Discovery of GS-9688 (Selgantolimod) as a Potent and Selective Oral Toll-Like Receptor 8 Agonist for the Treatment of Chronic Hepatitis B

Article abstract of DOI:10.1021/acs.jmedchem.0c00100

Toll-like receptor 8 (TLR8) recognizes pathogen-derived single-stranded RNA fragments to trigger innate and adaptive immune responses. Chronic hepatitis B (CHB) is associated with a dysfunctional immune response, and therefore a selective TLR8 agonist may be an effective treatment option. Structure-based optimization of a dual TLR7/8 agonist led to the identification of the selective TLR8 clinical candidate (R)-2-((2-amino-7-fluoropyrido[3,2-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (GS-9688, (R)-7). Potent TLR8 agonism (IL-12p40 EC50 = 220 nM) and >100-fold TLR7 selectivity (IFN-α EC50 > 50 μM) was observed in human peripheral blood mononuclear cells (PBMCs). The TLR8-ectodomain:(R)-7 complex confirmed TLR8 binding and a direct ligand interaction with TLR8 residue Asp545. Oral (R)-7 had good absorption and high first pass clearance in preclinical species. A reduction in viral markers was observed in HBV-infected primary human hepatocytes treated with media from PBMCs stimulated with (R)-7, supporting the clinical development of (R)-7 for the treatment of CHB.

Full text of DOI:10.1021/acs.jmedchem.0c00100

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Drug Annotation
Discovery of GS-9688 (Selgantolimod), as a Potent and Selective Oral
Toll-like Receptor 8 Agonist for the Treatment of Chronic Hepatitis B
Richard L. Mackman, Michael Mish, Gregory Chin, Jason Perry, Todd C. Appleby, Vangelis
Aktoudianakis, Sammy Metobo, Peter Pyun, Congrong Niu, Stephane Daffis, Helen Yu,
Jim Zheng, Armando Garcia Villaseñor, Jeff Zablocki, Jason Chamberlain, Haolun Jin,
Gary Lee, Kimberly Suekawa-Pirrone, Rex Santos, William E. Delaney, and Simon Fletcher
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00100 • Publication Date (Web): 14 May 2020
Downloaded from pubs.acs.org on May 14, 2020
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Discovery of GS-9688 (Selgantolimod) as a Potent and Selective Oral Toll-Like Receptor 8
Agonist for the Treatment of Chronic Hepatitis B
Richard L. Mackman,†,* Michael Mish, Gregory Chin, Jason K. Perry, Todd Appleby,
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Vangelis Aktoudianakis, Sammy Metobo, Peter Pyun, Congrong Niu, Stephane Daffis,†
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Helen Yu, Jim Zheng, Armando G. Villasenor, Jeff Zablocki, Jason Chamberlain, Haolun
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Jin, Gary Lee, Kimberley Suekawa-Pirrone, Rex Santos, William E. Delaney IV, Simon P.
Fletcher†
†Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, California 94404, United States
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ABSTRACT
Toll-like receptor 8 (TLR8) recognizes pathogen-derived single stranded RNA fragments to
trigger innate and adaptive immune responses. Chronic hepatitis B (CHB) is associated with a
dysfunctional immune response, and therefore a selective TLR8 agonist may be an effective
treatment option. Structure based optimization of a dual TLR7/8 agonist led to the identification
of the selective TLR8 clinical candidate (R)-2-((2-amino-7-fluoropyrido[3,2-d]pyrimidin-4-
yl)amino)-2-methylhexan-1-ol (GS-9688, (R)-7). Potent TLR8 agonism (IL-12p40 EC =220
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nM) and >100-fold TLR7 selectivity (IFN-α EC >50 µM) was observed in human peripheral
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blood mononuclear cells (PBMCs). The TLR8-ectodomain: (R)-7 complex confirmed TLR8
binding and a direct ligand interaction with TLR8 residue Asp545. Oral (R)-7 had good
absorption and high first pass clearance in preclinical species. A reduction in viral markers was
observed in HBV-infected primary human hepatocytes treated with media from PBMCs
stimulated with (R)-7, supporting the clinical development of (R)-7 for the treatment of CHB.
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TOC GRAPHIC
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OH
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PBMC IL-12p40 EC50 = 1.9 M
PBMC IFN- EC50 = 2.2 M
PBMC IL-12p40 EC50 = 0.22 M
PBMC IFN- EC50 >50 M
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INTRODUCTION
Toll-like receptors (TLRs) are a family of membrane-bound molecular pattern
recognition receptors that play a central role in the detection of molecular fragments from
microorganisms. Activation of the TLRs leads to the initiation of innate and adaptive immune
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responses. Human TLR8 is located on the endosomal membrane of a subset of immune cells,
notably myeloid dendritic cells, monocytes, macrophages, and neutrophils, and recognizes single
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stranded RNA (ssRNA) fragments. A closely related toll-like receptor, TLR7 is also located on
the endosome membrane, and similarly recognizes ssRNA, but in contrast to TLR8, is
predominantly expressed in plasmacytoid dendritic cells and B cells. Importantly, this
orthogonal cell-specific expression pattern drives disparate immune responses upon either TLR7
or TLR8 receptor activation. TLR7 agonism predominantly induces the production of interferon-

(IFN-), whereas TLR8 agonism stimulates the production of a variety of immunomodulatory
and pro-inflammatory cytokines including interleukin-12 (IL-12), IL-18 and tumor necrosis
factor- (TNF-).
Chronic hepatitis B (CHB) is a major global health care challenge and one of the main
causes of chronic liver disease, cirrhosis and hepatocellular carcinoma. An estimated two billion
people worldwide have been acutely infected with hepatitis B (HBV), of which >250 million
have developed CHB, and an estimated 887,000 die annually from complications associated with
CHB.3,4 CHB is characterized by a dysfunctional or exhausted immune response and therefore it
is hypothesized that TLR8 activation with a small molecule agonist could stimulate a host
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immune response and lead to an effective new treatment option for CHB. In vitro, TLR8 small
+
molecule agonists have been shown to stimulate a CD8 T cell response to HBsAg, a viral
protein secreted from HBV-infected hepatocytes, and the TLR8-induced immunomodulatory
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cytokine, IL-12, has also been shown to rescue the antiviral function of exhausted HBV-specific
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CD8 T cells. IL-12 and IL-18 also strongly activate cytolytic and non-cytolytic functions of
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natural killer cells and mucosal-associated invariant T-cells. In addition, TLR8 strongly induces
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TNF-, which has been shown to drive intrahepatic CD8 T cell expansion and inhibit HBV in
an adenovirus mouse model.9 Finally, TLR8 induced cytokines including IFN-, IL-1 and
TNF-, have direct antiviral properties against HBV in vitro.10-12 Taken together, these data
show that TLR8 engages multiple arms of the immune system, and produces antiviral cytokines
that have been shown to inhibit HBV in vitro and in vivo.
