Small-Molecule Inhibitors of the CD40-CD40L Costimulatory Protein-Protein Interaction

Article abstract of DOI:10.1021/acs.jmedchem.7b01154

Costimulatory interactions are required for T cell activation and development of an effective immune response; hence, they are valuable therapeutic targets for immunomodulation. However, they, as all other protein-protein interactions, are difficult to target by small molecules. Here, we report the identification of novel small-molecule inhibitors of the CD40-CD40L interaction designed starting from the chemical space of organic dyes. For the most promising compounds such as DRI-C21045, activity (IC₅₀) in the low micromolar range has been confirmed in cell assays including inhibition of CD40L-induced activation in NF-κB sensor cells, THP-1 myeloid cells, and primary human B cells as well as in murine allogeneic skin transplant and alloantigen-induced T cell expansion in draining lymph node experiments. Specificity versus other TNF-superfamily interactions (TNF-R1-TNF-α) and lack of cytotoxicity have also been confirmed at these concentrations. These novel compounds provide proof-of-principle evidence for the possibility of small-molecule inhibition of costimulatory protein-protein interactions, establish the structural requirements needed for efficient CD40-CD40L inhibition, and serve to guide the search for such immune therapeutics.

Full text of DOI:10.1021/acs.jmedchem.7b01154

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Article
Small-Molecule Inhibitors of the CD40–CD40L
Costimulatory Protein-Protein Interaction
JINSHUI CHEN, Yun Song, Damir Bojadzic, Alejandro Tamayo-
Garcia, Ana Marie Landin, Bonnie B. Blomberg, and Peter Buchwald
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01154 • Publication Date (Web): 12 Oct 2017
Downloaded from http://pubs.acs.org on October 12, 2017
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Small-Molecule Inhibitors of the CD40–CD40L Costimulatory Protein-
Protein Interaction
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Jinshui Chen , Yun Song , Damir Bojadzic , Alejandro Tamayo-Garcia , Ana
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Marie Landin , Bonnie B. Blomberg , and Peter Buchwald *
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Diabetes Research Institute, Molecular and Cellular Pharmacology, and Microbiology and
Immunology, Miller School of Medicine, University of Miami, Miami, Florida 33136, USA
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These authors contributed equally to the present work.
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ABSTRACT
Costimulatory interactions are required for T cell activation and development of an effective
immune response; hence, they are valuable therapeutic targets for immunomodulation.
However, they, as all other protein-protein interactions, are difficult to target by small
molecules. Here, we report the identification of novel small-molecule inhibitors of the CD40–
CD40L interaction designed starting from the chemical space of organic dyes. For the most
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promising compounds such as DRI-C21045, activity (IC ) in the low micromolar range has been
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confirmed in cell assays including inhibition of CD40L-induced activation in NF-κB sensor cells,
THP-1 myeloid cells, and primary human B cells as well as in murine allogeneic skin transplant
and alloantigen-induced T cell expansion in draining lymph node experiments. Specificity versus
other TNF-superfamily interactions (TNF-R1–TNF-α) and lack of cytotoxicity have also been
confirmed at these concentrations. These novel compounds provide proof-of-principle evidence
for the possibility of small-molecule inhibition of costimulatory protein-protein interactions,
establish the structural requirements needed for efficient CD40–CD40L inhibition, and serve to
guide the search for such immune therapeutics.
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INTRODUCTION
It is now well-recognized that co-stimulatory and co-inhibitory signaling play crucial
roles in the activation of immune responses and in T cell biology as they determine the
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functional outcome of T cell receptor (TCR) signaling . In addition to the engagement of the
TCR by the MHC-peptide complex, T cell activation also requires the ligation of costimulatory
molecules on T cells with their respective ligands on antigen-presenting cells (APCs). If this
second signal is lacking, T cells will not be activated and will not undergo proliferation, cytokine
production, or further differentiation into effector cells. While the underlying mechanisms are
not entirely understood, it is generally believed that antigen recognition (TCR) in the absence of
costimulation can alter the immune response and lead to tolerance. Consequently,
costimulatory modulation could provide immunomodulatory (IM) agents that are activation-
and antigen-specific, thus, safer and more effective than existing IM therapies. Costimulatory
interactions have emerged as particularly valuable therapeutic targets in transplant recipients
2-8
and in autoimmune diseases for exactly these reasons . They are cell surface protein-protein
interactions (PPIs) that belong to two main families: the immunoglobulin superfamily (e.g.,
CD28/CTLA4–CD80/86 and ICOS–ICOS-L) and the TNF–TNFR superfamily (TNFSF).
Among TNFSF interactions, we are particularly interested in the blockade of the CD40–
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CD40L interaction (Figure 1)
because this seems to be particularly effective in islet
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transplantations
. This pathway is also a promising target in a number of autoimmune
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diseases
, including type 1 diabetes (T1D)
. In fact, CD40 (TNFRSF5) and CD40L (CD154,
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TNFSF5) were the first TNFSF costimulatory molecules identified , and this PPI is among the
most extensively studied TNFSF members. CD40 is a transmembrane glycoprotein constitutively
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expressed on APCs, whereas its ligand, CD40L, exists both as a transmembrane protein and a
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soluble form in the plasma
. The interaction between CD40L on T cells and its receptor CD40
on B cells is essential for lymphocyte signaling leading to T cell-dependent B cell proliferation,
immunoglobulin class switching, and B-cell maturation. Mutations of CD40L expressed on T
cells are known to result in X-linked hyper-IgM syndrome (XHIGM), a primary immunodeficiency
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characterized by an inability to produce immunoglobulins of IgG, IgA, and IgE isotypes ,
indicating the important role of CD40–CD40L in CSR (class switch recombination). In addition to
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the role played by the CD40–CD40L interaction in immune responses , there is increasing
evidence for its role in atherosclerosis, cardiovascular disease, acute coronary syndrome,
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thrombosis, inflammation, inflammatory bowel disease, and even metabolic syndrome
.
Intriguingly, recent evidence also indicates that the CD40–CD40L interaction plays an important
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role in glucolipotoxicity-induced β-cell death .
