09-7.05 (m, 2H), 5.79 (s, 2H), 5.68 (s, 2H), 3.11 (dd, J =

8.8, 6.4 Hz, 2H), 2.87-2.71 (m, 2H), 1.99 (t, J = 2.6 Hz, 1H). ¹³C

NMR (126 MHz, CDCl₃) δ 150.49, 149.42, 133.45, 132.82, 127.91,

126.75, 126.01, 125.19, 124.98, 124.08, 121.13, 118.33, 113.52,

81.16, 68.15, 47.46, 25.06, 15.63. MS (ESI) calcd for C₂₁H₁₈N₄,

m/z 326.15, found 327.16 (M þ H)^þ.

Synthesis of Compound 47: 1-Benzyl-2-pentyl-4-phenyl-1H-

imidazo[4,5-c]quinoline. Suzuki coupling of the compound 44

with phenyl boronic acid was performed. To a solution of 44 (70

mg, 0.19 mmol) in THF were added polystyrene-bound PPh₃-Pd

(9.5 mg, 0.00095 mmol), K₂CO₃(40 mg, 0.29 mmol) dissolved in

water, and phenyl boronic acid (28 mg, 0.23 mmol). The

reaction was heated at 80 °C for 10-12 h. The solution was

filtered to remove the solid resin, and the filtrate was evaporated

under vacuum to obtain the residue. This was purified using

column chromatography (30% EtOAc/hexane) to obtain the

1

compound 47 (25 mg, 33%). H NMR (400 MHz, CDCl₃) δ

8.81-8.77 (m, 1H), 8.33 (dd, J = 8.5, 0.8 Hz, 1H), 7.93 (dd, J =

8.4, 0.8 Hz, 1H), 7.64-7.58 (m, 3H), 7.55-7.49 (m, 1H), 7.36

(dddd, J = 9.8, 6.9, 6.1, 2.2 Hz, 4H), 7.12-7.07 (m, 2H), 5.84 (s,

2H), 3.03-2.85 (m, 2H), 1.91 (dt, J = 15.5, 7.6 Hz, 2H), 1.42

(ddt, J = 36.4, 21.9, 7.4 Hz, 4H), 0.92 (t, J = 7.2 Hz, 3H). ¹³

C

NMR (101 MHz, CDCl₃) δ 155.17, 151.02, 144.50, 138.18,

135.43, 135.10, 135.01, 130.96, 130.01, 129.31, 129.27, 128.37,

128.05, 126.74, 125.76, 125.52, 119.61, 117.17, 48.92, 31.58,

27.56, 27.48, 22.37, 13.97. MS (ESI) calcd for C₂₈H₂₇N₃, m/z

405.22, found 406.23 (M þ H)^þ.

Synthesis of Compound 48: N-(1-Benzyl-2-butyl-1H-imidazo-

[4,5-c]quinolin-4-yl)hydroxylamine. To a solution of 27 (100 mg,

0.35 mmol) in anhydrous THF were added triethylamine

(72 mg, 0.71 mmol) and valeryl chloride (51 mg, 0.42 mmol).

The reaction mixture was then stirred for 6-8 h, followed by

removal of the solvent under vacuum. The residue was dissolved

in EtOAc, washed with water and brine, and then dried over

Na₂SO₄to obtain the residue. This was dissolved in MeOH,

followed by the addition of calcium oxide. The reaction mixture

was then heated in microwave at 110 °C for 1 h. The solvent was

removed under vacuum, and the residue was purified using

column chromatography (3% MeOH/dichloromethane) to ob-

tain 45 (75 mg, 62%). To a solution of 45 (25 mg, 0.072 mmol) in

anhydrous MeOH were added triethylamine (72 mg, 0.72 mmol)