Our aim was to design an oral, selective small molecule agonist of TLR8 with good
absorption and high first-pass hepatic clearance as a rational approach to induce effective pre-
systemic antiviral immunity in CHB patients. Activation of TLR8 expressing immune cells in the
gut following oral administration would be expected to result in the secretion of immune
mediators into the portal vein that will in turn, stimulate immune cells in the liver, the organ of
HBV infection. In addition, intestinal absorption of the agonist and drug exposure in the liver
should lead to direct effects on intrahepatic TLR8-expressing immune cells. Provided the
compound demonstrates high first pass hepatic clearance in hepatocytes, systemic exposure of
the agonist would be minimized, thereby reducing undesirable side effects from systemic TLR8
agonism. TLR7 agonism with oral small molecule agents has also been hypothesized to be
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beneficial for CHB treatment and led to the discovery of vesatolimod from this group. The
design of a selective TLR8 agonist would therefore afford the flexibility to modulate both
pathways independently and allow for a combination approach if applicable. This is especially
important because the relative contributions of oral TLR7 and TLR8 agonism, and their disparate
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immune responses to the treatment of CHB, or indeed, tolerability limitations, were largely
unknown at the time.
The co-crystal X-ray structure of TLR8 ectodomain with the dual TLR7/TLR8 agonist
3M-002 (1, Figure 1) was recently reported and enabled a rational approach to design novel
TLR8-selective agonists.14 Compound 1 has marginal selectivity for TLR8, and is structurally-
related to the TLR7 agonist imiquimod which was approved in the 1990s for the treatment of
genital warts.15,16 In contrast, Compounds 2 and 3 (VTX-2337), that contain the 2-
aminoquinoline or 2-aminobenzazepine bicyclic cores have increased selectivity for TLR8 .17,18
These scaffolds illustrate that the tricyclic core of 1 can be truncated to a quinoline or
benzoazepine bicyclic heterocycle and yet retain TLR8 potency. During the course of this
program additional novel TLR8 agonists were reported in the literature including the monocyclic
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,4-diaminopyrimidines 4 and 5.19,20
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Figure 1. TLR8 agonists
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NH2
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6a
(R)-7, GS-9688
The pyrido[3,2-d]pyrimidine heterocycle and its preparation was familiar to this group
due to its earlier inclusion in compounds that demonstrated promising anti-HCV activity.21
Based on the same heterocycle, 6a was identified as a moderately potent dual TLR7/8 agonist
lead. Optimization of the C-4 and C-7 substituents on the heterocycle led to the identification of
the TLR8 selective clinical candidate (R)-7 (GS-9688, selgantolimod). The structure-activity
relationships that led to the identification of (R)-7 as a selective TLR8 agonist are described in
addition to the preclinical pharmacokinetic properties that demonstrate pre-systemic exposure
and minimal oral bioavailability due to high first pass clearance. The inhibition of viral
parameters in hepatitis B infected primary human hepatocytes (PHH), combined with the
previously reported antiviral efficacy in the woodchuck model of CHB, support the development
of (R)-7 as an orally delivered, first in class, selective TLR8 agonist for the treatment of CHB.22
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RESULTS AND DISCUSSION
A high throughput multiplex assay was developed, that quantified the levels of IL-12p40
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(TLR8 cytokine) and IFN-α (TLR7 cytokine) in the supernatant of human PBMCs. Provided IL-
12p40 induction was >50% of the TLR8 selective control compound 3, a concentration for 50%
maximal induction (EC ) of this cytokine was calculated from the computationally fitted dose
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response curve. IFN-α induction values were calculated in a similar manner using the TLR7
agonist, vesatolimod, as the positive control. A single PBMC donor was used consistently during
the SAR iteration to reduce assay variability. Human embryonic kidney (HEK) 293 reporter cell
lines stably expressing human TLR8 or TLR7 have also been reported in the literature as a
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primary screening tool for the identification of TLR7 or TLR8-selective agonists. However, an
advantage of the robust high throughput PBMC assay developed here is that both TLR7 and
TLR8 responses could be assessed in the same assay using non-transformed cells that do not
overexpress the TLR receptors. Therefore a more biologically relevant functional selectivity
window could be calculated based on the respective IL-12p40 and IFN-α responses in a single
assay.
The active TLR8 homodimer has two equivalent agonist binding sites at the interface of
the ectodomain monomers. A common feature for all the TLR8 agonists shown in Figure 1 is the
presence of a weakly basic heterocyclic nitrogen, with a range of weakly basic pKa values e.g.
pKa = 6.3 and 7.3 for 1 and 3 respectively. In the low pH ~4-6 environment of the endosomal
compartment where TLR8 is functional, this weakly basic group is protonated. The co-crystal
X-ray structure of the TLR8-ectodomain homodimer:1 complex indicates the weakly basic group
forms an ionic interaction with Asp543 of one TLR8 monomer (Chain A, Figure 2A). The
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lipophilic propyl chain extends into a narrow but deep hydrophobic pocket formed by residues
Gly572, and Val573 from chain A, and Phe346, Tyr348, Gly376, Val378, Ile403, and Phe405
from chain B of the second TLR8 monomer. All the compounds in Figure 1 have a lipophilic
group that likely occupies this pocket illustrating its importance for binding of small molecule
agonists. The lipophilic pocket of TLR7 based on homology modeling was expected to be very
similar to that of TLR8. Thus, the major structural differences between TLR7 and TLR8 within
the ligand binding site, are residues Gly351 (Chain B) and Asp545 (Chain A), located at the front
of the pocket (Figure 2A). These residues correspond to Gln354 and Leu557, respectively, in
TLR7. Lead 6a was modeled into the binding site of TLR8 to establish the same charged
interaction with Asp543, and orientate the N-alkyl sidechain into the lipophilic pocket (Figure
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B). The model also suggested that the α-carbon of the alkyl sidechain was the most proximal
atom from which to explore substitutions directed toward the front of the pocket in an attempt to
engage Asp545 of TLR8. It was reasoned that polar groups could lead to a water mediated or
direct hydrogen bond to Asp545 in TLR8, but would be unfavorable toward TLR7, due to
presence of the lipophilic Leu557 sidechain, thereby generating TLR8 selectivity.
Figure 2. A. TLR8-Ectodomain:1 X-ray complex with protons added to the ligand by modeling.
Lighter green shading indicates Chain A surface of one TLR8 monomer and darker green is
Chain B of the second TLR8 monomer; B. Model of 6a; C. Model of (R)-6d with a bridging
water hydrogen bond to Asp545; D. Model of (R)-6d with a direct hydrogen bond to Asp545; E.
