Diprovocims: A New and Exceptionally Potent Class of Toll-like Receptor Agonists

Article abstract of DOI:10.1021/jacs.8b09223

A screen conducted with nearly 100000 compounds and a surrogate functional assay for stimulation of an immune response that measured the release of TNF-α from treated human THP-1 myeloid cells differentiated along the macrophage line led to the discovery of the diprovocims. Unique to these efforts and of special interest, the screening leads for this new class of activators of an immune response came from a compound library designed to promote cell-surface receptor dimerization. Subsequent comprehensive structure-activity relationship studies improved the potency 800-fold over that of the screening leads, providing diprovocim-1 and diprovocim-2. The diprovocims act by inducing cell-surface toll-like receptor (TLR)-2 dimerization and activation with TLR1 (TLR1/TLR2 agonist), bear no structural similarity to any known natural or synthetic TLR agonist, and are easy to prepare and synthetically modify, and selected members are active in both human and murine systems. The most potent diprovocim (3, diprovocim-1) elicits full agonist activity at extraordinarily low concentrations (EC₅₀= 110 pM) in human THP-1 cells, being more potent than the naturally derived TLR1/TLR2 agonist Pam3CSK4 or any other known small molecule TLR agonist.

Full text of DOI:10.1021/jacs.8b09223

Subscriber access provided by Kaohsiung Medical University
Article
Diprovocims: A New and Exceptionally
Potent Class of Toll-like Receptor Agonists
Matthew D Morin, Ying Wang, Brian T. Jones, Yuto Mifune, Lijing Su, Hexin
Shi, Eva Marie Y Moresco, Hong Zhang, Bruce Beutler, and Dale L. Boger
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b09223 • Publication Date (Web): 01 Oct 2018
Downloaded from http://pubs.acs.org on October 2, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 32
Journal of the American Chemical Society
Diprovocims: A New and Exceptionally Potent Class of Toll-like Receptor
Agonists
1
2
3
4
5
6
7
8
9
Matthew D. Morin,1 Ying Wang,2 Brian T. Jones,1 Yuto Mifune,1 Lijing Su,2 Hexin Shi,2 Eva Marie Y.
Moresco,2 Hong Zhang,2 Bruce Beutler,2,* and Dale L. Boger1,*
1Department of Chemistry and the Skaggs Institute of Chemical Biology, The Scripps Research
Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037 USA
2Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas,
TX 75390 USA
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Abstract. A screen conducted with nearly 100,000
compounds and a surrogate functional assay for
stimulation of an immune response that measured the
release of TNF- from treated human THP-1 myeloid
cells differentiated along the macrophage line led to
the discovery of the diprovocims. Unique to these
efforts and of special interest, the screening leads for
this new class of activators of an immune response came from a compound library designed to
promote cell surface receptor dimerization. Subsequent comprehensive structure–activity relationship
studies improved the potency 800-fold over that of the screening leads, providing diprovocim-1 and
diprovocim-2. The diprovocims act by inducing cell surface toll-like receptor(TLR)-2 dimerization and
activation with TLR1 (TLR1/TLR2 agonist), bear no structural similarity to any known natural or
synthetic TLR agonist, are easy to prepare and synthetically modify, and selected members are
active in both human and murine systems. The most potent diprovocim (3, diprovocim-1) elicits full
agonist activity at extraordinarily low concentrations (EC50 = 110 pM) in human THP-1 cells, being
more potent than the naturally-derived TLR1/TLR2 agonist Pam3CSK4 or any other known small
molecule TLR agonist.
Introduction
The human immune system is composed of two components, the innate and adaptive immune
systems. The innate immune system is encoded into the germline of an organism and constitutes the
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 32
first line of defense in mammals. It responds to pathogens and abnormal cells through initiation of
intracellular signaling cascades that lead to the activation of transcription factors which trigger the
production of cytokines and chemokines with participation of multiple cell types, including dendritic
cells, macrophages, neutrophils, and natural killer cells.1 The subsequent activation of the adaptive
immune system involves antigen-specific T cell and B cell responses mediated by antigen-presenting
dendritic cells. It serves to provide long term host protection through the action of T cells and
antibodies to neutralize the pathogens and abnormal cells.1,2 The Toll-like receptors (TLRs)3-6 are the
most widely recognized set of the pathogen-associated or damage-associated molecular pattern
(PAMPs and DAMPs) receptors that recognize molecular components of pathogens or abnormal cells
and marshal the initial innate immune response.3,5 They also induce the adaptive immune response,6
and the action of vaccines is often due in part to the activation of the TLR system.7 As such, TLR
agonists are attractive as new vaccine adjuvants for both infectious diseases and oncology that act by
well-defined mechanisms,7-10 as prophylactics against pathogen exposure (e.g.; biodefense),11 or as
immunostimulators alone or in combination with other drugs,11 particularly in oncology.8-11 A recent
exciting application lies in the field of cancer immunotherapy, where the adaptive immune system is
exploited to kill cancer cells based on their expression of neo-antigens.12 The potential of such cancer
immunotherapy results not only from the initial killing of tumor cells, but also from a long lasting
systemic anti-tumor memory response (antigen-specific anti-tumor immunity). However, most known
TLR agonists are mimics or modifications of microbial components that have unattractive structural,
physical, or stability properties,13 and only a limited number of small molecule classes have been
found to behave as TLR agonists.8-10 Notable examples of such small molecules include the TLR7
agonists imiquimod,14 isotoribine and 8-oxo-9-benzyladenine,14 as well as the TLR7/8 agonist
resiquimod15 that today still serve as the inspiration for nearly all such work.16,17 Recent interest in the
area has led to the discovery of a multi-Toll-like receptor agonist that acts on TLR 3, 8 and 9,18 to the
intense study of the STING agonist cGAMP,19 and to our own disclosure of the neoseptins,20 a new
class of mouse TLR4 agonists21-24 that bear no structural similarity to bacterial lipopolysaccharide
(LPS) or its active core Lipid A (LPA).21
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Herein, we report full details of the discovery of the diprovocims, a new and especially
attractive synthetic small molecule class of human TLR agonists (Figure 1). Studies decades ago
discovered the immune activating N-terminal segments of bacterial triacylated lipoproteins and
lipopeptides (e.g.; Pam3CSK4,25,26 Figure 1), which were shown later to act by heterodimerization of
TLR1/TLR2 at the cell surface.27,28 Such agonists based on the lipoproteins are effective vaccine
adjuvants when admixed or covalently-linked with antigens25,26 and continue to be widely studied
today.29 Among the TLRs, TLR2 requires heterodimerization with either TLR1 or TLR6 for activation.
Bacterial triacylated lipoproteins or lipopeptides are the most widely recognized agonists that activate
ACS Paragon Plus Environment

Page 3 of 32
Journal of the American Chemical Society
TLR1/TLR2 (e.g.; Pam3CSK4) and bacterial diacylated lipopolypeptides stimulate TLR2/TLR6 (e.g.;
MALP-230, Figure 1).31 Complementary to recent studies that have disclosed the only other known
small molecule TLR2/TLR1 agonists,32 the diprovocims are a new and exceptionally potent class of
synthetic small molecule TLR2/TLR1 agonists and they emerged from screening a unique chemical
library designed to promote cell surface receptor dimerization.33 The most potent of the diprovocims
(compound 3) elicits full agonist activity at extraordinarily low concentrations (EC50 = 110 pM) in
human cells, being more potent than Pam3CSK4 or any other known small molecule TLR agonist.