Consequently, inhibition of CD40 signaling can be beneficial in the pathogenic processes
of chronic inflammatory diseases, such as autoimmune diseases, neurodegenerative disorders,
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graft-versus-host disease, cancer, and atherosclerosis . Multiple antibodies targeting this
interaction have been developed and are in preclinical or clinical development – for example,
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bleselumab, lucatumumab, dacetuzumab, and others . Clinical trials of ruplizumab (hu5c8), an
anti-CD40L humanized antibody, looked promising; however, they have been halted because of
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possible thrombolytic side effects
, and development is no longer supported . Activated
platelets express CD40L; however in platelet-rich plasma, the 5c8 antibody by itself did not
induce platelet aggregation per se and did not significantly affect maximal aggregation. This
side effect may be partly due to the antibody nature of the therapy, and a pro-aggregation
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effect of the antibody by a mechanism involving the mAb Fc domain has been suggested .
Along these lines, more recently developed so-called Fc-silent domain antibodies (dAbs) that do
not bind to FcgRIIa, including letolizumab, were found to retain immunomodulatory activity,
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but do not activate platelets
.
Antibodies (immunoglobulins) can be highly specific for their targets and quite stable in
human serum; however, being protein therapeutics, their development is often hindered by the
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possibility of a strong immune response mounted against them . Even among novel
therapeutics approved by the FDA, post-market safety events were found to be significantly
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more frequent among biologics . Development of IM biologics is further complicated by the
high likelihood of unwanted adverse reactions that include serious infections, malignancy,
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cytokine release syndrome, anaphylaxis, hypersensitivity, and immunogenicity . Out of 40
licensed IM biologics, 18 have been associated with serious infections, including reactivation of
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bacterial, viral, fungal, and opportunistic infections . Peptide inhibitors have been explored as
possible alternatives. For example, a CD40–CD40L blocking cyclic heptapetide (CLPTRHMAC)
has been shown not to prime human platelet activation and aggregation in in vitro platelet
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activation studies, contrary to the anti-CD40L mAb also tested . More recently, a CD40-
targeting peptide (VLQWAKKGYYTMKSN designated as mouse KGYY ), which was designed
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from the CD40L domain critical for interaction with CD40, has been shown to be effective in
preventing T1D in NOD (non-obese diabetic) mice, a well-known animal model of this disease
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In addition to CD40, CD40L also binds to integrins, such as α β and Mac-1 (α β ),
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mediating different inflammatory pathologies including atherosclerosis, and these interactions
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could also be blocked by peptide inhibitors
. However, peptides, just as antibodies, are
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unlikely to be formulated for oral administration, which is an important consideration for
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prospective T1D preventive therapies that have to be sufficiently patient friendly (to allow
the long-term adherence and compliance needed for a successful preventive therapy). An IM
therapy that could prevent or revert recent-onset T1D is of particular interest as all existing IM
therapies tested to date in large-scale human clinical trials have failed to stop the progressive
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decline of the function of insulin-producing pancreatic β-cells
. Even the most successful
immunological interventions (e.g., cyclosporine, teplizumab, otelixizumab, rituximab, and
abatacept) achieved no more than a few months delay in the decline of insulin production, and
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prevention with parenteral insulin or GAD-vaccine did no better
. Currently, T1D is one of
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the few remaining autoimmune diseases without any approved immunological treatment .
We decided, therefore, to focus on identifying small-molecule compounds that can
interfere with the CD40–CD40L costimulatory PPI. However, PPI inhibition with small-molecule
compounds is challenging and, for a long time, has been considered unlikely to be successful.
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PPIs usually involve relatively large protein surfaces (1,500–3,000 Å )
and lack the well-
defined binding pockets present on traditional targets of most existing drugs, such as G-protein
coupled receptors, ion channels, and enzymes, making it difficult for small molecules to bind
with high enough energy / affinity. Within the last few years, however, sufficiently effective
small-molecule inhibitors have been identified for several important PPIs, and there are now
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>
40 PPIs targeted by small molecules that are in preclinical development
. Venetoclax,
which was identified from a small-molecule series designed to target PPIs in the B cell
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lymphoma 2 (BCL-2) family , has just received FDA approval for clinical use . Lifitegrast, a
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small molecule LFA-1–ICAM-1 inhibitor , has also been approved recently by the FDA for the
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treatment of dry eye . Focusing on the CD40–CD40L PPI, we have first identified suramin as a
small-molecule inhibitor with a half-maximal inhibitory concentration (IC ) in the 50 ꢀM range
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and then a set of organic dyes such as those illustrated by structures 1–4 (Figure 2) that
concentration-dependently inhibited this costimulatory PPI with activities in the low
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micromolar range
. Among organic dyes, we have also identified small-molecule inhibitors
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of the OX40–OX40L costimulatory PPI as well as the first promiscuous small-molecule PPI
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inhibitor
. Here, we present a set of new and more potent small-molecule inhibitors of the
CD40–CD40L interaction (5–11) that were designed starting from the chemical space of these
organic dyes (1–4, Figure 2), but are more drug-like and no longer contain color-causing
chromophores.
RESULTS
Design
To identify novel small-molecule compounds that can inhibit the CD40–CD40L
costimulatory protein-protein interaction (PPI) (Figure 1), we started from our previously
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identified organic dye inhibitors such as 1 or 4 (Figure 2)
and synthesized new chemical
entities that maintain the main molecular framework, but lack the aromatic azo chromophores
responsible for their vivid color. Compounds explored here (5–11, Figure 3) maintain the
aromatic ring scaffold, but the azo linkers are replaced with amide ones. We used an iterative
design, synthesis, testing, and redesign approach, and found that several structural elements
need to be maintained for activity. We originally started with structures with a single ring linker
(
such as in 5, Figure 3), but found that use of a two ring linker (such as in 6) significantly
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improved activity as illustrated by the three orders of magnitude improvement in IC s (0.3 vs.
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substitutions on both terminal aromatic rings (R , R ) also showed considerably diminished
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activity. Hence, structures tested here all have two-ring linkers (i.e., 6 – 11, Figure 3).