Cross section view of (R)-6d occupying the lipophilic pocket; F. Model of (S)-6d with a bridging
water hydrogen bond to Asp545. All modeled compounds in B-F were docked into the TLR8 x-
ray structure, PDB:3W3M
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Scheme 1 illustrates the preparation of a variety of C-4 modified pyrido[3,2-d]pyrimidine
compounds starting from the commercially available 2, 4-dichloropyrido[3,2-d]pyrimidine 8.
Nucleophilic addition of butan-1-ol or amines 11a-h at C-4 was followed by addition of 2, 4-
dimethoxybenzyl (Dmb) amine at C-2 to afford the intermediates 9 and 12a-h. Acid
deprotection of the Dmb group afforded the target compounds 10 and 6a-f. Ester 12g was
reduced to the alcohol before Dmb removal to yield (R)-6g, and ester 12h was first hydrolysed to
the acid followed by Dmb removal to afford (R)-6h. The amides (R)-6i and (R)-6j were
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generated from the above acid intermediate using a HATU mediated coupling prior to Dmb
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
4
a
Scheme 1. Synthesis of N - and O -substituted pyrido[3,2-d]pyrimidin-2-amines
Cl
R
N
R1
N
NH2
N
Cl
11a R = H, R1 = H
1
1b R = CH , R = H
8
3
1
(
R)-11c R = CH OH, R = H
2 1
(
S)-11c R = CH2OH, R1 = H
(R)-11d R = CH OH, R = CH3
2 1
(i)-(ii)
11a-c (iv)-(v)
11d-f
(iv)-(v)
11g-h
R1
(iv)-(v)
HO
(S)-11d R = CH2OH, R1 = CH3
R)-11e R = CH2CH2OH, R1 = CH3
(
(R)-11f R = CH2NPhth, R1 = CH3
(R)-11g R = CO Me, R = CH CH
3
R
2
1
2
OMe
O
N
HN
R
HN
N
HN
N
(R)-11h R = CO Me, R = CH3
2 1
N
N
N
N
O
N
N
N
N
NHDmb
N
NHDmb
NHDmb
NHDmb
9
12a-c
12d-f
(vi)
12g R1
CH2CH3
(vii), (viii)
=
12h R1 = CH3
(
ix), (x) for (R)-6h
(
iii)
(vi)
HN
(ix), (xi), (xii) for (R)-6i/j
R
OH
R
O
R
HN
N
HN (R)
HN (R)
N
N
N
N
N
O
N
N
N
N
N
N
N
NH2
N
NH2
NH2
NH2
N
NH2
10
6a R = H
b R = CH3
R)-6c R = CH OH
(R)-6d R = OH
(S)-6d R = OH
(R)-6e R = CH2OH
(R)-6f R = NH2
(R)-
6g
(R)-6h R = OH
(R)-6i R = NH2
(R)-6j R = NHMe
6
(
2
(S)-6c R = CH2OH
aReagents and Conditions. (i) Butan-1-ol, NaH (60% disp.), 71%; (ii) 2,4-
dimethoxybenzyl(Dmb)-NH , iPr NEt, 100°C, 37%; (iii) TFA, 9%; (iv) amine 11a-h, iPr NEt;
2
2
2
rt, (100°C for (R)-11e); (v) Dmb-NH , iPr NEt, 100-135°C, 52% for 12g, 68% for 12h; (vi)
2
2
TFA, 11-73% over 3 steps; (vii) LAH; (viii) TFA, 5% over 2 steps; (ix) KOH(aq.), MeOH,
quant.; (x) TFA, quant.; (xi) HATU, iPr NEt, NMP, ammonia (14%) or methylamine (18%);
2
(xii) TFA, 22-42%.
4
4
Table 1. TLR activity of N - and O -substituted pyrido[3,2-d]pyrimidin-2-amines
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
R
N
N
N
NH2
IL-12p40
IFN-α
Cpd.
R
EC50
EC50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
a
(µM)
(µM)
1
-
-
4.9
18
(
3M-002)
3
1.3
>50
1.9
>30
>50
2.2
(
VTX 2337)
H
1
0
O
H
6a
NH
6
b
3.1
5.1
NH
(
R)
OH
(
R)-6c
S)-6c
R)-6d
NH
19
>50
2.00
>50
0.91
>50
>50
>20
>50
>50
>50
(
S)
OH
(
NH
0.43
0.79
0.42
4.3
(R)
OH
(
NH
(S)
OH
(S)-6d
NH
OH
(R)
(
R)-6e
R)-6f
R)-6g
R)-6h
(R)-6i
R)-6j
aAverage of n ≥ 2 results
NH
(R)
NH2
(
NH
25
(
R)
OH
(
3.33
>50
36
NH
O
(
(R)
OH
NH
O
(R)
NH2
NH
O
(
>50
(R)
N
H
NH
The induction of IL-12p40 (TLR8 cytokine) and IFN-α (TLR7 cytokine) in PBMCs for
4
compounds 6a-j is reported in Table 1. The lack of TLR activity for O -linked analog 10
established that the corresponding amine linkage in 6a was critical, and the similar potency of
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the α-branched analog, 6b, indicated that α-carbon branching was tolerated by both TLRs.
Further extension of the α-branching group by the addition of a hydroxyl group resulted in 5-fold
improved TLR8 potency and 5-fold selectivity for (S)-6c toward TLR8, whilst the corresponding
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(R)-6c isomer was 10-fold less potent toward TLR8. A significant advance was observed for
isomers (R)-6d and (S)-6d that extend the α-alkyl chain to 4 carbons from the branch point. The
(
S)-6d isomer had comparable potency and selectivity to the shorter chain analog (S)-6c, but the
R)-6d isomer was > 20-fold more potent than (R)-6c toward TLR8 and had no TLR7 mediated
(
IFN-α induction up to 50 µM, leading to the first selective TLR8 agonist in the series. Extending
the α-alkyl chain with another methylene, analog (R)-6g, reduced potency while extending the
hydroxyl group further from the branch point by one methylene, analog (R)-6e, was also less
potent. Modification of the hydroxyl in (R)-6d to an amine, (R)-6f, or other groups capable of
hydrogen bond donation e.g. 6h-j, afforded only weak activity. Taken together, the SAR
established that the length and R stereochemistry of the lipophilic sidechain in (R)-6d, together
with the hydroxyl group were all required for optimal TLR8 potency and selectivity.