The diprovocims act by a well-defined mechanism (TLR1/TLR2 agonist)34 and selected members are
active in both human and murine systems. As shown herein, the class exhibits exquisite structure–
activity (SAR) relationships. The efficacy of the class matches that of natural TLR agonists such as
LPS or the lipopeptides and selected members exhibit a potency that matches or exceeds that of the
natural agonists. Compound 3 (diprovocim-1) has been shown to be active as an adjuvant in mice
when co-administered by conventional intramuscular (i.m.) injection (vaccination) with an antigen
(OVA).34 It was also shown to act synergistically with a checkpoint inhibitor (anti-PD-L1), where the
combination treatment cured or prolonged survival of mice implanted with an immunogen bearing
murine melanoma (B16-OVA) and protected mice from tumor rechallenge.34 Notably, the latter
impressive in vivo activity was observed with diprovocim/OVA co-administration i.m., rather than with
intratumor adjuvant administration as has been a convention in recent studies. Finally, the class
bears no structural similarity to the TLR1/TLR2 lipoprotein agonists or any other natural or synthetic
TLR agonist, and members of the class are easy to prepare and synthetically modify.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Structures of the known TLR1/TLR2 (Pam3CSK4) and TLR2/TLR6 (MALP-2) agonists and
diprovocim-1 and diprovocim-2.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 32
Results
1
2
3
4
5
6
7
8
9
Screen and Results. The discovery of the role of the TLRs emerged in genetic studies in
whole organisms with impaired innate immune responses caused by spontaneous or induced nucleic
acid mutations in genes impacting the immune system (lack of sensitivity to LPS due to a mutated
and disabled TLR4).5,35 Complementary to such efforts, we explored the use of an unbiased chemical
genetics approach,35 screening libraries of compounds in surrogate cell-based functional assays for
activation of an immune response. A screening campaign was conducted with nearly 100,000
compounds,36 using a functional assay that measured the stimulated release of TNF- from treated
human THP-1 myeloid cells partially differentiated along the macrophage line by PMA pretreatment
(Supporting Information, Figure S1).34 The functional activity measured by this assay is both a rare
activation event (activator/agonist vs inhibitor/antagonist) and is sensitive, such that even weak
stimulation of TNF- release proved easily detectable. By design, subsequent assay of active
compounds against macrophages from available mice that bear germline genetic defects or
knockouts of the genes encoding each of the TLRs, other molecular pattern recognition receptors, or
downstream signaling molecules would be used to establish whether the activity was derived from
known or presently unrecognized targets.20 The chemical libraries screened in the efforts represent a
unique compound collection populated by nontraditional compounds36 designed to target protein–
protein37,38 or protein–DNA interactions39 as well as the major enzyme classes.40 Within the full
100,000 compound library that was screened, the lead compounds detailed herein emerged from a
previously unreported subset of the library designed to promote cell surface receptor dimerization (ca.
6,000 compounds), with each half monomer of the C2-symmetrical compounds designed to bind each
protein receptor monomer.33 The sub-library from which the leads were identified was prepared as
100 mixtures of 10 compounds on the trans-pyrrolidine-3,4-dicarboxylate core template (Supporting
Information Figure S2).38 It was composed of all individual combinations of ten defined R1 and
another ten defined R2 substituents, and a mixture of ten linkers (denoted by X, Figure 2).33,41 The
screening results were remarkable, providing pools of 10 compounds that displayed activity each and
every time the R2 subunit was either 4-fluorophenethylamine (residue 4, well X4) or trans-2-
phenylcyclopropylamine (residue 9, well X9) in the tested pools independent of the structure of R1
(Figure 2). Analogous libraries with different cores (e.g.; iminodiacetic acid and isoindoline-4,5-
dicarboxylic acid vs pyrrolidine-3,4-dicrboxylic acid),33 other R1 and R2 substituents,33 alternative
linker groupings,33 and even higher order displays (e.g.; trimer, tetramer)33 failed to provide activity in
the screen. Even more remarkable was the fact that a large number of closely compounds with
related R1 and R2 substituents within the sub-library were inactive, including phenethylamine, (4-
methoxyphen)-ethylamine, (4-hydroxyphen)ethylamine, (3-methoxyphen)ethylamine, benzylamine
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 5 of 32
Journal of the American Chemical Society
(Supporting Information Figure S2). Thus, it was also determined that even small deviations from the
structure resulted in no detection of activity, providing a first level structure–activity relationship (SAR)
study. Not only was the activity robust and structurally specific, but the repetitive observation with
each appearance of residue 4 or residue 9 in the compound mixtures regardless of the identity of R1
indicated that the activity was not the result of an unrecognized artefact (Figure 2).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Top: Screening results for the receptor dimerization library (plate 39 G–H wells and plate 40 all wells)
measuring stimulated TNF- release from PMA-differentiated human THP-1 myeloid cells (10 compound
mixtures tested at 50 M). The compound mixtures in the starred wells were prepared as individual
compounds. Bottom: Structures of active mixtures containing R2 residues 4 and 9 where R1 is single defined
substituent and X is a mixture of ten linkers, see Supporting Information Figure S2.
Diprovocim-1. The ten compounds in two of the active wells (plate 39G4 and plate 39G9),
containing either the residue 4 or residue 9 substituent, were prepared and tested individually,
identifying only two active linkers. Benzene-1,4-dicarboxylic acid was by far the most potent of the
two active linkers and this is illustrated in Figure 3 with the individual compounds found in plate 39G9.
Structurally specific activity was observed and even dicarboxylic acid linkers spatially or structurally
related to benzene-1,4-dicarboxylic acid were inactive (e.g.; C5 vs C4 or C9 and C10).41 For
simplicity, the same studies conducted concurrently with plate 39G4, containing the R2 residue 4 (4-
fluorophen)ethylamine substituent are detailed separately following the discussion of chronological
work conducted on plate 39G9.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 32
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Top: Synthesis of the individual compounds in plate 39G9 used to identify the linker of active
compounds. Bottom: Activity of individual compounds (labeled by the linker C1–C10) in the plate 39G9 mixture
of the receptor dimerization library tested alongside controls (LPS and 0 = no compound) measuring stimulated
TNF- release from differentiated human THP-1 cells (tested at 50 M unless indicated otherwise).