Synthesis
All new compounds were synthesized following a similar route that involved a general
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procedure of amide coupling (Scheme 1). The synthesis of compound 6 (DRI-C21041) is
shown for illustration. It involved three steps consisting of two amide couplings (using a
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modified version of the procedure of Venkatraj and co-workers ) and one hydrogenation
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(
using a modified version of the procedure of Kassack and co-workers ) (Scheme 1). The first
amide coupling of the commercially available amine 12 and acid 13 gave the nitro derivative 14.
The following hydrogenation provided the amine derivative 15. The second amide coupling of
the amine 15 and the acid 16 furnished the final product 6 (total yield 49%). The synthesis of
most other compounds was achieved via the same three steps using the biphenyl as an
aromatic linker in the first step (a) and different acids as coupling partners in the third step (c).
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Compound 8 (DRI-C21042) was simply synthesized in one step by hydrogenation of 6.
Compounds 7 and 11 were assembled using the corresponding differently substituted naphthyl
rings. Details are summarized in the experimental section.
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Binding Inhibition
CD40–CD40L
The ability of compounds 5–11 (Figure 3; Table 1) to inhibit the CD40–CD40L interaction
was quantified using a cell-free in vitro binding inhibition assay measuring the amount of
soluble CD40L (CD154) bound to plate-coated CD40 in the presence of increasing
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concentrations of test compounds as described before
. Organic dye 1 from our previous
work was used as reference control. As the binding inhibition data show (Figure 4A),
compounds 6, 7, 9–11 concentration-dependently inhibited the CD40–CD40L interaction in this
assay with activities in the high nanomolar to low micromolar range as indicated by the
corresponding IC values. In general, concentration-dependencies were adequately described
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by a standard log inhibitor vs. response model (i.e., a classical sigmoid binding function with a
Hill slope of 1). All IC values shown in Table 1 were obtained by fitting this model. Compounds
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6
, 10 (DRI-C21045), and 11 (DRI-C25441) showed particularly promising activities with IC50
values of 0.31, 0.17, and 0.36 ꢀM, respectively – about an order of magnitude better than our
reference dye compound 1 (direct red 80). Meanwhile, compounds 5, with a single ring linker,
and 8, with an amine substituent, showed very little activity (IC > 300 ꢀM) – clear evidence of
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structural selectivity at the binding site (see Discussion). Because polymolecular
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conglomeration
/ aggregation
is a frequent cause of promiscuous inhibition in
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screening assays , we have checked for this by adding a non-ionic detergent (Triton-X 100,
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.01%) to the binding inhibitory assay, as recommended for the detection of such effects .
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This already has been shown to have no effect for our original leads , not even for the
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promiscuous PPI inhibitor erythrosine B (ErB) . We have rechecked again here for the present
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compounds, and the presence of the detergent caused slight, but no significant shifts in the
IC s of all compounds, e.g., 10 (1.2×), 11 (1.9×), or ErB (1.04×) with the exception of 6, our least
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soluble compound, which was more strongly shifted (8.0×). An illustrative example is included
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in Supporting Information Figure S7.
Selectivity/specificity
In addition to adequate activity, sufficient selectivity and specificity are also desirable.
Typically, small-molecule compounds are considered to be promising enough leads if they show
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30–100-fold selectivity over other possible pharmacological targets of interest
. To check
for this, we assessed the ability of these compounds to inhibit the TNF-R1–TNF-α interaction,
which is part of the same TNF superfamily receptor-ligand interaction (Figure 4B). All active new
compounds showed >100 fold selectivity in their ability to inhibit the CD40 vs. the TNF
interaction (Table 1), which further supports structural specificity and argues against a possible
promiscuous (e.g., aggregation-related) effect. Because in addition to its CD40 receptor, CD40L
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also binds to integrins such as Mac-1 (α β ) , we tested the ability of the present compounds
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2
to inhibit this interaction using a similar assay. Most of the tested compounds showed similar
activity (Supporting Information Figure S1) with the dye control 1 and the amine 8 showing
about 10-fold increased inhibitory potency.
In Vitro Activity
Inhibition of CD40L-induced NF-κB activation
To confirm the activity of our compounds in a cell-based model, we used sensor cells
containing a secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control
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of a promoter fused to an NF-κB binding site and transfected with CD40. In these cells (HEK
Blue CD40) the secretion of SEAP is specifically induced by the binding CD40L to its cell-surface
receptor (CD40) and the downstream activation of the corresponding NF-κB pathway. A
monoclonal anti-CD40L antibody was used as a positive control. All tested compounds (1 as well
as 6, 10, and 11) showed concentration-dependent inhibition of the activation of NF-κB in these
cells that was induced by CD40L as shown in Figure 5 with activities in the low micromolar
range (Table 1). For all compounds (including the reference compound 1), the corresponding
IC s are about an order of magnitude higher than those determined in the cell-free binding
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inhibition assay (Table 1), a loss of activity most likely due to the presence of proteins and cells
in this assay causing some loss by non-specific binding.
Inhibition of CD40L-induced marker expression in THP cells
CD40L also induces various responses in dendritic cells, human myeloid THP-1 cells, and
other human acute myeloid leukemia (AML) cells. These responses include, for example,
morphological changes, cell proliferation, rescue from apoptosis, secretion of IL-2 and other
cytokines, as well as the up-regulation of the expression of adhesion and costimulatory
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molecules such as CD54, CD86, CD80, MHC-II, and CD40 itself . As before , as an additional in
vitro cell assay to assess the inhibitory potential of the present compounds, we quantified their
effect on the expression of CD40L-induced MHC-II upregulation in THP-1 cells, which can serve
as surrogate dendritic cells (Supporting Information Figure S2). CD40L alone indeed induced
increased expression, and this effect was concentration-dependently inhibited by the tested
compounds (6, 10, and 11) (Supporting Information Figure S2).