Modeling was used in an attempt rationalize the TLR8 potency SAR and also the
differential selectivity toward TLR7 of the two isomers (R)-6d and (S)-6d. One pose for the (R)-
6
d analog in TLR8 (Figure 2C) suggested the hydroxyl was interacting via a water molecule
4
with Asp545 and the N -alkyl group was rotated 180° relative to its orientation in 6a. A second
4
pose (Figure 2D) maintained the orientation of the N -alkyl group relative to 6a, but led to the
displacement of the bridging water molecule and the formation of a direct hydrogen bond to
Asp545. Both possibilities were considered, but it wasn’t until an x-ray co-crystal structure of
compound (R)-7 was solved that the direct hydrogen bond model was deemed to be correct (vide
infra). Figure 2E shows a cross section of the lipophilic pocket with the 4 carbon chain of (R)-6d
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
fully occupying the space in the lipophilic pocket. The total distance from the end of the 4
carbon chain to the hydroxyl group is ideal to occupy the lipophilic pocket whilst also enabling
the hydroxyl group to interact with Asp545. However, the shorter 3-carbon (R)-6c and longer 5-
carbon (R)-6g lengths are suboptimal. The 5-fold weaker activity of (R)-6e, which has an
identical length to (R)-6d, is presumed to be a combination of incomplete occupancy of the
lipophilic pocket from the α-branch point and the greater entropic penalty of the more flexible
hydroxyethyl group that forms the key interaction with Asp545. The (S)-6d isomer was modeled
in TLR8 in a pose similar to 6a and is presumed to derive its TLR8 potency from a similar water
mediated interaction with Asp545 (Figure 2F). However, the fact that (S)-6d and indeed the
shorter (S)-6c analog had comparable TLR7 and TLR8 potency is harder to rationalize, but does
suggest that other binding poses are present in the TLR7 binding site that allow these S-isomers
to generate favorable interactions with Leu557 despite the presence of the hydroxyl group. More
thorough X-ray studies using TLR7 and TLR8 constructs will be required to tease out the
binding modes of the S isomers in TLR7, the feasibility of which has now increased given the
recent publication of TLR7 x-ray small molecule complex structures.23
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The pyrido[3,2-d]pyrimidine heterocycle forms a π-stacking interaction with the aromatic
ring of Phe405 (Figure 2A) so it was of interest to explore the effects of alternate heterocycle
cores. The corresponding quinazolines of both 6d isomers, (R)-14 and (S)-14, and quinoline
isomers, (R)-16 and (S)-16, were therefore prepared according to Scheme 2. The quinazoline
2
4
analogs have recently been shown to be TLR8 selective in HEK reporter assays. Commercial
13 was converted to isomers 14 via a BOP mediated coupling with the amine isomers 11d, while
the corresponding quinolines 16 were prepared in a palladium cross coupling of quinoline 15
with amine isomers 11d.
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a
Scheme 2. Synthesis of N -substituted quinazoline-2-amines, 14, and quinoline-2-amines, 16
OH
OH
HN
(i)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
11d
N
NH2
N
NH2
13
(R)-14
S)-14
(
Cl
OH
HN
(ii)
11d
N
NH2
N
NH2
15
(R)-16
(
S)-16
a
t
Reagents and Conditions. (i) DMF, DBU, BOP, 11d, 62-71%; (ii) Pd(dba) , NaO Bu, (R)-
2
BINAP, 11d, 135°C, 7-16%.
4
Table 2. TLR activity of N -substituted quinazolin-2-amines and quinolin-2-amines
IL-12p40
IFN-α
EC50 (µM)
Cpd.
Structure
a
a
EC50 (µM)
(
R)-14
1.84
0.71
>50
>50
>50
0.57
>50
>50
OH
HN
N
N
(
S)-14
(R)-16
S)-16
NH2
OH
HN
N
(
NH2
aAverage of n≥2 results
The quinazoline isomers 14 had parallel SAR to their corresponding pyrido[3,2-
d]pyrimidin-2-amines albeit 2-fold less potent in each case toward TLR8 (Table 2). In contrast
removal of the additional nitrogen in the core, quinolines 16, resulted in complete loss of
activity. The measured pKa for (R)-6d was 7.0, 8.2 for quinazoline (R)-14, and calculated to be
2
5
even higher for the quinoline (R)-16 , thus protonation state in the endosome was not the cause
of lower activity. This loss of activity for the quinoline (R)-16 is also surprising in light of the
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
observation that quinoline 2, is a potent TLR8 agonist, and suggests that the 2-aminoquinoline
core binds differently to these other cores in the TLR8 pocket leading to suboptimal positioning
4
of the N -group into the lipophilic pocket.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cytotoxicity screening of the TLR8 selective lead (R)-6d indicated a narrow cell-based
toxicity index, ~10-fold (MT-4 CC /IL-12p40 EC ) in the rapidly proliferating MT-4 cell line
5
0
50
due to ‘off-target’ effects. Continued optimization was therefore conducted and focused on
exploring substitution at the remaining α-carbon position leading to α, α-substituted analogs. In
the models of (R)-6d and (S)-6d (Figures 2C, 2D) there is a small unoccupied space near the
unsubstituted α-site. To support this effort several chiral α, α-substituted amino acid esters and
alcohols were required and their preparation is described in Scheme 3. The amino acid esters
18a-b and 18g were generated from commercially available unnatural amino acids through
esterification of the acid followed by protecting group removal as needed. The tertiary amino
alcohol (S)-18c was derived from the acid (S)-19 via a borane reduction, and alcohols (R)-18c-f
26
by stereospecific synthesis from the 5,6-dihydro-2H-1,4-oxazin-2-one intermediate (R)-20. The
oxazin-2-one route not only facilitated control of stereochemistry, but also allowed for rapid
generation of amino alcohols with modified alkyl chains e.g. (R)-18f.
Scheme 3. Stereoselective synthesis of α, α-substituted amino acid esters and alcoholsa
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R
R
R1
O
OH
OH
XHN
NH2
O
(
(
R)-17a X = FMoc, R = CH2CH=CH2
R)-17c X = H, R = (CH2)3CH3
1 2 3 3
17b R = R = (CH ) CH
(
(
i), (ii) for (R)-17a
i) for (R)-17c
(i)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R
R1
O
OMe
NH2
(R)-18a R = CH2CH=CH2, R1 = CH3
8b R = R1 = (CH2)3CH3
R)-18g R = (CH2)3CH3, R1 = CH3
1
(
(iii)
OH
OH
NH2
NH2
O
(S)-19
(S)-18c
Ph
Ph
O
R
(iv)
(v)-(vi)
OH
N
HN
H2N
O
O
R
O
(
R)-20
21a R = (CH ) CH
3
(R)-18c R = (CH2)3CH3
(R)-18d R = (CH2)4CH3
(R)-18e R = (CH2)5CH3
(R)-18f R = (CH2)3OH
2
3
2
2
2
1b R = (CH2)4CH3
1c R = (CH2)5CH3
1d R = (CH2)4OBn
a
Reagents and Conditions. (i) TMSCHN , MeOH, quant.; (ii) Piperidine, THF, 85% over 2
2
steps; (iii) BH ·THF; (iv) BF ·OEt , -78°C, RMgCl, 60-76% (v) LiBH , 0°C-rt, quant.; (vi)
3
3
2
4
Pd(OH) /C, EtOH, HCl, H , 70-75°C, quant.