and hydroxylamine hydrochloride (76 mg, 1.08 mmol). The

reaction mixture was heated at 50 °C for 10-12 h. The solvent

was then removed under vacuum, and the residue was dissolved

in EtOAc, washed with water, and dried over Na₂SO₄. The

EtOAc fraction was then evaporated under vacuum, and the

residue was purified using basic-alumina (pH > 7) column

chromatography (30% MeOH/dichloromethane) to obtain the

compound 48 (10 mg, 40%). ¹H NMR (500 MHz, CDCl₃) δ 7.41

(d, J = 8.1 Hz, 1H), 7.32 (dq, J = 25.0, 7.2 Hz, 4H), 7.21 (s, 1H),

7.06 (d, J = 7.1 Hz, 2H), 6.88 (dd, J = 10.6, 5.7 Hz, 1H), 5.61 (s,

2H), 2.84-2.77 (m, 2H), 1.78 (dt, J = 15.5, 7.7 Hz, 2H), 1.40 (dt,

J = 15.0, 7.4 Hz, 2H), 0.90-0.86 (m, 3H). ¹³C NMR (126 MHz,

Synthesis of Compound 44: 1-Benzyl-4-chloro-2-pentyl-1H-

imidazo[4,5-c]quinoline. To a solution 27 (200 mg, 0.71 mmol)

in anhydrous THF were added triethylamine (179 mg, 1.77

mmol) and hexanoyl chloride (143 mg, 1.06 mmol). The reaction

mixture was then stirred for 6-8 h, followed by removal of the

solvent under vacuum. The residue was dissolved in EtOAc,

washed with water and brine and then dried over Na₂SO₄to

obtain the intermediate amide compound. This was dissolved in

MeOH, followed by the addition of calcium oxide. The reaction

mixture was then heated in microwave at 110 °C for 1 h. The

solvent was removed under vacuum, and the residue was

purified using column chromatography (3% MeOH/dichloro-

methane) to obtain the compound 44 (170 mg, 66%). ¹H

NMR (400 MHz, CDCl₃) δ 8.16 (d, J = 8.4 Hz, 1H), 7.88

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4463

CDCl₃) δ 151.78, 133.18, 127.13, 125.87, 125.13, 123.30, 119.10,

117.94, 46.50, 27.68, 24.81, 20.34, 11.58. MS (ESI) calcd for

C₂₁H₂₂N₄O, m/z 346.18, found 347.19 (M þ H)^þ.

was dissolved in 5 mL of trifluoroacetic acid and stirred for

30 min, followed by removal of the solvent by purging nitrogen

and drying under vacuum to obtain the trifluoroacetate salt of

the compound 52 (26 mg, 20%). ¹H NMR (400 MHz, MeOD) δ

8.02 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.69 (t, J =

7.8 Hz, 1H), 7.49-7.32 (m, 4H), 7.15 (d, J = 7.6 Hz, 2H), 6.00 (s,

2H), 4.71 (s, 2H), 3.38 (q, J = 7.3 Hz, 2H), 1.45 (t, J = 7.3 Hz,

3H). ¹³C NMR (101 MHz, MeOD) δ 149.52, 148.45, 136.86,

134.46, 134.03, 130.18, 129.15, 128.12, 125.29, 125.15, 124.97,

121.76, 118.34, 112.47, 48.94, 43.17, 42.36, 10.06. MS (ESI)

calcd for C₂₀H₂₁N₅, m/z 331.18, found 332.19 (M þ H)^þ.

Synthesis of Compound 54: 1-(4-Amino-2-butyl-1H-imidazo-

[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol. To a solution of 20

(100 mg, 0.43 mmol) in anhydrous THF were added triethyl-

amine (66 mg, 0.65 mmol) and valeryl chloride (62 mg, 0.52

mmol). The reaction mixture was then stirred for 6-8 h,

followed by removal of the solvent under vacuum. The residue

was dissolved in EtOAc, washed with water and brine, and then

dried over Na₂SO₄to obtain the intermediate amide compound.

This was dissolved in MeOH, followed by the addition of

calcium oxide, and was heated in microwave at 110 °C for 1 h.

The solvent was then removed and the residue was purified using

column chromatography (9% MeOH/dichloromethane) to ob-

tain the compound 53 (58 mg). To a solution of 53 in a solvent

mixture of MeOH:dichloromethane:chloroform (0.1:1:1) was

added 3-chloroperoxybenzoic acid (84 mg, 0.49 mmol), and the

solution was refluxed at 45-50 °C for 40 min. The solvent was

then removed and the residue was purified using column chro-

matography (20% MeOH/dichloromethane) to obtain the N-

oxide derivative (55 mg). This was then dissolved in anhydrous

dichloromethane, followed by the addition of benzoyl isocya-

nate (39 mg, 0.26 mmol) and heated at 45 °C for 15 min. The

solvent was then removed under vacuum, and the residue was

dissolved in anhydrous MeOH, followed by the addition of

excess sodium methoxide. The reaction mixture was then heated

at 80 °C for an hour. The solvent was removed under vacuum,

and the residue was purified using column chromatography

(11% MeOH/dichloromethane) to obtain the compound 54 (40

mg, 30%). ¹H NMR (400 MHz, MeOD) δ 8.26 (d, J = 8.3 Hz,

1H), 7.69 (d, J = 8.4 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.31 (t,

J = 7.7 Hz, 1H), 4.63 (s, 2H), 3.20-3.02 (m, 2H), 1.98-1.80 (m,

2H), 1.62-1.45 (m, 2H), 1.27 (s, 6H), 1.04 (t, J = 7.4 Hz, 3H).