Because the R2 side chain was key to the activity, we prepared a set of five compounds,
containing the identified linker (benzene-1,4-dicarboxylic acid), to systematically establish the
importance of the presence and number of 2-phenylcyclopropylamine (R2) versus phenethylamine
(R1) side chains present in the compounds in plate 39G9 (Figure 4).41 The five compounds were
prepared from the three pyrrolidine-3,4-dicarboxamides 5–7 and simultaneous (8, 10 and 12)
coupling with benzene-1,4-dicarboxylic acid (2 equiv PyBrOP, i-Pr2NEt, DMF, 23 oC) or sequential (9
and 11) couplings starting with the mono methyl ester of benzene-1,4-dicarboxylic acid with an
intermediate hydrolysis of the methyl ester (4 equiv LiOH, THF:MeOH:H2O 4:1:1, 23 oC). The results
of their assessment were stunning. Although the compounds that contained no or one 2-
phenylcyclopropylamine substituents were inactive (8 and 9), each added 2-phenylcyclopropylamine
ACS Paragon Plus Environment

Page 7 of 32
Journal of the American Chemical Society
substituent (two as found in library, three, and four) incrementally (3–5-fold each) increased the
potency (potency with number of residue 9 side chains: 0 and 1 < 2 < 3 < 4). The compounds
containing three and four 2-phenylcyclopropropylamine substituents (11 and 12) were more potent
than the original lead 10 (EC50 = 80 nM) in the library and compound 12, containing four such
substituents, was exceptionally potent (EC50 = 10 nM) and equally efficacious with LPS. Just as
significantly and although the leads that emerged from the screening library were inactive in
stimulating the release of TNF- from mouse macrophages, compound 12 was active at doing so,
albeit at a lower potency (EC50 = 100 nM). Although a lack of activity in the murine system would not
be detrimental to their advancement for human studies, the observation of activity in mouse
macrophages with 12 and subsequent related compounds is especially useful in conducting
pharmacological in vivo studies in mouse models.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Compounds that established the importance of the presence and number of 2-phenyl-
cyclopropylamine residues derived from active lead (10) in plate 39G9. EC50’s derived from dose-response
curves for the stimulated release of TNF- from differentiated THP-1 cells or mouse macrophages by 8–12.
This activity of 12 is even more impressive since it was prepared with racemic trans-
pyrrolidine-3,4-dicarboxylic acid and racemic trans-2-phenylcyclopropylamine as found in the original
library. As a consequence and even though each trans stereochemical relationship in the pyrrolidine
cores and the side chain substituents is fixed, 12 was still a mixture of all possible 21 diastereomers
and enantiomers arising from the use of racemic materials. As daunting as this may seem, the
unraveling of the activity within this complex diastereomeric mixture proved surprisingly
straightforward. Each enantiomer of 2-phenylcyclopropylamine (1S,2R and 1R,2S) is commercially
available42 and a reported resolution43 was used to obtain the two enantiomers (S,S and R,R) of N-
Boc-pyrrolidine-3,4-dicaboxylic acid.43 These were used to prepare four pyrrolidine/side chain
subunits 15–18, representing all combinations of each enantiomer of N-Boc-pyrrolidine-3,4-
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 32
dicaboxylic acid independently substituted with each enantiomer of 2-phenylcyclopropylamine (same
enantiomer at each of two sites) (Figure 5). Initially and for simplicity, we did not examine
combinations where the two substitution sites on the pyrrolidine core contained the two different
enantiomers (1S,2R and 1R,2S) of 2-phenylcyclopropylamine. The four substituted pyrrolidine core
monomers 15–18 were then N-Boc deprotected (4 N HCl, dioxane, 23 oC, 2 h) and simultaneously or
sequentially coupled to benzene-1,4-dicarboxylic acid to provide the linked dimers, containing each
and all combinations of the individual enantiomerically defined monomers (10 compounds).
Consistent with a specific receptor interaction, the results of their assessment were crystal clear.
Activity was observed only in compounds that contained the (S,S)-pyrrolidine-3,4-dicarboxylic acid
core and only when the (1S,2R)-2-phenylcyclopropylamine side chain was present. In fact, the activity
increased as the number of (1S,2R)- versus (1R,2S)-2-phenylcyclopropylamine substituents
increased in much the same manner as was observed in the side chain series 8–12. This further
indicated that the incorporation of the inactive (1R,2S)-2-phenylcyclopropylamine enantiomer in
pyrrolidine monomers also containing the active 1S,2R enantiomer would likely be unproductive
(remaining 11 compounds). Moreover, this activity spanned a huge range with nearly all the
compounds being less potent than the all racemate or inactive (Figure 5). Compound 3 (diprovocim-
1), composed of a single enantiomer of the pyrrolidine core dicarboxylic acid (S,S) and a single
enantiomer of the 2-phenylcyclopropylamine (1S,2R) proved to be by far the most active
diastereomer, exhibiting a stunning potency (EC50 = 110 pM) for stimulating the release of TNF-
from human differentiated THP-1 cells. This activity proved to be 100-fold more potent than the all
racemate 12 (EC50 = 10 nM), consistent with it being responsible for essentially all the activity
observed in the original racemic mixture (21 diastereomers). Compound 3 was also found to be
roughly >30-fold more potent than any other diastereomer. In addition, diprovocim-1 (3) was potent
(EC50 = 1.3 nM) at stimulating the release of TNF- from mouse macrophages. Here, it not only
proved to be more potent than the diastereomeric mixture 12, but all other isomers were much less
active and none approached or matched the activity of the all racemate (Figure 5). Finally,
diprovocim-1 (3) proved to be equally efficacious but 5–10 times more potent than Pam3CSK4 (1,
EC50 = 0.91 nM) in a direct side-by-side comparison in the assay for the stimulated release of TNF-
from differentiated human THP-1 cells (Supporting Information Figure S3).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 9 of 32
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Ten Diastereomers prepared of the all racemate 12, identifying the active component. EC50’s derived
from dose-response curves for the stimulated release of TNF- from differentiated human THP-1 cells and
mouse macrophages by 3 (diprovocim-1) and 19–27.
Compound 8 was also prepared as the single all (S)-enantiomer from 15 and was found to be
inactive (EC50 >5 M) like the all racemate mixture, failing to stimulate the release of TNF- from
either partially differentiated THP-1 cells or mouse macrophages. Just as significantly, it was found to
be incapable of acting as an antagonist, inhibiting the activity of diprovocim-1 (3), when co-
administered at 5 M. This indicates that the lack of agonist activity with 8, and presumably related
compounds, is due to ineffective binding to the target TLRs and is not due to effective binding but
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 32
failure to induce the active dimer receptor conformation. To further establish the importance of each
(1S,2R)-2-phenylcyclopropylamine amide substituent and to rule out an inhibitory effect of the
presence an alternative substituent, we prepared a series of deletion analogues (29–34) of
diprovocim-1 with sequential removal of each side chain or key structural feature (Supporting
Information Figure S4).44 From these studies, it was established that the presence and
stereochemistry of each (1S,2R)-2-phenylcyclopropylamine amide substituent, the presence and
stereochemistry of the two intact (S,S)-pyrrolidine-3.4-dicarboxylate cores, and the benzene-1,4-
dicarboxylate linker are all integral to the expressed activity of diprovocim-1 (3).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
At this stage, we reexamined the compound linkers found in the original library (35–43), along
with five additional closely related dicarboxylic acid linkers (44–48), but now with only the active
enantiomer of the substituted pyrrolidine-3,4-dicarboxamide 15 and bearing four (1S,2R)-2-
phenylcyclopropylamine amide substituents in place of the all racemate (Figure 6). The latter series
44–48 placed the two carboxylic acids on phenyl or naphthyl aromatic rings, but located spatially in
positions slightly altered from that found in diprovocim-1 or at the same position but with an attached
fused ring (45). Each was prepared in a single coupling step from 15 and the linker dicarboxylic acid
o
o
(4 N HCl, dioxane–THF, 23 C, 3 h; 2 equiv PyBrOP, i-Pr2NEt, DMF, 23 C, 18 h). Not surprisingly,
benzene-1,4-dicarboxylic acid remained the most potent linker in the original series (3 vs 35–43).