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Cyto- and genotoxicity
In parallel with the above experiments, cytotoxicities were also evaluated using a
standard MTT assay; on one hand, to assess the safety of these compounds, and on the other,
to ensure that the effects seen are not due to cell toxicity effects. Except for the amine
substituted compound 8, which had no significant CD40–CD40L inhibitory activity anyway, and
the carboxylic acid substituted 11, which was the only compound lacking an aromatic sulfonic
acid substituent, the other compounds showed no signs of cytotoxicity for concentrations of up
to 100 and 200 ꢀM. Among the most active compounds that were also tested in cell-based
assays (6, 10, and 11), only 11 showed a significant effect on viability at 100 ꢀM (Supporting
Information Figure S3). The mutagenic potential was also assessed for two compounds (6 and
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0) using the Muta-Chromplate assay, which is a 96-well micro-plate version of the Salmonella
typhimurium Ames test. As shown in Supporting Information Figure S4, neither of these
compounds was found to have genotoxic potential as assessed by this assay for the
concentrations tested of up to 500 ꢀM.
Inhibition of CD40L-induced B cell proliferation
CD40 stimulation is an important signal for B cell proliferation, and soluble CD40L can
67, 68
induce proliferation of human B cells in a concentration-dependent manner. As before
, we
used a CD40L-induced proliferation of primary human B cells assay to assess the inhibitory
effect of our new small-molecule compounds. As Supporting Information Figure S5 shows,
CD40L-induced B cell proliferation was concentration-dependently inhibited by compounds 6,
0, and 11 in statistically significant manner at concentrations higher than 10 ꢀM. As expected,
the CD40L antibody included as a positive control showed inhibitory activity with a half-
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maximal inhibitory concentration of about 1 nM in a manner consistent with our previous
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results .
Inhibition of CD40L-induced B cell activation
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As an even more relevant activity assessment, we tested the ability of compounds 6, 10,
and 11 to block CD40L-induced functional activation of primary B cells. Immunoglobulin class
switch DNA recombination (CSR) and somatic hypermutation (SHM), which are critical for the
maturation of antibody responses to antigens in B cells, are tightly controlled by the activity of
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activation-induced cytidine deaminase (AID) . AID is encoded by the AICDA gene that is
exclusively expressed in B cells and is necessary for opening the DNA molecule in the switch (S)
regions in order to allow CSR. The expression of AID is tightly regulated by multiple repressive
and enhancing elements. Engagement of CD40, which is constitutively expressed on the surface
of B cells, with CD40L on T cells has been shown to positively regulate the expression of AID in B
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cells through activation of NF-κB signaling pathway ( and references therein). In particular,
CD40-initiated NF-κB activation has been shown to synergize with interleukin-4 (IL-4) mediated
8
6
STAT6 signaling to induce optimal AID expression . To further validate the activity of our
CD40–CD40L small-molecule inhibitors, we generated an aicda-lentiviral construct containing
the promoter and enhancer region of aicda fused to the reporter Ds-Red, and used it to
transfect human B cells isolated from healthy donors. In these cells, regulation of AID
expression through CD40–CD40L signaling following incubation at different conditions can be
evaluated by Ds-Red level assessed via flow cytometry. As shown in Figure 6, IL-4 alone failed to
induce AID expression (<5% Ds-Red positive cells), but addition of CD40L produced good
activation (>25%). This was blocked by the CD40L antibody (mAb) used as a positive control (at
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a single concentration), and it was concentration-dependently blocked by compounds 6, 10,
and 11 with IC s of 13.2, 4.5, and 15.9 ꢀM, respectively (Figure 6, Table 1). A set of
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representative flow cytometry histograms illustrating the concentration-dependent response
obtained for compound 10 are shown in Supporting Information Figure S6. These are
particularly encouraging results as they were achieved using a true functional activity readout
and they were obtained in primary human cells.
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In Vivo Activity
Skin transplant model
A brief pharmacokinetic evaluation of compound 10 in mice indicated that, even if
elimination is relatively fast (elimination half-life, t , in plasma of approximately 2 h), effective
1
/2
concentrations can be reached following systemic administration (Figure 7). Consequently, as a
first in vivo test of the immunomodulating ability of the present compounds, we examined if
compound 10 could cause prolongation of skin allograft survival using a model similar to that of
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Pinelli and co-workers . Skin transplants are among the most stringent transplants as they are
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known to generate higher immune response than, liver, heart, or kidney transplants . Skin
grafts from Balb/c mice were transplanted in C57BL/6 mice that were treated with CTLA4-Ig
plus either a CD40L antibody (MR-1) as a positive control or compound 10 (30 mg/kg) (daily,
s.c.; in 20% hydroxypropyl-β-cyclodextrin, HPβCD). Mice treated with CTLA4-Ig alone rejected
skin grafts with a mean survival time (MST) of 12.0 days. Addition of 10 prolonged survival in a
statistically significant manner (MST of 15 days), but less than the positive control MR-1 (MST of
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9 days) (Figure 8). There were no signs of toxicity during the two week chronic treatment with
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Inhibition of alloantigen-induced immune response
The local administration of alloantigens induces T cell expansion in the draining lymph
nodes (DLNs), and the ability of test compounds to inhibit this is an indication of their
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immunosuppressive potency. To test the activity of our compounds, we used a model in
which splenocytes isolated from DBA-2 mice were injected in the footpad of Balb/c recipients
causing a significant (approximately eight-fold) increase in the number of cells in the draining
popliteal lymph node by day 3. Compound 10 (20–60 mg/kg; s.c.; b.i.d.) significantly inhibited
this in a dose-responsive manner and approaching the efficacy of the MR-1 anti-CD40L antibody
used as a positive control (Figure 9).