2
2
The subsequent coupling of the α, α-substituted amines to the pyrido[3,2-d]pyrimidine-2-
core 8 is described in Scheme 4. Unlike the mono α-substituted series the more hindered α, α-
substituted amines typically required elevated temperatures in the nucleophilic substitution
reaction. In examples where Dmb protecting groups were present, their removal proceeded as
described previously, along with any ester or carbon-carbon double bond reductions, to afford
23a-e. The same methods were also applicable to the nucleophilic substitution on the 7-F and 7-
Cl heterocycle intermediates, 25 and 27 respectively. 7-Chloro intermediate 28 could also be
converted to the 7-methyl analog (R)-30 using methylboronic acid in a Suzuki cross coupling.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Finally, preparation of (S)-7 illustrates, an alternative approach to 7-fluoro analogs via a simple
two step method from readily available fluoropicolinic acid 31.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
a
Scheme 4. Synthesis of N -α, α-substituted pyrido[3,2-d]pyrimidin-2-amines
Cl
Cl
N
N
Br
N
CO2H
NH2
N
N
N
N
F
NH2
F
N
Cl
Cl
Cl
24
27
31
8
(
i)-(iii) 18a-b
18c-e
(i)-(ii), (v)
(vi)-(ix))
(i)-(ii)
(xii)
(R)-18c
R
R1
R
OH
OH
HN
HN
OH
N
N
Cl
N
OH
N
N
HN
N
N
N
N
N
N
N
NHDmb
N
NH2
N
F
N
NH2
F
Cl
Cl
NHDmb
(
(
(
R)-23c R = (CH2)3CH3
S)-23c R = (CH2)3CH3
R)-23d R = (CH2)4CH3
(
R)-22a R = CH2CH=CH2, R1 = CH3
22b R = R1 =(CH2)3CH3
25
28
32
(
i)-(ii), (v)
R)-18c/f/g
(v) for (R)-29
(xiii)
(S)-
18c
(R)-23e R = (CH2)5CH3
(xi), (v) for (R)-30
(
(
iv), (v) for (R)-23a
v) for 23b
(
R1
R
R1
OH
OH
OH
HN
HN
N
R
HN
N
HN
N
N
N
N
N
N
N
N
F
NH2
R
NH2
F
N
NH2
N
NH2
(R)-23a R = CH2CH2CH3, R1 = CH3
3b R = R1 = (CH2)3CH3
(S)-7
(
R)-7 R = CH2OH, R1 = H
(R)-29 R = Cl
(R)-30 R = Me
CO2H
OH
OH
2
O
(R)-26a R = CH2OH, R1 = OH
(R)-26b R = CO2H, R1 = H
(R)-26c R = CH2O-glu, R1 = H
glu =
(x)
HO
a
Reagents and Conditions. (i) amine 18a-e, iPr NEt, 80°C; (ii) Dmb-NH , iPr NEt (K CO for
2
2
2
2
3
(R)-23e and (R)-26a), 85-120°C, 17-66% over 2 steps; (iii) LAH, 29-100%, (over 2 steps, (R)-
2
3a (45%) and (R)-30 (82%); over 3 steps (S)-23c (26%) and (R)-23d (7%)); (iv) Pd/C, H ;(v)
2
TFA, 7%-quant.; (vi) Pd(Ph ) , ZnCN , DMF, 115°C, quant.; (vii) K CO , H O , 0°C, 97% over
3
4
2
2
3
2
2
2
steps; (viii) (COCl ) , 110°C, 90%; (ix) PCl , POCl , 110°C, 75%; (x) Fermentation; (xi)
2 3 5 3
CH B(OH) , Pd(Ph ) , K PO , 150°C; (xii) ClC(=NH)-NH , (CH ) SO , 160°C, 24%; (xiii)
3
2
3 4
3
4
2
3 2
2
NMP, BOP, DBU, (S)-18c, 20%.
4
Table 3. N -α, α-substituted pyrido[3,2-d]pyrimidin-2-amines
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R
N
N
X
N
NH2
Caco-2
MS r/d/c/h
CL pred.
(L/h/kg)
IL-12p40
EC50 (µM)
IFNα
MT-4
CC50
Log
D
-6
Cpd.
R
X
pKa
(10
a
EC50 (µM)a CC50 (µM)a Index
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
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0
1
2
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5
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9
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1
2
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5
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7
8
9
0
1
2
3
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
-1
cms )
(R)
OH
OH
H
H
H
H
H
H
H
F
0.79
2.4
>50
>50
>50
15
7.8
>50
38
10
>21
-
6.8
-
-
-
(
R)-6d
NH
(
R)-23a
3b
(R)-23c
S)-23c
R)-23d
R)-23e
S)-7
(R)-7
R)-26a
R)-26b
R)-26c
R)-29
(R)-30
-
1.1
2.9
1.4
1.4
1.8
2.5
-
-
-
(
R)
NH
2
>50
0.17
2.4
-
-
-
OH
NH
(R)
OH
31
182
11
29
0.4
0.2
77
-
-
-
29/29
3.5/1.4/1.3/0.8
NH
(
OH
OH
7.9
26
32/31
3.5/1.5/1.4/0.8
(S)
NH
(
(R)
0.58
10
20
17
-
-
-
-
-
-
-
NH
OH
(
(R)
>50
>50
>50
>50
>50
>50
>42
>50
3.6
0.79
17
-
NH
(
OH
3.3
-
(S)
NH
(R)
OH
OH
F
0.22
>50
>50
>50
0.27
0.16
6.3
-
2.0
-
7.9/11 3.0/1.7/1.5/1.0
NH
HO
(
(R)
F
>50
>50
>50
22
-
-
NH
O
(
F
-
-
-
-
-
(R)
OH
NH
Glu
(
(R)
O
F
-
-
-
-
-
NH
(
OH
OH
Cl
81
144
6.4
7.1
2.6
1.9
32/31
31/29
3.4/1.8/1.4/1.0
3.8/1.1/1.5/0.5
(R)
NH
M
e
23
(R)
NH
a
Average of n≥2 results. Cytotoxicity (CC50) index, defined as MT-4 CC50 / IL-12p40 PBMC EC50; AB, apical to
basolateral; BA, basolateral to apical; MS, microsomes; r, rat; d, dog; c, cyno; h, human; CL pred., predicted
clearance.
The α, α-substituted analogs 23a-e were evaluated across a wide range of assays
including TLR8 potency and selectivity, cytotoxicity index CC (MT-4 CC / IL-12p40 EC ),
5
0
50
50
pKa, log D, caco-2 cell permeability and in vitro microsomal metabolism (Table 3). The α-
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methyl analog (R)-23c, not only improved the TLR8 potency by 5-fold compared to the
corresponding mono α-substituted analog (R)-6d, but also retained >80-fold selectivity over
TLR7, and improved the MT-4 cytotoxicity window to 182-fold. The S isomer, (S)-23c was
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10-fold less active toward TLR8 than (R)-23c and less selective over TLR7 reinforcing the
preference for the R isomer series. Like the previous mono α-substituted series, extending the α-
alkyl chain analogs, (R)-23d/e, or shortening, (R)-23a, was less potent although the one
methylene extended (R)-23d was only 3-fold less potent. The achiral analog 23b was devoid of
activity suggesting the second α-substitutent needs to be sterically small, consistent with the
modeling prediction.