Moreover, no compound in the series 44–48 displayed activity close to diprovocim-1. All except 44
were inactive, and even 44 was >10-fold less active than diprovocim-1. However, what is remarkable
in these comparisons is how specific the activity remained for the benzene-1,4-dicarboxylic acid
linker. All other linkers were >10-fold less active, with most displaying no activity even at
concentrations >50,000-fold higher. Clearly, the linker contribution to the activity of 3 is just as specific
and integral as that of the substituted pyrrolidine-3,4-dicarboxamide monomers.
ACS Paragon Plus Environment

Page 11 of 32
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. EC50’s derived from dose-response curves for the stimulated release of TNF- from differentiated
human THP-1 cells or mouse macrophages by 3 (diprovocim-1) and 35–48.
With this appreciation of the importance of the precise spacing of the dicarboxylic acid in the
structure of the linker, a more targeted examination of alternative linkers was conducted (Figure 7).
Isosteric replacements of the benzene ring (49), six-membered heterocyclic dicarboxylic acids (50,
51) that might alter the physical properties (e.g.; solubility) or define an orientation of the adjacent
pyrrolidine-3,4-dicarboxamide, an acyclic unsaturated dicarboxylic acid linker that might flexibly
substitute for benzene (52), a series of further substituted benzene-1,4-dicaboxylic acids introducing
functionality for use in conjugation to candidate antigens (53–58), and those used to explore
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 12 of 32
disubstitution (59–63) were examined. The latter series was especially interesting because of the
substituents marked influence on the adoption of preferred conformational orientations of the adjacent
pyrrolidine-3,4-dicarboxamides through destabilizing steric interactions or stabilizing hydrogen
bonding. Each was prepared by acid-catalyzed Boc deprotection of 15 (4 N HCl, dioxane, 23 oC) and
a single coupling step with the linker dicarboxylic acid (2 equiv PyBrOP, i-Pr2NEt, DMF, 23 oC, 18 h).
Remarkably and as benign as many of the changes are, nearly all such compounds examined were
less potent than diprovocim-1, highlighting the unique role the linker plays in the expression of the
biological activity. The exception to this generalization was the phenol 53, which nearly matched the
activity of diprovocom-1 itself. Although the number of compounds on which to base the conclusions
is small, the potency appears to smoothly decrease as the size of the (adjacent) substituents increase
(e.g.; potency: 3 > 53 > 55 > 58 > 54, 57 and 3 > 60 > 61 > 62 > 63). Although not required for their
applications, compounds (53 and 55) within this series still exhibit excellent potency sufficient to
confidently establish that functionalization sites on the linker core are available for conjugation with
candidate antigens when warranted.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 13 of 32
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Figure 7. Linker modifications. EC50’s were derived from dose-response curves for the stimulated release of
TNF- from differentiated human THP-1 cells and mouse macrophages by 49–63.
51
52
53
54
55
56
57
58
59
60
Diprovocim-2. A more straightforward series of studies was conducted with the active plate
39G4 well. Examination of the 10 individual compounds in the well mixture identified the same active
linker (benzene-1,4-dicarboxylic acid, not shown). Because the R2 side chain was key to the
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 14 of 32
observation of the repetitive activity seen in the original screen independent of the structure of R1
(Figure 2), a set of five compounds was prepared with the active benzene-1,4-dicarboxylic acid linker
to establish the importance of the presence and number of 4-fluorophenethylamine (R2) versus
phenethylamine (R1) side chains present in the compounds in the plate 39G9 well (Figure 8).41 The
five compounds were prepared from the three pyrrolidine-3,4-dicarboxamides 6, 64 and 65 and
simultaneous (8, 67 and 69) or sequential (66 and 68) coupling with benzene-1,4-dicarboxylic acid or
its mono methyl ester, respectively. The results of their assessment were clear. Activity was
enhanced with the addition of each 4-fluorophenethylamine side chain (ca. 3–5-fold) and 69,
containing four, was the most potent (EC50 = 100 nM, racemate) compound in the set.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 8. Synthesis of compounds that established the importance of the presence and number of 4-
fluorophenethylamine side chains.
Since the 4-fluoro substituent on the 4-fluorophenethylamide uniquely conveyed activity to the
compounds (phenethylamide inactive), a series of alternative substituents were examined in which
the 4-fluoro group was moved to the 3- and 2-positions, conservatively replaced with other halogens
(Cl and Br), replaced with a series of small electron-donating or electron-withdrawing groups with
some capable of serving as hydrogen bond donors or acceptors, replaced with a large phenyl group,
ACS Paragon Plus Environment

Page 15 of 32
Journal of the American Chemical Society
or with incorporation of a 2-naphthyl aromatic ring (Figure 9). Each was prepared as a racemate by
coupling racemic N-Boc trans-pyrrolidine-3,4-dicarboxylic acid with the arylethylamine (2 equiv, EDCI,
HOAt, 2,6-lutidine, DMF, 23 oC, 16 h) followed by N-Boc deprotection of 70–83 (4 N HCl, dioxane 23
oC, 3 h) and subsequent coupling with benzene-1,4-dicarboxylic acid (2 equiv PyBrOP, i-Pr2NEt,
1
2
3
4
5
6
7
8
o
DMF, 23 C, 18–24 h). Stunningly, all were inactive at stimulating the release of TNF- from human
9
differentiated THP-1 cells at concentrations up to 5 M. The sole exception was 84 (EC50 = 1 M,
>10-fold less active), which incorporated the most conservative change of moving the fluorine
substituent from the p- to m-position (3- vs 4-fluorophenethylamide). Clearly the fluorine substituent
and the 4-fluorophenethylamide side chains are essential and remarkably specific to the expression
of the biological activity of the compounds.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 9. Aryl substituent effects on activity of diprovocim-2 (4).