¹³C NMR (101 MHz, MeOD) δ 156.33, 151.22, 143.80, 134.53,

126.75, 125.41, 125.12, 121.49, 120.93, 115.31, 71.16, 54.80,

29.72, 27.12, 26.52, 22.24, 12.86. MS (ESI) calcd for C₁₈H₂₄-

N₄O, m/z 312.20, found 313.20 (M þ H)^þ.

Synthesis of Compound 49: 1-Benzyl-2-butyl-4-hydrazinyl-1H-

imidazo[4,5-c]quinoline. To a solution of 45 (60 mg, 0.17 mmol)

in anhydrous MeOH was added tert-butyl carbazate (57 mg,

0.43 mmol). After 15 h, the solvent was removed under vacuum

and the residue was purified using column chromatography (4%

MeOH/dichloromethane) to obtain the intermediate N-Boc

derivative. This was then dissolved in 5 mL of trifluoroacetic

acid and stirred for 30 min, followed by removal of the solvent

by purging nitrogen and drying under vacuum to obtain the

trifluoroacetate salt of the compound 49 (55 mg, 57%). ¹H

NMR (500 MHz, MeOD) δ 7.82 (d, J = 8.4 Hz, 1H), 7.79 (d,

J = 8.3 Hz, 1H), 7.45-7.40 (m, 1H), 7.28 (dt, J = 25.5, 7.2 Hz,

3H), 7.15-7.10 (m, 1H), 7.03 (d, J = 7.4 Hz, 2H), 5.82 (s, 2H),

2.94-2.90 (m, 2H), 1.79-1.69 (m, 2H), 1.40 (dq, J = 14.8, 7.4

Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, MeOD)

δ 154.65, 151.25, 143.19, 135.64, 133.54, 128.81, 128.68, 127.41,

126.96, 125.31, 125.10, 124.68, 122.23, 120.02, 114.47, 48.23,

29.28, 26.26, 21.84, 12.51. MS (ESI) calcd for C₂₁H₂₃N₅, m/z

345.20, found 346.20 (M þ H)^þ.

Synthesis of Compound 50: 1-Benzyl-2-butyl-1H-imidazo[4,5-c]-

quinoline. To a solution of 41 (300 mg, 1.221 mmol) in an-

hydrous THF were added triethylamine (183 mg, 1.82 mmol)

and valeryl chloride (174 mg, 1.45 mmol). The reaction mixture

was then stirred for 6-8 h, followed by removal of the solvent

under vacuum. The residue was dissolved in EtOAc, washed

with water and brine, and then dried over Na₂SO₄to obtain the

intermediate amide compound. This was dissolved in MeOH,

followed by the addition of calcium oxide. The reaction mixture

was then heated in microwave at 110 °C for an hour. The solvent

was removed under vacuum, and the residue was purified using

column chromatography (9% MeOH/dichloromethane) to ob-

1

tain the compound 50 (360 mg, 94%). H NMR (400 MHz,

CDCl₃) δ 9.38 (s, 1H), 8.25 (d, J = 8.1 Hz, 1H), 7.93 (d, J = 8.5

Hz, 1H), 7.66-7.57 (m, 1H), 7.42 (dd, J = 11.2, 4.1 Hz, 1H),

7.40-7.31 (m, 3H), 7.06 (d, J = 6.6 Hz, 2H), 5.80 (s, 2H),

3.02-2.85 (m, 2H), 1.90 (dt, J = 15.4, 7.7 Hz, 2H), 1.57-1.39

(m, 2H), 0.96 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃)

δ 155.81, 144.98, 144.75, 136.53, 135.14, 134.10, 130.83, 129.35,

128.15, 126.72, 126.29, 125.47, 119.88, 117.58, 48.94, 29.57,

27.18, 22.53, 13.77. MS (ESI) calcd for C₂₁H₂₁N₃, m/z 315.17,

found 316.18 (M þ H)^þ.