Turning attention to the pharmacokinetic properties, the low molecular weight, moderate
logD, and good solubility in aqueous media resulted in excellent passive permeability in caco-2
cells for both (R)- and (S)-23c with no apparent efflux ratio (efflux ratio = BA/AB). High first
pass clearance was predicted using microsomal incubations for both 23c isomers from multiple
preclinical species. Metabolite identification studies in hepatocytes indicated extensive
heterocycle core metabolism which raised the concern that some of the oxidized core metabolites
could be immunologically active in vivo, leading to a convoluted in vivo profile. Therefore, in
an attempt to suppress core metabolism, the core was modified at several positions of which the
most favorable was 7-substitution, analogs (R)-7 (7-F), (R)-29 (7-Cl) and (R)-30 (7-Me)
respectively. The pKa of the heterocycle nitrogen was lowered by only 0.5 units for the 7-F and
7
-Cl analogs to a range of 6.3-7.1, higher than the endosome, and therefore not expected to
impact potency (Table 3). Indeed, all three analogs demonstrated comparable potency to the 7-
unsubstituted analog (R)-23c, but TLR8 selectivity improved to >185 fold, and the CC index in
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clearance across species was noted except for a lower predicted human clearance of the C7-
methyl analog (R)-30. The risk of potentially lower first pass clearance for this analog was
sufficient to rule it out of contention for selection. Metabolite identification following incubation
of (R)-7 in human hepatocytes indicated that core oxidation had indeed been suppressed and the
most dominant metabolites were lipophilic chain oxidation, hydroxyl oxidation to the acid, and
glucuronidation. Although the specific site of oxidation on the lipophilic chain was not
rigorously identified, the SAR for small changes along this chain was extremely narrow, and
even small changes such as fluorine led to weaker activity (data not shown). Although it was
extremely unlikely oxidation along the four carbon chain would result in an active metabolite,
the terminal hydroxyl (R)-26a was prepared and tested as one potential metabolite to verify lack
of TLR7/8 activity (Table 3). The acid (R)-26b was also prepared and found to be inactive to
both TLRs, as was the O-glucuruonide (R)-26c that was structurally confirmed by NMR
following isolation from microbial fermentation mixtures. Compound (R)-7 sufficiently de-
risked the potential metabolite concerns and was more potent than (R)-29 so it was selected for in
vivo evaluation. In each non-clinical species, high clearance and low oral bioavailability was
observed consistent with the in vitro prediction of high first pass metabolism (Table 4). A bile
duct cannulated study in rat detected minimal parent compound in bile and urine, confirming
metabolism was the dominant mechanism of clearance in rat. Oral absorption was confirmed
through analysis of portal vein samples in dog and cynomolgus monkey following oral dosing,
and these data indicated at least 28% absorption in dog and ~100% in cynomolgus monkey.
Taken together, the in vitro predictions of good oral absorption and high first pass clearance were
confirmed in vivo in the non-clinical species. The predicted first pass clearance of (R)-7 in
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humans is high, close to that of liver blood flow, so (R)-7 was expected to be a well absorbed
high clearance compound in humans meeting the desired target profile (Table 3).
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0
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Table 4. Mean plasma pharmacokinetic parameters of (R)-7 following IV and oral
administration
IV1
Oral2
CL
L/h/kg)
V
(L/kg)
ss
t
½
AUCinf
(nM•h)
C
max
(nM)
Species
F
(
(h)
Sprague Dawley Rat
Beagle Dog
1.8 ± 0.2
1.2 ± 0.13
6.2 ± 1.8
1.1 ± 0.0
1.01 ± 0.13
2.1 ± 1.5
1.45 ± 0.12 827 ± 540 517 ± 224 8.8% ± 5.7%
1.32 ± 0.18 11.8 ± 7.3 9.7 ± 7.7 0.8% ± 0.5%
0.67 ± 0.07 4.7 ± 4.2 1.2 ± 0.5 0.3% ± 0.2%
Cynomolgus Monkey
130-min intravenous infusion to Sprague Dawley (SD) rats (1 mg/kg), beagle dogs (0.25 mg/kg) and cynomolgus
2
monkeys (0.1 mg/kg) (mean ± SD, n = 3). Oral administration to SD rats (5 mg/kg), beagle dogs (0.5 mg/kg) and
cynomolgus monkeys (3 mg/kg), formulation was 2% ethanol, 40% PEG 300 and 58% 0.01N HCl.
Evidence for direct binding of (R)-7 to TLR8 was attained by solving the ectodomain
TLR8:(R)-7 co-crystal structure (Figure 3). The structure indicates that the compound binds with
a direct hydrogen bond to Asp545, the basic group interacts with Asp543, and the lipophilic N4-
alkyl chain occupies the narrow lipophilic pocket. However, Asp545 shifted slightly relative to
the published structure of 1 to provide a more optimal direct hydrogen bond to the hydroxyl
group of (R)-7. This supports the premise that the Asp545 interaction is the key driver of TLR8
potency and selectivity. The α-methyl group is directed to the top of the binding site and nicely
occupies the space proximal to Tyr348, Val378 and Gly351 which results in increased binding
potency.
Figure 3. Single co-crystal structure of TLR8 ectodomain:(R)-7 complex. PDB Code: 6WML
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The broader selectivity profile of (R)-7 toward immune receptors was assessed in reporter
gene assays using HEK293 cells, stably expressing individual human TLRs or other innate
immune receptors. The minimum effective concentration (MEC) of (R)-7 that resulted in a 3-
fold increase above background was 49 nM for human TLR8 (Figure 4). In contrast, the MEC
for human TLR7 was 5197 nM further supporting a mechanism of selective engagement of (R)-7
on human TLR8 over human TLR7. Moreover, (R)-7 did not stimulate HEK293 cells stably
expressing other human TLRs including TLR2, TLR3, TLR4, TLR5, TLR9 and other receptors
including Nod-like receptors (NLRs; NOD1 and NOD2), RIG-I -like receptors (RLRs; RIG-I
and MDA5) or C-type lectin receptors (CLRs; Dectin1a, Dectin 1b and Mincle) (data not
shown). This data, taken together with the structural data, supports the direct and selective
engagement and activation of TLR8 by (R)-7.
Figure 4. TLR8 and TLR7 HEK293 reporter assay response to (R)-7
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3
.5
Human TLR8
Human TLR7
3.0
2.5
2
1
1
0
.0
.5
.0
.5
0
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0.0
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0.001 0.01 0.1
(R)-7 (M)
1
10
In HBV-infected PHH viral RNAs are transcribed from the viral genome (covalent closed
circular DNA, cccDNA) leading to the expression of the viral proteins e.g. HBeAg and HBsAg.