Because the library and 69 were prepared from racemic trans-pyrrolidine-3.4-dicarboxylic acid,
they were a mixture of enantiomers and diastereomers. Compound 69 was a mixture of three
compounds (two enantiomers and one meso compound), making the establishment of the active
enantiomer in the mixture straightforward. Each of the two enantiomers (S,S and R,R) of N-Boc-
pyrrolidine-3,4-dicaboxylic acid43 was coupled with 4-fluorophenethylamine (EDCI, HOAt, 2,6-lutidine,
o
23 C, 16 h) to prepare the two possible enantiomeric pyrrolidine-3,4-dicarboxamides (S,S)- and
(R,R)-65 (Figure 10). These were independently but simultaneously coupled with benzene-1,4-
dicarboxylic acid (2 equiv PyBrOP, i-Pr2NEt, DMF, 23 oC, 18–24 h) to provide the two enantiomers 4
and 98, bearing the all S or all R configurations, respectively. Similarly, sequential coupling (PyBrOP,
i-Pr2NEt, DMF, 23 oC) of (S,S)- and (R,R)-65 with the mono methyl ester of benzene-1,4-dicarboxylic
o
acid with an intermediate hydrolysis of the methyl ester (LiOH, THF/MeOH/H2O, 23 C) provided the
meso isomer 99. The all S enantiomer 4 (EC50 = 50 nM, diprovocim-2) proved to be 3-fold more
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 16 of 32
potent than the all racemate (EC50 = 150 nM), whereas the all R enantiomer 98 was inactive (EC50 =
>5 M, >100-fold less potent). Interestingly, the meso isomer 99 (EC50 = 70 nM), containing both a
S,S- and R,R-pyrrolidine-3,4-dicarboxamide, was also active and nearly matched the potency of the
all S enantiomer. Despite this potent activity in human THP-1 cells, no compound in this series
stimulated the release of TNF- from mouse macrophages.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 10. Synthesis of the three enantiopure compounds found in the all racemate 69, identifying the active
components. EC50’s were derived from dose-response curves for the stimulated release of TNF- from
differentiated human THP-1 cells by 4 (diprovocim-2) and 98–99.
With use of the enantiopure (S,S)-pyrrolidine-3,4-dicarboxamide 65, the dicarboxylic linkers in
the original library (C1–C10) as well as a subset of those that proved most interesting with
diprovocim-1 were re-examined now with compounds that bear all four 4-fluorophenethylamide side
chains (Figure 11). Each was prepared in a single coupling step (2 equiv PyBrOP, i-Pr2NEt, DMF, 23
oC, 18–24 h) of the linker dicarboxylic acid following acid-catalyzed deprotection of enantiopure (S,S)-
65. In this series, none of the alternative linkers provided compounds that were active in either human
ACS Paragon Plus Environment

Page 17 of 32
Journal of the American Chemical Society
THP-1 cells or mouse macrophages and only the phenol 110, like the behavior of 53 versus
1
2
3
diprovocim-1, nearly matched the activity diprovocim-2.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 11. Synthesis of compounds that contain the active pyrrolidine-3,4-dicarboxamide enantiomer (S,S)-65
and the original library 10 linkers or additional selected dicarboxylic acid linkers. Bottom: EC50’s derived from
dose-response curves for the stimulated release of TNF- from differentiated human THP-1 cells or mouse
macrophages by 4 (diprovocim-2) and 100–110.
Hybrid structures of diprovocim-1 and diprovocim-2 and additional key compounds.
Given the unique behavior that only two of the examined side chains displayed and their importance
to the functional activity of 3 and 4, compounds containing a combination of the two side chains were
explored (111–113) and prepared as the all racemate diastereomeric mixtures (Supporting
Information Figure S5). The compounds displayed a consistent trend where replacement of the
phenethylamine side chains with 4-fluorophenethylamine sided chains improved activity and where a
2-phenylcyclopropylamine replacement of the 4-fluorophenethylamine side chains further improved
activity. Thus, the two side chains can be used interchangeably with predictable influences on the
biological potency (trans-2-phenylcyclopropylamine > 4-fluorophenethylamine) indicating they are
binding at the same site influencing activity in a similar manner at its target. The most potent of these
were prepared as single enantiomers, bearing the identified active enantiomer derived from 15 and
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 18 of 32
either (S,S)- and (R,R)-65 (Figure 12). These studies established that the hybrid structure 114
(diprovocim-4), possessing the two (S,S)-pyrrolidine cores, is an effective TLR1/TLR2 agonist active
in both human THP-1 cells and mouse macrophages. Thus, 114 displays properties like those of
diprovocim-1. In addition, it is less potent than diprovocim-1 but more active than diprovocim-2,
displaying a potency between that of the compounds from it is derived. The analogous hybrid
structure 115 (diprovocim-5), containing one subunit derived from (R,R)-65, was 5-fold less active in
human THP-1 cells and inactive in mouse macrophages, reflecting both a lower potency and greater
species difference.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 12. Diprovocim-4 and diprovocim-5. EC50’s derived from dose-response curves for the stimulated
release of TNF- from differentiated human THP-1 cells or mouse macrophages.
Among the most interesting of the modifications made to the diprovocims was the
incorporation of the fluorine group found in diprovocim-2 (4) into the side chain substituent of
diprovocim-1 (3) (Figure 13). Thus, coupling of (1S,2R)-2-(4-fluorophenyl)cyclopropylamine45 (116, 2
equiv) with (S,S)-14 (EDCI, HOAt, 2,6-lutidine, DMF, 23 oC, 16 h) followed by Boc deprotection of 117
(4 N HCl, dioxane, 23 oC, 3 h) and coupling of the resulting amine with benzene-1,4-dicarboxylic acid
o
(PyBrOP, i-Pr2NEt, DMF, 23 C, 18–24 h) provided 118 (diprovocim-3). Its assessment for the
stimulated release of TNF- from differentiated human THP-1 cells (EC50 = 130 pM) and mouse
macrophages (EC50 = 1.2 nM) revealed that it was indistinguishable from diprovocim-1 (3), displaying
exceptionally potent activity. Thus, the addition of the p-fluoro group did not further enhance the
potency of 3, but its presence also did not disrupt the activity of 3. Another way of viewing this result
is that introduction of the four rigidifying and stereochemically-defined cyclopropanes into diprovocim-
ACS Paragon Plus Environment

Page 19 of 32
Journal of the American Chemical Society
2 (4) improved the activity 400-fold. The unique behavior of the 4-fluoro substituent in 118 was
established with the examination of a small but key series of additional compounds that contain
alternative C4 phenyl substituents (X = Cl, Br, Me, OMe and CN vs F, Figure 13). The set contains
conservative but larger halogen replacements (X = Cl, Br), a conservative but larger hydrophobic
substituent replacement (X = Me), and both strong yet small electron-donating (X = OMe) and
electron-withdrawing (X = CN) substituent replacements. Compounds 119–123 were prepared by the
route outlined for 118, using the corresponding 4-substituted (1S,2R)-2-(phenyl)cyclopropylamines.45
Remarkably, all compounds in this expanded series exhibited substantially reduced activity that
correlates with the progressively increased size or extended length of the phenyl C4 substituent
(activity: H = F > Cl > Me > Br > OMe, CN). The most active of these compounds (119, X = Cl) proved
to be 300-fold less potent in THP-1 cells and >1000-fold less potent in mouse macrophages.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 13. Diprovocim-3 (118) and related aryl substitution analogues 119–123 of diprovocim-1 (3).