DISCUSSION
Identification of small-molecules capable of costimulatory modulation as possible IM
agents is of considerable interest. They could offer alternatives to antibodies and make oral
administration possible. Several peptides interfering with the CD40–CD40L interaction with
1
9, 39, 89-91
varying degrees of efficacy have been reported
; however, they are also unlikely to
lead to therapeutics with adequate oral bioavailability. Finding small molecules that can
interfere with costimulatory PPIs is undeniably challenging, but for TNFSF PPIs, several have
been identified recently that provide at least proof-of-principle evidence that such modulation
6
2
is possible, even if none have yet advanced to clinical development . Here, we report the first
drug-like small molecules that have been shown to inhibit the CD40–CD40L interaction with low
micromolar potency. In the cell-free binding inhibitory assay (Figure 4), several compounds
showed less than micromolar potency, e.g., IC values of less than 0.5 ꢀM for 6, 10, and 11
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(
Table 1), which were selected for further testing. For comparison, we have also tested a set of
CD40-targeting inhibitory peptides (KGYY ) that have been reported to be effective in
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preventing T1D in NODs . In our human CD40–CD40L binding assay, mouse KGYY15
(VLQWAKKGYYTMKSN), which was used in the NOD prevention study, showed no inhibitory
activity. Human KGYY (VLQWAEKGYYTMSNN) showed some inhibition, but only with an IC of
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154 ꢀM (Supporting Information Figure S7).
Previous small-molecule CD40–CD40L inhibitors include organic dyes such as 1–4
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identified by us
and (S)-2-(2’-(biphenyl-3-ylmethylcarbamoyl)-6,6’-bis((S)-2-(pyrrolidin-1-
ylmethyl)pyrrolidine-1-carbonyl)-4,4’-bipyridine-2-carboxamido)-2-cyclohexylacetic acid
BIO8898), a large structure with a 4,4’-bipyridine core and four arms, identified by Silvian and
(
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co-workers at Biogen . With compounds of the present study (5–11), there is evidence of
adequate selectivity and specificity (Table 1), and several structural elements required for
CD40–CD40L inhibitory activity have been identified. Among others, they include a chain of
aromatic rings, which is often seen in PPI inhibitors in general. While a single-ring linker seemed
to work in some of the azo dyes (e.g., 3 and 4), a linker with two aromatic rings (i.e., chain of
four aromatic rings) seems to be needed for our new amide-linked structures (e.g., 6 vs 5).
Presence of polar substituents at both ends is also needed for activity: compounds with an
unsubstituted or chlorine-substituted naphthalene ring as well as compounds without a polar
substituent on the terminal phenyl ring at the opposite end were inactive (data not shown). For
the sulfonyl substituent, position 1 seems to be somewhat favored (e.g., 6 vs. 7), and –COOH
could be an adequate substitution (cf. 11). At the other 4’ end, several substituents can
maintain activity, e.g., nitro (6), triazole (9), or ester (10) (Table 1), but amine (8) or halogen
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substituted compound completely lose CD40L inhibitory activity. All these agree well with the
general observation that biphenyl scaffolds and acidic (e.g., –COOH) moieties seem to be
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present with high frequency among good protein binders . It has also been suggested that
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chains of aromatic rings could act as α-helix mimetics and so are particularly well suited to
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interfere with PPIs
. For our previous inhibitory compounds, including 1–4, we have shown
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8
that they bind to CD40L and not to CD40 , most likely in an allosteric manner interfering with
the trimeric structure of these proteins (all TNFSF receptors and ligands bind as trimers; Figure
1
). Considering their overall structural similarity (cf. Figure 3 and Figure 2) and their ability to
inhibit the binding of CD40L to Mac-1 as well (Supporting Information Figure S1), it is
reasonable to assume that the present compounds (6–11) also bind to CD40L.
All tested compounds concentration-dependently inhibited the CD40L-mediated
activation of NF-κB sensor cells (Figure 5), of THP-1 cells, which can serve as surrogate dendritic
cells (Supporting Information Figure S2), as well as the proliferation and activation of primary
human B cells (Supporting Information Figure S5, Figure 6) and did so at concentration levels
that are below cytotoxic levels (Supporting Information Figure S3, Table 1). While activity seems
to be less than in the cell-free binding assay, most likely due to nonspecific binding to proteins,
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activities obtained are still better than those obtained for the dye compounds before .
Hydrophobicity might also cause solubility limitations; measured water solubilities indicate that
this could be a possible issue for compound 6 and 11 (with solubilities of 7 and 17 ꢀM,
respectively), but not 10, which has a water solubility of 240 ꢀM. Immunomodulatory activity
has also been confirmed in vivo in a rodent skin transplant experiment (Figure 8) and in an
alloantigen-induced T cell expansion model (draining popliteal lymph node; Figure 9).
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Prolongation of the skin graft was less than that obtained with the CD40 murine antibody, but
promising enough for a once-a-day administration treatment regimen considering the relatively
fast elimination of the tested compound (10). Notably, the same compound (10) was able to
inhibit the alloantigen-induced T cell response in a draining lymph node in a dose responsive
manner when administered b.i.d. for three days. These results are particularly encouraging for
prospective IM treatments since CD40 stimulation is an important signal for many cells involved
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in immune responses including T cells, B cells, or dendritic cells, which act as APCs . Results
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obtained here also confirm our original hypothesis
that the chemical space of organic dyes
can serve as a good starting point to identify PPI inhibitors in general. Such dyes are likely to
show good protein binding affinity; many of them are used for tissue staining and can often
achieve tissue specificity. Most azo dyes are likely to be within the chemical space of ‘privileged
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structures’ for protein binding (to the degree that such a space exists)
. Because of their
strong color, however, they are unsuitable for therapeutic development, and they are also
susceptible to quick metabolic degradation by intestinal microorganisms and hepatic enzymes
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.
The present results confirm that ‘colorless’ versions can be developed (e.g., by replacing
the azo chromophores with amide linkers) while still maintaining good PPI inhibitory activity. It
is also noteworthy that the present compounds achieve CD40–CD40L inhibitory activity with
relatively small structures (molecular weights in the 500–600 dalton range). Typical PPI
inhibitors tend to require larger structures to achieve sufficient activity with molecular weight
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that are in flagrant violation of the Lipinski rule-of-five (i.e., MW < 500) , which serves as a
widely used guide to achieve oral bioavailability and an adequate pharmacokinetic profile.