Direct stimulation of HBV infected PHH with (R)-7 did not result in any antiviral effects
consistent with the lack of robust TLR8 expression in human hepatocytes (data not shown). In an
indirect experiment, designed to assess the antiviral effect of TLR8 induced immune mediators
generated in PBMCs, HBV-infected PHH were treated with media from human PBMCs
stimulated with (R)-7 ((R)-7-CM). Exposure of HBV-infected PHH to (R)-7-CM over 10 days
resulted in a decrease in HBV, DNA HBV RNA, HBeAg and HBsAg levels by >50% relative to
no compound control (Figure 5). However, (R)-7-CM did not reduce levels of cccDNA
demonstrating that, in this in vitro system, TLR8-induced cytokines are insufficient to induce
non-cytolytic degradation of cccDNA (data not shown).
Figure 5. Antiviral activity in HBV-infected PHH treated with (R)-7-CM.
1
1
20
00
HBV RNA
HBV DNA
HBeAg
8
6
4
2
0
0
0
0
0
HBsAg
Cell Viability
Mock-CM
(R)-7-CM
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The woodchuck hepatitis virus (WHV) is a hepadnavirus closely related to HBV and
2
7
therefore the woodchuck model has been used to evaluate TLR agonists. The TLR7 agonist
vesatolimod (GS-9620) demonstrated anti-WHV activity in the woodchuck model prompting this
program to conduct a similar model to evaluate oral (R)-7 following local IACUC guidelines.
The results from oral dosing of (R)-7 have recently been published and will only be summarized
1
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here. Once weekly oral dosing of (R)-7 at 3 mg/kg for 8 weeks reduced serum WHV DNA and
WHBsAg to below the limit of detection in 3 out of 6 treated woodchucks. These responses were
not conclusively associated with baseline levels of WHV DNA or WHBsAg. The responses were
sustained following cessation of dosing in these 3 animals. Strikingly, >95% reductions in
intrahepatic WHV RNA, WHV DNA and WHV cccDNA were also observed in these 3
sustained antiviral responders. Pharmacokinetic sampling was limited during the study to a
single timepoint at 0.5 h following dosing. There was a trend to higher exposures in the
responding animals in this study but this trend was not reproduced in a larger follow-on study.28
The lack of a clear correlation between systemic drug exposure and efficacy suggests that the
response is not driven from systemic activation of TLR8 but more likely, pre-systemic gut and
liver TLR8 activation. Understanding the molecular basis for the differential responses in
2
8
woodchucks is now the focus of future experiments. In conclusion this striking result observed
in several woodchucks strongly supports the potential for (R)-7 to be an effective new agent for
the treatment of CHB.
Conclusion
The clinical compound (R)-7 is an orally active selective TLR8 agonist, that was discovered
through structure guided evolution of the C-4 substituent on an initial pyrido[3,2-d]pyrimid-2-
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amine lead. (R)-7 demonstrated EC =220 nM for induction of IL-12p40 in cryopreserved
5
0
human PBMCs and >100-fold selectivity for TLR8 against TLR7 in PBMCs and HEK293 cells.
In vivo, (R)-7 demonstrated good oral absorption properties across several preclinical species
and a high first pass clearance that limited systemic exposure levels. Treatment of HBV-infected
PHH with cytokines obtained from (R)-7-stimulated PBMCs decreased the levels of HBV DNA
and RNA in addition to other viral proteins. These findings combined with the promising in vivo
efficacy in the woodchuck model of CHB, supports the potential of (R)-7 to be an effective new
treatment option for CHB. (R)-7 is currently in multiple phase 2 studies for the treatment of
CHB.
0
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Experimental Section
All organic compounds were synthesized at Gilead Sciences, Inc. (Foster City, CA) unless
otherwise noted. Commercially available solvents and reagents were used as received without
further purification. Nuclear magnetic resonance (NMR) spectra were recorded on a Varian
Mercury Plus 400 MHz at room temperature, with tetramethylsilane as an internal standard.
Proton nuclear magnetic resonance spectra are reported in parts per million (ppm) on the δ scale
and are referenced from the residual protium in the NMR solvent (CHCl -d : δ 7.26, MeOH-d : δ
3
1
4
3
.31, DMSO-d : δ 2.50, CH CN-d : δ 1.94). Data is reported as follows: chemical shift
6 3 3
(multiplicity [s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, h = heptet, m =
multiplet, br = broad, app = apparent], coupling constants (J) in Hertz, integration). See
Supporting Information for NMR spectra of final compounds. Preparative normal phase silica
gel chromatography was carried out using a Teledyne ISCO CombiFlash Companion instrument
with silica gel cartridges. Preparative HPLC purification is described in the experimental
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method. Purities of the final compounds were determined by analytical high-performance liquid
chromatography (HPLC), and were greater than 95% unless otherwise noted (See Supporting
Information for HPLC spectra and methods). LC/MS was conducted on a Thermo Finnigan MSQ
1
1
1
1
1
1
1
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1
2
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1
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3
4
5
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7
8
9
0
+
–
Std using electrospray positive and negative [M+1] and [M-1] , and a Dionex Summit HPLC
System (model: P680A HPG) equipped with a Gemini 5 µ C18 110A column (30 mm × 4.60
mm), eluting with CH CN containing 0.1% formic acid, and water containing 0.1% formic acid;
3
0
min-1.8 min 2-100% CH CN, 1.8 min-1.85 min 100%-2% CH CN, 1.85 min-2.00 min 2%
3 3
CH CN at 1800µL/min.
3
CAUTION: The compounds generated using these methods are potent
immunomodulators and should be handled with appropriate personal protective equipment
practices, including double glove, lab coat disposable arm bands, and disposable footwear
booties. A full mask respirator should be used when handling the solid form of the compounds in
amounts exceeding 10 mg.
The synthesis, characterization data, and associated references for the amines 11e-g and
18a-g are provided in supporting information.