Hybrid structures with incorporation of the lipid side chains found in natural TLR2/TLR1
and TLR2/TLR6 agonists. Among the TLRs, TLR2 requires heterodimerization with either TLR1 or
TLR6 for activation. Bacterial triacylated lipoproteins are the most widely-recognized agonists
activating TLR1/TLR2 (e.g.; Pam3CSK425-29), whereas bacterial diacylated lipopolypeptides bind and
activate TLR2/TLR6 (e.g.; MALP-230, see Figure 1).31 TLR1/TLR2 preferentially binds triacyl
lipopeptides and binds diacyl lipopeptides only weakly, whereas TLR2/TLR6 only binds diacyl
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 20 of 32
lipopeptides. X-ray crystallographic structures of Pam3CSK4 (three lipid chains) bound to
hTLR1/TLR2 and Pam2CSK4 (two lipid chains) bound to mTRL2/TLR6 have been disclosed.28 These
studies revealed that the amide lipid chain of Pam3CSK4 inserts into a hydrophobic pocket in TLR1,
while the remaining two ester lipid chains bind in a TLR2 hydrophobic channel, filling a long
continuous hydrophobic pocket spanning both proteins at the TLR1/TLR2 heterodimer interface. In
comparison, Pam2CSK4 binds with both ester lipid chains also bound to TLR2, but does not contain
or require the amide lipid binding to TLR6 to promote active TLR2/TLR6 dimer formation. In fact,
TLR6 lacks the lipid binding pocket needed to accommodate the third amide lipid chain of the triacyl
lipopeptides and proteins. Within the diprovocims, the amide side chains are serving the role of the
lipid chains, extending into the hydrophobic pockets spanning TLR1/TLR2. The distinction being that
the symmetrical diprovocims contain four, not three, such groups. As a result, we examined a range
of lipid amide replacements for the trans-2-phenylcyclopropylamine and 4-fluorophenethylamine
derived amides and explored hybrid structures containing three as well as four side chains. The first
series examined was composed of compounds that contain three side chains, linking one half of
diprovocim-1 with a single lipid side chain amide attached either directly to the benzene-1,4-
dicarboxylic acid linker (126–129) or as the amide substituent on (R)- or (S)-pyrrolidine-3-carboxylic
acid (130–137) (Figure 14). These were prepared by coupling 125 with the corresponding alkyl amine
or the corresponding (R)- or (S)-pyrrolidine-3-carboxamide (EDCI, HOAt, 2,6-lutidine, DMF–CH2Cl2,
25 oC, 3–4 h). Their assessment for the stimulated release of TNF- from differentiated human THP-1
cells and mouse macrophages revealed that no compound in the series 126129 exhibited agonist
activity. In contrast, the series in which the lipid amide substituent was attached via a pyrrolidine-3-
carboxamide stimulated the release of TNF- from both human THP-1 cells and mouse macrophages
(Figure 14). Like diprovocim-1, each was more effective in the human THP-1 cells than mouse
macrophages (>10-fold, typically ca. 100-fold). Little distinction was observed whether the lipid chain
was attached via the (R)- or (S)-pyrrolidine-3-carboxamide. A preference for the lipid chain length was
observed with the intermediate lengths displaying the greatest potency (131/135 > 132/136) and the
longest chain examined (130/134) seemed to display a uniquely detrimental effect in mouse versus
human cells, likely representing species distinctions in the signaling receptors. The best in the series
were >20-fold less potent than diprovocim-1 in human THP-1 cells and >200-fold less effective in
mouse macrophages, comparable in their activity with diprovocim-4 or Pam2CSK4 and more effective
than diprovocim-2.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 21 of 32
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 14. Hybrid structures containing one lipid side chain and a total of three hydrophobic amide side chains.
EC50’s derived from dose-response curves for the stimulated release of TNF- from differentiated human THP-
1 cells or mouse macrophages.
An even more significant set of observations were made with hybrid structures that contained
two such lipid side chains. The first such series examined is composed of compounds that contain
four side chains, linking one half of diprovocim-1 with symmetrical diamides derived from (S,S)-
pyrrolidine-3,4-dicarboxylic acid that bear two lipid side chain amides (Figure 15). Single step
preparation of the (S,S)-pyrrolidine-3,4-dicarboxamides, obtained by coupling (S,S)-14 with the
corresponding alkyl amines (EDCI, HOAt, 2,6-lutidine, DMF–CH2Cl2, 25 oC), followed by Boc removal
o
(4 N HCl, dioxane, 25 C) and coupling of the resultant amine with 125 (EDCI, HOAt, 2,6-lutidine,
DMF–CH2Cl2, 25 oC, 3–4 h) provided the series of hybrid structures 138–144 containing two lipid side
chains. Because of the activity observed, a comprehensive set of lipid side chain lengths was
prepared and examined. Three compounds in this series (141–143), containing the intermediate lipid
chain lengths (6–10 carbons), exhibited remarkably potent and robust activity (EC50 = 280–170 pM,
THP-1 cells), being more potent than Pam3CSK4 and approaching the activity of diprovocim-1. The
compounds with the longer lipid chain lengths, although still very potent, displayed progressively less
potent activity (140 > 139 > 138) and the compound with the shortest chain length (144) was notably
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 22 of 32
even less potent. The potency difference between stimulation of human versus mouse cell release of
TNF- smoothly and progressively diminished as the chain length was shortened with 143 exhibiting
the most potent activity in both cell lines and a 20-fold differential. The behavior of 143, containing
two C6 lipid side chains that closely approximates the length and number of carbon atoms found the
diprovocim-1 and diprovocim-2 side chains, nearly matches that of diprovocim-1 in both the human
THP-1 cells and mouse macrophages (EC50 = 180 pM and 4 nM, respectively), being only 2-fold and
4-fold less potent. Because of this lipid chain length of 6 carbons, and its incidental chronological
discovery, it is a compound we now refer to as diprovocim-6 (143).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A second series of such hybrid structures was prepared and examined in which two lipid side
chain amides were incorporated at opposite ends of the diprovocim-1 structure, each replacing one of
the (1S,2R)-2-phenylcyclopropylamine amide substituents. The compound in this series (147) that
contained the intermediate lipid chain length (six carbons), most closely approximating the length of
the 2-phenylcyclopropylamine amide substituents, exhibited excellent activity (EC50 = 800 pM, THP-1
cells), being slightly more potent than Pam3CSK4 and only 8-fold less active than diprovocim-1
(Figure 15). This compound series also exhibited a smooth and pronounced trend of diminished
activity as the chain length of the lipid amide substituent increased (potency: 147 > 148 > 149). In
addition, the activity of 147–149 in mouse macrophages was more significantly reduced relative to
diprovocim-1 (ca. 1000-fold) and the activity differential in human THP-1 cells versus mouse
macrophages increased (30–1000 vs 10-fold). Notably, the synthetic precursors 145 and 146 from
which 147–149 were prepared, which lack two of the four hydrophobic side chain amides, were found
to be inactive.
ACS Paragon Plus Environment

Page 23 of 32
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Figure 15. Hybrid structures containing two lipid side chains and a total of four hydrophobic amide side chains.
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Additionally, a series of compounds in which all four side chains of diprovocim-1 were replaced
with a comprehensive set of lipid side chain amides was prepared and examined (Figure 16). The
(S,S)-pyrrolidine-3,4-dicarboxamides that contained the two amide lipid side chains (16–4 carbons)
were coupled in a single step with benzene-1,4-dicarboxylic acid (EDCI, HOAt, 2,6-lutidine, DMF–
CH2Cl2, 25 oC, 4–5 h) to provide 150–156. No compound in this series proved capable of stimulating
the release of TNF- from either in the human THP-1 cells or mouse macrophages (EC50 >5 M).