Synthesis of Compound 52: 1-Benzyl-2-((ethylamino)methyl)-

1H-imidazo[4,5-c]quinolin-4-amine. To a solution of 2-(tert-

butoxycarbonyl(ethyl)amino)acetic acid (90 mg, 0.44 mmol) in

anhydrous DMF were added HBTU (167 mg, 0.44 mmol),

triethylamine (53 mg, 0.52 mmol), a catalytic amount of DMAP,

and 41 (100 mg, 0.4 mmol). The reaction mixture was then

heated to 70 °C for 18 h. The solvent was then removed under

vacuum. The residue was dissolved in EtOAc, washed with

water, dried using Na₂SO₄, and then concentrated to obtain

the residue, which was purified using column chromatography

(6% MeOH/dichloromethane) to obtain the compound 51 (133

mg). To a solution of 51 in a solvent mixture of MeOH:

dichloromethane:chloroform (0.1:1:1), 3-chloroperoxybenzoic

acid (138 mg, 0.8 mmol) was added and the solution was

refluxed at 45-50 °C for 40 min. The solvent was then removed

under vacuum, and the residue was purified using column

chromatography (10% MeOH/dichloromethane) to obtain the

N-oxide derivative (31 mg). This was then dissolved in anhy-

drous dichloromethane, followed by the addition of benzoyl

isocyanate (16 mg, 0.11 mmol) and heated at 45 °C for 15 min.

The solvent was then removed under vacuum and the residue

was dissolved in anhydrous MeOH, followed by the addition of

excess sodium methoxide and heated at 80 °C for 1 h. The

solvent was then removed under vacuum, and the residue was

purified using column chromatography (8% MeOH/dichloro-

methane) to obtain the intermediate N-Boc derivative. This

Synthesis of Compound 55: 1-Benzyl-4-chloro-1H-[1,2,3]triazolo-

[4,5-c]quinoline. To a solution of 27 (60 mg, 0.21 mmol) in 1.5 mL

of 1:1 acetic acid/water at 0 °C was added 0.5N aqueous solution of

sodium nitrite (0.56 mL, 0.28 mmol) and the reaction was stirred

for 2 h. The solvent was then removed under vacuum. The residue

was dissolved in EtOAc, washed with water, and dried over

Na₂SO₄. The EtOAc fraction was then concentrated under va-

cuum to obtain the compound 55 (58 mg, 94%). ¹H NMR (500

MHz, CDCl₃) δ 8.12 (dd, J = 8.4, 0.7 Hz, 1H), 7.96 (dd, J = 8.2,

0.9 Hz, 1H), 7.70 (ddd, J = 8.4, 7.2, 1.4 Hz, 1H), 7.52 (ddd, J =

8.3, 7.2, 1.2 Hz, 1H), 7.30-7.21 (m, 3H), 7.12-7.09 (m, 2H), 6.19

(s, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 142.94, 141.16, 136.74,

132.71, 131.61, 128.30, 127.80, 127.24, 126.61, 125.80, 124.24,

124.20, 120.01, 112.71, 52.00. MS (ESI) calcd for C₁₆H₁₁ClN₄,

m/z 294.07, found 317.06 (M þ Na^þ) and 612.11 (M þ M þ Na^þ).

Synthesis of Compound 56: 1-Benzyl-1H-[1,2,3]triazolo[4,5-

c]quinolin-4-amine. Compound 55 (46 mg, 0.16 mmol) was

dissolved in 0.1 mL of 2 M ammonia solution in MeOH, and

the reaction was heated in a microwave at 150 °C for 1 h. The

solvent was then removed under vacuum, and the residue was

purified using column chromatography (65% EtOAc/hexane)

to obtain the compound 56 (35 mg, 82%). ¹H NMR (500 MHz,

TFA) δ 8.25-8.13 (m, 1H), 7.87 (d, J = 7.2 Hz, 1H), 7.79 (d, J =

8.0 Hz, 1H), 7.63 (t, J = 6.1 Hz, 1H), 7.39 (s, 3H), 7.24 (s, 2H),

4464 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11

Shukla et al.