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The relatively small structures here (compared to other PPI inhibitors) also mean
improved ligand binding efficiency. Ligand efficiency (LE) is defined as the binding energy per
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unit size, typically, the binding free energy per non-hydrogen atom: LE = ꢁG /N
. For small-
nHa
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molecule PPI inhibitors, LE tends to be in the 1.0 kJ/atom range
– less than the average
. For reference
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LE of around 1.5 kJ/atom seen for usual protein-ligand interactions
purposes, an LE of 1.5 kJ/atom means an approximate doubling of affinity (halving of K or IC )
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with the addition of each non-hydrogen atom. For the present compounds (6, 10, and 11), LE is
in the 0.91–0.93 kJ/atom range, similar to that of typical PPI inhibitors, and much better than LE
of the larger and less potent 1 (0.4 kJ/atom).
CONCLUSIONS
In conclusion, by starting from the chemical space of organic dyes and using an iterative
design, synthesize, test, and re-design approach, we have identified a set of new drug-like
small-molecule inhibitors of the CD40–CD40L interaction (e.g., 6 or 10). Their ability to inhibit
CD40L-induced activation has been confirmed in cell-based assays including primary human B
cells and in a rodent skin transplant experiment. Activities are in the low-micromolar range,
which might require further optimization; nevertheless, the present work provides clear proof-
of-principle evidence that the CD40–CD40L costimulatory interaction, an important
immunomodulatory and anti-inflammatory target, is susceptible to small-molecule inhibition.
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EXPERIMENTAL SECTION
Materials
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Chemicals and reagents used were obtained from Sigma-Aldrich (St. Louis, MO) except as
indicated. Direct red 80 was from Santa Cruz Biotechnology (Dallas, TX). Recombinant receptors
(hCD40:Fc, CD40:COMP, and TNF-R1:Fc) and tagged ligands (hCD154, hCD154:Fc, MegaCD40L,
and TNF-α), were obtained from Enzo Life Sciences (San Diego, CA). Mac-1 (recombinant
human integrin α β protein) and the monoclonal anti-human CD154 (clone 40804) and anti-
M
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human TNF-α antibodies (clone 1825) were obtained from R&D Systems (Minneapolis, MN).
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Inhibitory peptides KGYY15 were purchased from New England Peptide (Gardner, MA, USA).
Chemistry. General Methods
Commercial grade reagents and solvents were purchased from VWR (Radnor, PA) and
Sigma-Aldrich (St. Louis, MO) and directly used without further purification. 4’-Nitro[1,1’-
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biphenyl]-4-carboxylic acid was synthesized by two steps as described in the literature . All
reactions were carried out in oven- or flame-dried glassware under an atmosphere of dry argon
unless otherwise noted. Except as otherwise indicated, all reactions were magnetically stirred
and monitored by analytical thin-layer chromatography (TLC) using Merck (Kenilworth, NJ) pre-
coated silica gel plates with F254 indicator. Visualization was accomplished by UV light (256 nm)
with a combination of potassium permanganate and/or vanillin solution as an indicator. Flash
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column chromatography was performed according to the method of Still using silica gel 60
(mesh 230-400; EMD Milipore, Billerica, MA).
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All newly synthesized compounds were characterized with H NMR, C NMR, high-
resolution mass spectrometry (HRMS), and infrared (IR) spectroscopy – detailed data are
provided below. Chemical shifts are reported in ppm relative to TMS. DMSO-d (2.50 ppm) was
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used as a solvent for H NMR and C NMR. H NMR and C NMR spectra were recorded on
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Bruker Avance 300 (300 MHz H), 400 (400 MHz H, 100 MHz C), and 500 (500 MHz H, 125
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MHz C). Chemical shift values (δ) are reported in ppm relative to Me Si (δ 0.0 ppm) unless
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otherwise noted. Proton spectra are reported as δ (multiplicity, coupling constant J, number of
protons). Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), p
(
quintet), h (septet), m (multiplet), and br (broad). IR spectra were recorded with a FT-IR
spectrophotometer Paragon 1000 (Perkin Elmer). Mass spectra were obtained at the Mass
Spectrometry Laboratory, Department of Chemistry, University of Florida (Gainesville, FL). Low-
resolution ES (electron spray) mass spectra were carried out with Finnigan LCQ DECA/Agilent
1
100 LC/MS mass spectrometer (Thermo Fisher Scientific, Waltham, MA). High-resolution mass
spectra were recorded on an Agilent 6220 ESI TOF (Santa Clara, CA) mass spectrometer.
Analysis of sample purity was performed on an Agilent (Palo Alto, CA) 1100 series HPLC system
with a Thermoscientific Hypurity C8 (5 μm; 2.1 × 100 mm + guard column). HPLC conditions
were as follows: solvent A = water with 2 mM ammonium acetate, solvent B = methanol with 2
mM ammonium acetate, and flow rate = 0.2 mL/min. Compounds were eluted with a gradient
of A:B = 80:20 at 0 min to 0:100 at 50 min. Purity was determined via integration of UV spectra
at 254 nm, and all tested compounds have a purity of ≥95%. All target compounds 5−11 were
tested as triethylamine salts unless otherwise stated.
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Water solubilities were determined by preparing saturated solutions in distilled water
and measuring their concentrations by HPLC using calibration solutions prepared by dissolving
known amounts of compounds in 20% (w/v) 2-hydroxypropyl-β-cyclodextrin (HPβCD) aqueous
solution. Analyses for this and for plasma concentrations were performed on a Hitachi (Tokyo,
Japan) L-2000 series HPLC system with a Sigma-Aldrich Discovery C18 column (5 ꢀm; 4.6 x 150
mm + Supelco guard column) using a 60:40 mobile phase of water:acetonitrile (both with 2.0
mM ammonium acetate, flow rate 0.25 mL/min) by injection of 5 ꢀL samples using a Hitachi L-
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2
200 auto-sampler and with detection at 254 nm.