However, this series of compounds also displayed especially poor solubility properties and we cannot
rule out that this contributed to the lack of activity observed in the cell-based functional assays.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 24 of 32
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 16. Compounds that contain a full set of four lipid chain amides.
A final series of diprovocim-1 analogues was examined that was composed of compounds in
which a single amide substituent was replaced with a lipid amide (Figure 17). These were prepared
o
by coupling of 125 (EDCI, HOAt, 2,6-lutidine, DMF–CH2Cl2, 25 C, 3–4 h, 51%) to provide the mono
methyl ester 157. Hydrolysis of the methyl ester to provide the carboxylic acid 158 (LiOH,
o
THF/MeOH/H2O, 25 C, 2.5 h, 99%) followed by its coupling with the corresponding alkyl amines
provided 159–166. These compounds also displayed well-defined activity trends, but one where the
stimulated release of TNF- from human THP-1 cells increased as the lipid chain length increased
through and up to C14 (potency: 166 (C16) < 165 (C14) > 164 (C12), 163 (C10) > 162 (C8), 161 (C6)
> 160 (C4) > 159 (C2)). Compound 164 matches and 165 slightly exceeds the stunning potency of
diprovocim-1.
ACS Paragon Plus Environment

Page 25 of 32
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 17. Hybrid diprovocims incorporating a single lipid chain amide.
Discussion and Conclusions
Diprovocim-1 has been examined in vitro and in vivo and the results of these studies have
been reported elsewhere.34 Although the target identification for leads that emerge from an undirected
functional assay such as the one used in this work is usually challenging, especially if the lead
compounds bear no structural resemblance to known effector molecules, it proved straightforward for
3 and 4. By design, it was recognized that subsequent assay of active compounds against
macrophages from available mice that bear germline genetic defects or knockouts of the genes
encoding each of the TLRs, other pattern recognition receptors, or downstream signaling molecules
could be used to establish whether the activity was derived from known or unrecognized targets.46 By
using this approach, the TNF- production induced by 3 was established to be solely dependent on
both TLR1 and TLR2, independent of all other TLRs including TLR6 (Figure 18), and independent of
other molecular pattern recognition receptors (not shown).34 The activity of diprovocim-1 was further
shown to be dependent on TLR1/TLR2 signaling through MyD88, TIRAP, and IRAK4 (Figure 18) and
3 has been shown to induce the phosphorylation and activation of IKK, IKK, p38, JNK, and ERK,
activating conventional TLR1/TLR2 downstream signaling through both the MAPK and NF-B
pathways.34 Diprovocim-1 (3) acts as an adjuvant in mice when administered by conventional means
(intramuscular, i.m.) with an antigen (OVA) and it was established that this in vivo activity (i.e.;
vaccination) is dependent on both TLR1 and TLR2 (inactive in both TLR1 and TLR2 deficient mice).34
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 26 of 32
Most significantly, diprovocim-1 was shown to act synergistically with checkpoint blockade (anti-PD-
L1) where the combined treatment, but neither alone, cured or prolonged the survival of mice
implanted with antigen bearing murine melanoma (B16-OVA) and systemically protected mice from
tumor re-challenge in an antigen specific manner (antigen-specific anti-tumor immunity).34 The in vivo
activity was observed with the anti-cancer vaccine (diprovocim-adjuvanted OVA) administration
intramuscularly (i.m.) and distal from the tumor, not intratumoral as has been a convention in recent
related studies.19d,48 Diprovocim-1 displayed no intrinsic cytotoxic activity in cell culture (IC50 >10 M),
not only exhibiting no cell growth inhibition against mouse macrophages or human THP-1 cells used
in the assays, but also when tested against L1210 mouse leukemia, HCT116 human colon cancer,
B16 mouse melanoma, and human foreskin (HFF) or mouse (NIH/3T3) fibroblast cells in culture (not
shown).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 18. Top: Assay for stimulated TNF- release upon treatment with 3 (500 nM) using macrophages from
mice containing disabling germline mutations or knockouts of genes encoding key mTLRs and downstream
signaling proteins. Bottom: Assay for stimulated TNF- release upon treatment with 3 (250 pM) versus controls
without compound using differentiated THP-1 cells that is blocked by antibodies that bind either TLR1 or TLR2.
All results are representative of two independent experiments and error bars represent SEM.
As detailed herein, it was a screen conducted with nearly 100,000 compounds and a surrogate
functional assay for stimulation of an immune response that led to the discovery of the diprovocims.
ACS Paragon Plus Environment

Page 27 of 32
Journal of the American Chemical Society
Unique to these efforts, the screening leads for this new class of activators of an immune response
came from a compound library specifically designed to promote cell surface receptor dimerization.33
Such receptor dimerizers,33 a subclass of chemical inducers of dimerization (CID),49 are composed of
two linked monomers, where each monomer is incorporated and functions to bind one of the two
receptor proteins, promoting or stabilizing receptor dimerization (Figure 19). With the diprovocims
(dimer provocation of an immune response) and TLR1/2, this is also accomplished with receptor
dimer adoption of an activated, signaling receptor complex (agonist vs antagonist). X-ray
crystallographic studies of a (diprovocim-1)2/(TLR2)2 complex, computational modeling of diprovocim-
1 binding to TLR1/TLR2 heterodimer, and subsequent TLR1 and TLR2 mutagenesis studies indicate
binding of diprovocim-1 at the site of and in a manner analogous to Pam3CSK4.47 One amide side
chain substituent extends into the hydrophobic pocket in TLR1 and two adjacent amide substituents
bind in the TLR2 hydrophobic channel, filling a long continuous hydrophobic pocket spanning both
proteins at the TLR1/TLR2 heterodimer interface analogous to the three lipid chains of Pam3CSK4.
The remaining fourth amide substituent of diprovocim-1 binds along and further stabilizes the
TLR1/TLR2 dimer interface. This is further supported by the results of the studies detailed herein
where certain lipid amides can effectively replace the diprovocim side chain amides in selected
instances (e.g.; 165).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 19. One half of diprovocim each binds a receptor protein, promoting receptor dimerization and
activation.