6.38 (d, J = 23.6 Hz, 2H). ¹³C NMR (126 MHz, TFA) δ 134.33,

133.27, 131.52, 129.18, 129.06, 127.08, 125.83, 123.58, 118.48,

117.54, 115.29, 113.04, 110.78, 54.87. MS (ESI) calcd for

C₁₆H₁₁N₅, m/z 275.12, found 276.12 (M þ H)^þ.

N. TLR2 engagement on CD8 T cells lowers the threshold for

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Biochim. Biophys. Acta 2002, 1589, 1–13.

Synthesis of Compound 57: 1-Benzyl-2-oxo-N-propyl-1H-

imidazo[4,5-c]quinoline-3(2H)-carboxamide. To a solution of

41 (30 mg, 0.12 mmol) in anhydrous THF were added triethyl-

amine (15 mg, 0.14 mmol) and n-propyl isocyanate (33 mg, 0.39

mmol); the n-propylamine released as a consequence of cycliza-

tion en route to the cyclic urea was found to consume n-propyl

isocyanate, necessitating the use of additional equivalents of the

reagent. The reaction was heated in microwave at 100 °C for 1 h.

The solvent was then removed under vacuum, and the residue

was purified using column chromatography (6% MeOH/

dichloromethane) to obtain the compound 57 (26 mg, 61%).

¹H NMR (500 MHz, CDCl₃) δ 9.90 (s, 1H), 8.79 (s, J = 5.4 Hz,

1H), 8.15 (d, J = 8.5, 0.6 Hz, 1H), 7.91 (d, J = 8.6, 0.6 Hz, 1H),

7.60 (t, J = 8.4, 6.9, 1.3 Hz, 1H), 7.40 (t, J = 8.3, 6.9, 1.2 Hz,

1H), 7.37-7.32 (m, 2H), 7.31-7.28 (m, 1H), 7.26 (d, J = 6.7, 5.4

Hz, 2H), 5.60 (s, 2H), 3.49-3.45 (m, J = 7.1, 5.8 Hz, 2H),

1.78-1.68 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H). ¹³C NMR (126

MHz, CDCl₃) δ 151.08, 148.67, 143.44, 136.25, 132.69, 128.54,

127.08, 127.02, 125.87, 125.74, 124.61, 123.68, 118.43, 117.87,

112.78, 44.45, 39.68, 20.57, 9.22. MS (ESI) calcd for C₂₁H₂₀-

N₄O₂, m/z 360.16, found 361.17 (M þ H)^þ.

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NF-KB Induction. The induction of NF-κB was quantified

using HEK-Blue-7 cells and HEK-Blue-8 cells as previously

described by us.^8,24HEK293 cells were stably transfected with

human TLR7 (or human TLR8), MD2, and secreted alkaline

phosphatase (sAP) and were maintained in HEK-Blue selection

medium containing zeocin and normocin. Stable expression of

secreted alkaline phosphatase (sAP) under control of NF-

κB/AP-1 promoters is inducible by the TLR7 (or TLR8) agonists,

and extracellular sAP in the supernatant is proportional to NF-

κB induction. HEK-Blue cells were incubated at a density of

∼10⁵cells/mL in a volume of 80 μL/well, in 384-well, flat-

bottomed, cell culture-treated microtiter plates until confluency

was achieved and, subsequently, graded concentrations of sti-

muli. sAP was assayed spectrophotometrically using an alkaline

phosphatase-specific chromogen (present in HEK-detection

medium as supplied by the vendor) at 620 nm.

IFN-r Induction in Whole Human Blood. Aliquots (3 mL) of

heparin-anticoagulated blood obtained from healthy human

donors after informed consent and stimulated for 12 h with

graded concentrations of test compounds. The plasma was

isolated by centrifugation, diluted 1:20, and IFN-R was assayed

in triplicate using a high-sensitivity human IFN-R-specific

ELISA kit (PBL Interferon Source, Piscataway, NJ).

Acknowledgment. This work was supported by NIH/

NIAID contract HHSN272200900033C.

Supporting Information Available: Characterization of inter-

mediates and final compounds (¹H, ¹³C, mass spectra). This

material is available free of charge via the Internet at http://

pubs.acs.org.

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Article Doi

Structure-activity relationships in human toll-like receptor 7-active imidazoquinoline analogues

DOI: 10.1021/jm100358c

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Article abstract of DOI:10.1021/jm100358c

Full text of DOI:10.1021/jm100358c

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