4
N -butylpyrido[3,2-d]pyrimidine-2,4-diamine (6a). Compound 8 (CAS# 39551-54-7,
Astatech, Inc., Bristol, PA) (50 mg, 0.25 mmol) in THF (2 mL) was treated with butan-1-amine
1
1a (0.03 mL, 0.28 mmol) and N,N-diisopropylethylamine (0.13 mL, 0.75 mmol). The mixture
was stirred for 30 min and then Dmb-NH (0.19 mL, 1.25 mmol) and N,N-diisopropylethylamine
2
(0.13 mL, 0.75 mmol) were added. The mixture was heated at 100°C for 16 h, cooled to rt,
diluted with EtOAc (20 mL), washed with water (20 mL), brine (20 mL), and then concentrated
under reduced pressure. The residue was subjected to silica gel chromatography eluting with 0-
100% EtOAc-hexanes to provide 12a. LC/MS (m/z) 368.14 [M+1]. 12a was treated with TFA (3
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mL), stirred for 30 min, diluted with water and MeOH and then stirred for a further 60 min. The
mixture was then concentrated under reduced pressure and the residue co-evaporated with
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MeOH (3 x). The solid was collected by filtration to provide 6a (51 mg, 62%) as a TFA salt. H
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NMR (400 MHz, MeOH-d ) δ 8.59 (dd, J = 4.4, 1.4 Hz, 1H), 7.82 (dd, J = 8.5, 1.4 Hz, 1H), 7.72
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(dd, J = 8.4, 4.4 Hz, 1H), 3.66 (t, J = 7.3 Hz, 2H), 1.76 – 1.64 (m, 2H), 1.43 (dq, J = 14.7, 7.4
Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H). LC/MS (m/z) 218.1 [M+1].
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N -(pentan-2-yl)pyrido[3,2-d]pyrimidine-2,4-diamine (6b). The same procedure as 6a,
except replacing 11a with (+/-)-pentan-2-amine 11b, provided 6b (63 mg, 73 %) as a TFA salt.
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H NMR (400 MHz, MeOH-d ) δ 8.61 (dd, J = 4.4, 1.4 Hz, 1H), 7.84 (dd, J = 8.5, 1.4 Hz, 1H),
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.74 (dd, J = 8.5, 4.4 Hz, 1H), 4.60 – 4.45 (m, 1H), 1.74 (dtd, J = 13.5, 8.3, 6.7 Hz, 1H), 1.68 –
1.55 (m, 1H), 1.41 (app h, J = 7.5 Hz, 2H), 1.32 (d, J = 6.6 Hz, 3H), 0.95 (t, J = 7.4 Hz, 3H).
LC/MS (m/z) 232.1 [M+1].
(R)-2-((2-aminopyrido[3,2-d]pyrimidin-4-yl)amino)pentan-1-ol ((R)-6c). The same
procedure as 6a, except replacing 11a with (R)-2-aminopentan-1-ol (R)-11c, provided (R)-6c (40
1
mg, 36 %) as a TFA salt. H NMR (400 MHz, MeOH-d ) δ 8.64 (dd, J = 4.4, 1.4 Hz, 1H), 7.83
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(dd, J = 8.5, 1.5 Hz, 1H), 7.76 (dd, J = 8.5, 4.4 Hz, 1H), 4.55 (dq, J = 7.4, 5.4 Hz, 1H), 3.78 –
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.69 (m, 2H), 1.77 – 1.65 (m, 2H), 1.52 – 1.36 (m, 2H), 0.98 (t, J = 7.3 Hz, 3H). LC/MS (m/z)
48.1 [M+1].
(S)-2-((2-amino-[3,2-d]pyrimidin-4-yl)amino)pentan-1-ol
((S)-6c).
The
same
procedure as 6a, except replacing 11a with (S)-2-aminopentan-1-ol (S)-11c, provided (S)-6c (3
mg, 11 %) as a TFA salt with the exception that, after the addition of the Dmb-NH the mixture
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was heated at 135 ̊C in a microwave reactor for 30 min and the final compound (S)-6c was
subjected to purification via reverse phase preparative HPLC (10-70% CH CN in water with
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.1% TFA using a Hydro-RP column over a 20 min gradient). H NMR (400 MHz, MeOH-d ) δ
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.37 (dd, J = 4.2, 1.5 Hz, 1H), 7.62 (dd, J = 8.5, 1.5 Hz, 1H), 7.52 (dd, J = 8.5, 4.2 Hz, 1H), 4.37
(dq, J = 10.1, 5.0 Hz, 1H), 3.69 (d, J = 4.9 Hz, 2H), 1.78 – 1.62 (m, 2H), 1.44 (dq, J = 14.5, 6.9
Hz, 2H), 0.97 (t, J = 7.3 Hz, 3H). LC/MS (m/z) 248.2 [M+1].
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(
R)-2-((2-aminopyrido[3,2-d]pyrimidin-4-yl)amino)hexan-1-ol ((R)-6d). The same
procedure as 6a, except replacing 11a with (R)-2-aminohexan-1-ol (R)-11d, provided (R)-6d (50
1
mg, 55 %) as a TFA salt. H NMR (400 MHz, MeOH-d ) δ 8.64 (dd, J = 4.3, 1.5 Hz, 1H), 7.82
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(
dd, J = 8.5, 1.5 Hz, 1H), 7.77 (dd, J = 8.5, 4.3 Hz, 1H), 4.53 (m, 1H), 3.73 (d, J = 5.3 Hz, 2H),
.85 – 1.59 (m, 2H), 1.39 (m, 4H), 0.91 (m, 3H). LC/MS (m/z) 262.2 [M+1].
S)-2-((2-aminopyrido[3,2-d]pyrimidin-4-yl)amino)hexan-1-ol ((S)-6d). The same
1
(
procedure as 6a, except replacing 11a with (S)-2-aminohexan-1-ol (S)-11d, provided (S)-6d (46
1
mg, 50 %) as a TFA salt. H NMR (400 MHz, MeOH-d ) δ 8.63 (dd, J = 4.4, 1.4 Hz, 1H), 7.84
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(
dd, J = 8.5, 1.4 Hz, 1H), 7.76 (dd, J = 8.5, 4.4 Hz, 1H), 4.53 (m, 1H), 3.73 (d, J = 5.3 Hz, 2H),
.87 – 1.61 (m, 2H), 1.52 – 1.31 (m, 4H), 1.01 – 0.85 (m, 3H). LC/MS (m/z) 262.2 [M+1].
R)-3-((2-aminopyrido[3,2-d]pyrimidin-4-yl)amino)heptan-1-ol ((R)-6e). The same
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(
procedure as 6a, except replacing 11a with (R)-3-aminoheptan-1-ol (R)-11e, and heating at 100°
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for 1 h prior to the Dmb-NH addition, provided (R)-6e (19 mg, 19%) as a TFA salt. H NMR
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(
400 MHz, MeOH-d ) δ 8.63 (dd, J = 4.4, 1.5 Hz, 1H), 7.82 (dd, J = 8.5, 1.5 Hz, 1H), 7.76 (dd, J
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=
8.5, 4.4 Hz, 1H), 4.65 – 4.62 (m, 1H), 3.72 – 3.59 (m, 2H), 1.99 – 1.83 (m, 2H), 1.81 – 1.66
(m, 2H), 1.46 – 1.29 (m, 4H), 0.97 – 0.82 (m, 3H). LC/MS (m/z) 276.1 [M+1].
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(R)-N -(1-aminohexan-2-yl)pyrido[3,2-d]pyrimidine-2,4-diamine ((R)-6f). The same
procedure as 6a, except replacing 11a with (R)-11f and heating the reaction after the Dmb-NH2
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