The systematic SAR studies detailed herein improved the potency 800-fold over that of the
lead compounds, providing diprovocim-1 and diprovocim-2. The diprovocims act by a well-defined
mechanism (TLR1/TLR2 agonist), bear no structural similarity to any known natural or synthetic TLR
agonist, are easy to prepare and synthetically modify, and selected members are active in both
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 28 of 32
human and murine systems. The most potent diprovocim (3, diprovocim-1) elicits full agonist activity
at extraordinarily low concentrations (EC50 = 110 pM) in human THP-1 cells, being more potent than
any other known small molecule TLR agonist, including the naturally-derived TLR1/TLR2 agonist
Pam3CSK4. Complementary to the studies conducted to date with diprovocim-1,34 further studies of
this class of synthetic TLR1/TLR2 agonists as new organic vaccine adjuvants in infectious diseases
and oncology, as adjuvants in vaccine prophylactics against pathogen exposure (e.g.; biodefense),
and as immunostimulators alone or in combination with other treatments, particularly in oncology, are
underway. It is worth highlighting that the characteristics of such an immune stimulator are expectedly
different than those of a conventional drug.17 It is administered infrequently as part of prime and boost
vaccinations, it is typically administered i.m., the vaccination not only provides long term systemic
protection but does so with a localized low dose administration, and the acceptable chemical (e.g.;
Mwt), physical (e.g.; low solubility and high cLogP), PK (e.g.; short vs long circulating half-life in
blood), permeability (low vs high), and metabolic stability properties are distinct from those of a
traditional drug.17 Most notably and because of the importance of the field, the diprovocims represent
a welcomed new addition to the limited number of known TLR agonists. Most are mimics or
modifications of microbial components (e.g.; LPS, lipopeptides, nucleic acids) and represent
unattractive starting points for drug discovery, many are difficult to structurally modify (e.g.; covalent
antigen-adjuvant construct) and chemically prepare, and only a limited number of other synthetic
small molecule classes have been found to behave as TLR agonists. As a result, we expect many
others will find the diprovocims especially attractive to work with.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ASSOCIATED CONTENT
Supporting Information
Full experimental details. This material is available free of charge via the internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*D.L.B.: e-mail, dale.boger@outlook.com.
*B.B.: e-mail, bruce.beutler@UTSouthwestern.edu.
Note
Competing interests: B.B., D.L.B. and H.Z. have financial interests in Tollbridge Therapeutics, which
has licensed the patent on the diprovocims.
ACKNOWLEDGMENT
We gratefully acknowledge the financial support of the National Institutes of Health (AI082657 and
CA042056, D.L.B.; AI082657, AI100627, and AI125581, B.B.; GM104496, H.Z.). We thank
ACS Paragon Plus Environment

Page 29 of 32
Journal of the American Chemical Society
Aleksandar Radakovic and Jelena Momirov for conducting the screening for cytotoxic activity and
Kevin Litwin for the Figure 19 and TOC graphics.
1
2
3
4
5
References
1. Beutler, B. Mol. Immunol. 2004, 40, 845–859.
2. Hoebe, K.; Janssen, E.; Beutler, B. Nat. Immunol. 2004, 5, 971–974.
6
7
8
9
3. (a) Moresco, E. M. Y.; LaVine, D.; Beutler, B. Curr. Biol. 2011, 21, R488–R493. (b) Blasius, A. L.;
Beutler, B. Immunity 2010, 32, 305–315. (c) Kawai, T.; Akira, S. Nat. Immunol. 2010, 13, 373–384.
(d) Beutler, B. Blood 2009, 113, 1399–1407.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4. (a) Hashimoto, C.; Hudson, K. L.; Anderson, K. V. Cell 1988, 52, 269–279. (b) Lemaitre, B.;
Nicolas, E.; Michaut, L.; Reichhart, J. M.; Hoffmann, J. A. Cell 1996, 86, 973–983.
5. (a) Poltorak, A.; He, X.; Smirnova, I.; Liu, M.-Y.; Van Huffel, C.; Du, X.; Birdwell, D.; Alejos, E.;
Silva, M.; Galanos, C.; Freudenberg, M. A.; Ricciardi-Castagnoli, P.; Layton, B.; Beutler, B. Science
1998, 282, 2085–2088. (b) Beutler, B.; Du, X.; Poltorak, A. J. Endotoxin Res. 2001, 7, 277–280. (c)
Beutler, B.; Rietschel, E. T. Nat. Rev. Immunol. 2003, 3, 169–176.
6. Medzhitov, R.; Preston-Hurlburt, P.; Janeway, C. A. Jr. Nature 1997, 388, 394–397.
7. (a) Vogel, F. R. Clin. Infect. Dis. 2000, 30 (Suppl 3), S266–S270. (b) Guy, B. Nat. Rev. Microbiol.
2007, 5, 505–517. (c) Coffman, R. L.; Sher, A.; Seder, R. A. Immunity 2010, 33, 492–503.
8. (a) Czarniecki, M. J. Med. Chem. 2008, 51, 6621–6626. (b) Peri, F.; Calabrese, V. J. Med. Chem.
2014, 57, 3612–3622.
9. Wang, X.; Smith, C.; Yin, H. Chem. Soc. Rev. 2013, 42, 4859–4866.
10. (a) Hennessy, E. J.; Parker, A. E.; O’Neill, L. A. J. Nat. Rev. Drug Discovery 2010, 9, 293–301. (b)
Meyer, T.; Stockfleth, E. Expert Opin. Invest. Drugs 2008, 17, 1051–1065. (c) Hoebe, K.; Jiang, Z.;
Georgel, P.; Tabeta, K.; Janssen, E.; Du, X.; Beutler, B. Curr. Pharmaceut. Des. 2006, 12, 4123–
4134. (d) Kanzler, H.; Barrat, F. J.; Hessel, E. M.; Coffman, R. L. Nat. Med. 2007, 13, 552–559.
11. (a) Amlie-Lefond, C.; Paz, D. A.; Connelly, M. P.; Huffnagle, G. B.; Whelan, N. T.; Whelan, H. T.
J. Allergy Clin. Immunol. 2005, 116, 1334–1342. (b) O’Neill, L. A.; Bryant, C. E.; Doyle, S. L.
Pharmacol. Rev. 2009, 61, 177–197.
12. (a) Aldous, A. R.; Dong, J. Z. Bioorg. Med. Chem. 2018, 26, 2842–2849. (b) Sahin, U.; Tureci, O.
Science 2018, 359, 1355–1360. (c) Ribas, A.; Wolchok, J. D. Science 2018, 359, 1350–1354.
13. (a) Engel, A. L.; Holt, G. E.; Lu, H. Expert Rev. Clin. Pharmacol. 2011, 4, 275–289.
14. (a) Prins, R. M.; Craft, N.; Bruhn, K. W.; Khan-Farooqi, H.; Koya, R. C.; Stripecke, R.; Miller, J. F.;
Liau, L. M. J. Immunol. 2005, 176, 157–164. (b) Lee, J.; Chuang, T.-H.; Redecke, V.; She, L.; Pitha,
P. M.; Carson, D. A.; Raz, E.; Cottam, H. B. Proc. Natl. Acad. Sci. USA 2003, 100, 6646–6651. (c)
Lee, J.; Wu, C. C. N.; Lee, K. J.; Chuang, T.-H.; Katakura, K.; Liu, Y.T.; Chan, M.; Tawatao, R.;
Chung, M.; Shen, C.; Cottam, H. B.; Lai, M. M. C.; Raz, E.; Carson, D. A. Proc. Natl. Acad. Sci. USA
2006, 103, 1828–1833.
15. Smits, E. L. J. M.; Cools, N.; Lion, E.; Camp, K.; Ponsaerts, P.; Berneman, Z. N.; Van Tendeloo,
V. F. I. Cancer Immunol. Immunother. 2010, 59, 35–46.
16. Beesu, M.; Malladi, S. S.; Fox, L. M.; Jones, C. D.; Dixit, A.; David, S. A. J. Med. Chem. 2014, 57,
7325–7341.
ACS Paragon Plus Environment