8
-(4-(4-Nitrobenzamido)benzamido)naphthalene-1-sulfonic acid (5)
Preparation of compound 5 followed the general synthetic scheme of Scheme 1 via the
synthesis of specific intermediaries 17 and 18 as summarized in Supporting Information Scheme
S1. The general procedure for the coupling reaction as described below for 17 was followed
with 4-nitrobenzoic acid 16 (0.25 g, 1.47 mmol) and 8-(4-aminobenzamido)naphthalene-1-
sulfonic acid (18) (0.50 g, 1.13 mmol) to give the triethylamine salt of the title compound 5 as a
1
yellow solid (0.47 g, 70%) (>99.9% pure by HPLC analysis (UV spectra at 254 nm)). H NMR (500
MHz, DMSO-d ): δ 12.54 (s, 1H), 10.80 (s, 1H), 8.81 (br, 1H), 8.39 (d, J = 8.8 Hz, 2H), 8.31 (dd, J =
6
1
.3, 7.2 Hz, 1H), 8.26-8.16 (m, 5H), 8.00 (dd, J = 1.1, 8.2 Hz, 1H), 7.92 (d, J = 8.7 Hz, 2H), 7.80
(dd, J = 1.0, 8.1 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.47 (t, J = 7.6 Hz, 1H), 3.07 (q, J = 7.2 Hz, 6H),
1
3
1
1
4
.15 (t, J = 7.3 Hz, 9H); C NMR (125 MHz, DMSO-d ): δ 164.8, 164.2, 149.2, 141.8, 141.2, 140.5,
6
35.7, 133.4, 131.8, 131.0, 129.3, 128.9, 127.4, 125.8, 125.3, 124.0, 123.9, 123.6, 122.9, 119.3,
5.8, 8.6; FTIR (neat) νmax 3235, 2999, 2685, 1677, 1638, 1589, 1515, 1490, 1434, 1403, 1342,
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294, 1254, 1230, 1186, 1148, 1132, 1099, 1038, 1010, 917, 845, 835, 821, 759, 719, 706, 691,
–1
–
–
678 cm ; HRMS (ESI) [M-H] calcd. for C H N O S , 490.0714; found, 490.0715.
2
4 16 3 7
8
-(4’-(4-Nitrobenzamido)biphenyl-4-ylcarboxamido)naphthalene-1-sulfonic acid (6)
Preparation of compound 6 followed the general synthetic scheme of Scheme 1 via the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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53
54
55
56
57
58
59
60
synthesis of intermediaries 14 and 15. The general procedure for coupling as described below
for 17 was followed with 4-nitrobenzoic acid (16) (0.50 g, 3.0 mmol) and 8-(4’-aminobiphenyl-4-
ylcarboxamido)naphthalene-1-sulfonic acid (15) (1.20 g, 2.3 mmol) to give the triethylamine salt
of the title compound 6 as a yellow solid (1.35 g, 87%) (99.9% pure by HPLC analysis (UV spectra
1
at 254 nm)). H NMR (500 MHz, DMSO-d ): δ 12.64 (s, 1H), 10.75 (s, 1H), 8.85 (br, 1H), 8.39 (d, J
6
= 8.1 Hz, 2H), 8.36 - 8.16 (m, 6H), 8.02 (d, J = 8.2 Hz, 1H), 7.95 (d, J = 8.2 Hz, 2H), 7.91 - 7.81 (m,
5
H), 7.59 (t, J = 7.7 Hz, 1H), 7.49 (t, J = 7.5 Hz, 1H), 3.07 (q, J = 7.2 Hz, 6H), 1.15 (t, J = 7.1 Hz, 9H);
1
3
C NMR (125 MHz, DMSO-d ): δ 165.1, 164.1, 149.2, 142.0, 141.8, 140.6, 138.8, 135.8, 134.9,
6
1
34.2, 133.3, 132.0, 129.4, 128.8, 127.5, 127.3, 126.0, 125.9, 125.4, 124.4, 124.1, 123.7, 123.0,
120.8, 45.7, 8.7; FTIR (neat) νmax 3360, 3017, 2714, 1679, 1666, 1592, 1521, 1489, 1432, 1416,
398, 1340, 1321, 1279, 1235, 1194, 1152, 1131, 1102, 1038, 1009, 929, 895, 864, 852, 824,
1
–1
–
–
761, 708, 675, 661 cm ; HRMS (ESI) [M-H] calcd. for C H N O S , 566.1027; found, 566.1054.
3
0 20 3 7
Estimated water solubility: 4.7 ꢀg/mL (7.0 ꢀM).
4-(4’-(4-Nitrobenzamido)biphenyl-4-ylcarboxamido)naphthalene-1-sulfonic acid (7)
Preparation of compound 7 involved the synthesis of specific intermediaries 19 and 20
as summarized in Supporting Information Scheme S2. The general procedure for coupling as
described below for 17 was followed with 4’-(4-nitrobenzamido)biphenyl-4-carboxylic acid (20)
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(
0.26 g, 0.55 mmol) and 4-amino-1-naphthalenesulfonic acid (21) (112 mg, 0.5 mmol) to give
the triethylamine salt of the title compound 7 as a yellow solid (0.27 g, 80%) (>99.9% pure by
1
HPLC analysis (UV spectra at 254 nm)). H NMR (500 MHz, DMSO-d ): δ 10.72 (s, 1H), 10.49 (s,
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
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1H), 8.93 (dd, J = 2.1, 6.9 Hz, 1H), 8.83 (br, 1H), 8.40 (d, J = 8.7 Hz, 2H), 8.23 (d, J = 8.7 Hz, 2H),
8
.20 (d, J = 8.3 Hz, 2H), 8.04 - 7.98 (m, 2H), 7.96 (d, J = 8.6 Hz, 2H), 7.90 (d, J = 8.3 Hz, 2H), 7.85
1
3
(d, J = 8.6 Hz, 2H), 7.58 - 7.51 (m, 3H), 3.09 (q, J = 7.3 Hz, 6H), 1.17 (t, J = 7.3 Hz, 9H); C NMR
125 MHz, DMSO-d ): δ 165.8, 164.1, 149.3, 142.6, 142.4, 140.6, 138.9, 134.9, 134.7, 132.9,
(
6
129.8, 129.6, 129.4, 128.6, 128.0, 127.3, 126.3, 125.7, 125.6, 124.2, 123.7, 123.2, 122.5, 120.8,
5.7, 8.7; FTIR (neat) νmax 3304, 2999, 2703, 1649, 1599, 1562, 1527, 1482, 1455, 1418, 1345,
1327, 1306, 1262, 1220, 1177, 1160, 1104, 1040, 1004, 897, 866, 833, 796, 769, 758, 704, 684
4
–1
+
+
cm ; HRMS (ESI) [M+H] calcd. for C H N O S , 568.1173; found, 568.1161.
3
0 22 3 7
8-(4’-(4-Aminobenzamido)biphenyl-4-ylcarboxamido)naphthalene-1-sulfonic acid (8)
The general procedure for hydrogenation as described below for 18 was followed with
8-(4’-(4-nitrobenzamido)biphenyl-4-ylcarboxamido)naphthalene-1-sulfonic acid (6) (1.35 g, 2.0
mmol) to give the triethylamine salt of the title compound as a white solid (0.95 g, 75%) (98.4%
1
pure by HPLC analysis (UV spectra at 254 nm)). H NMR (500 MHz, DMSO-d ): δ 12.63 (s, 1H),
6
9
.91 (s, 1H), 8.80 (br, 1H), 8.32 (dd, J = 1.2, 7.2 Hz, 1H), 8.26 (d, J = 8.4 Hz, 2H), 8.20 (dd, J = 0.9,
7.6 Hz, 1H), 8.01 (dd, J = 1.1, 8.1 Hz, 1H), 7.92 (d, J = 8.6 Hz, 2H), 7.81 (d, J = 8.3 Hz, 3H), 7.76 (d,
J = 8.6 Hz, 4H), 7.58 (t, J = 7.8 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 6.64 (d, J = 8.6 Hz, 2H), 5.80 (br,
1
3
2H), 3.06 (q, J = 7.1 Hz, 6H), 1.14 (t, J = 7.3 Hz, 9H); C NMR (125 MHz, DMSO-d ): δ 165.3,
6
1
65.1, 152.1, 142.2, 141.8, 139.8, 135.8, 133.9, 133.6, 133.3, 131.8, 129.4, 128.7, 127.4, 126.9,
125.9, 125.7, 125.3, 124.2, 124.0, 123.0, 121.1, 120.3, 112.7, 45.8, 8.6; FTIR (neat) νmax 3412,
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344, 3243, 2990, 2689, 1635, 1600, 1558, 1534, 1475, 1430, 1415, 1397, 1338, 1297, 1278,
1237, 1198, 1173, 1134, 1102, 1040, 1011, 928, 904, 845, 831, 820, 790, 762, 740, 720, 696,
–1
–
–
6
70 cm ; HRMS (ESI) [M-H] calcd. for C H N O S , 536.1286; found, 536.1299.
30 22 3 5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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60
8-(4’-(1H-Benzo[d][1,2,3]triazole-5-carboxamido)biphenyl-4-ylcarboxamido)naphthalene-1-
sulfonic acid (9)
The general procedure for coupling reaction as described below for 17 was followed
with 1H-benzo[d][1,2,3]triazole-5-carboxylic acid (77 mg, 0.47 mmol) and 8-(4’-aminobiphenyl-
4-ylcarboxamido)naphthalene-1-sulfonic acid (15) (0.21 g, 0.39 mmol) to give the triethylamine
salt of the title compound as a white solid (180 mg, 69%) (99.0% pure by HPLC analysis (UV
1
spectra at 254 nm)). H NMR (500 MHz, DMSO-d ): δ 12.65 (s, 1H), 10.57 (s, 1H), 8.67 (s, 1H) ,
6
8
.32 (dd, J = 0.8, 7.2 Hz, 1H), 8.27 (d, J = 8.4, 2H), 8.21 (d, J = 7.1 Hz, 1H), 8.08 (d, J = 9.3 Hz, 1H),
8.05 - 7.96 (m, 4H), 7.87 - 7.80 (m, 5H), 7.58 (t, J = 7.8 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 3.34 (br,
13
2
H), 3.06 (q, J = 7.3 Hz, 6H), 1.15 (t, J = 7.3 Hz, 9H); C NMR (125 MHz, DMSO-d ): δ 165.2,
6
165.1, 142.0, 141.8, 139.2, 135.8, 134.5, 134.1, 133.3, 131.8, 128.8, 127.4, 127.1, 125.9, 125.8,
25.3, 124.2, 124.0, 123.0, 120.7, 45.8, 8.6; FTIR (neat) νmax 3294, 3027, 1654, 1593, 1562,
1528, 1477, 1412, 1400, 1345, 1321, 1302, 1277, 1247, 1209, 1196, 1164, 1129, 1043, 1013,
1
–1
–
9
35, 919, 881, 862, 837, 822, 799, 759, 742, 723, 662 cm ; HRMS (ESI) [M-H] calcd. for
–
C H N O S , 562.1191; found, 562.1197.
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-(4’-(4-(Methoxycarbonyl)benzamido)biphenyl-4-ylcarboxamido)naphthalene-1-sulfonic acid
(10)
The general procedure for coupling as described below for 17 was followed with mono-
0
1
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7
8
9
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methyl terephthalate (0.28 g, 15.7 mmol) and 8-(4’-aminobiphenyl-4-
ylcarboxamido)naphthalene-1-sulfonic acid (15) (0.63 g, 1.21 mmol) to give the triethylamine
salt of the title compound as a white solid (0.69 g, 84%) (97.7% pure by HPLC analysis (UV
1
spectra at 254 nm)). H NMR (500 MHz, DMSO-d ): δ 12.64 (s, 1H), 10.58 (s, 1H), 8.80 (br, 1H),
6
8.31 (dd, J = 1.1, 7.2 Hz, 1H), 8.27 (d, J = 8.4 Hz, 2H), 8.21 (d, J = 6.7 Hz, 1H), 8.12 (s, 4H), 8.01
(dd, J = 1.0, 8.1 Hz, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.89-7.77 (m, 5H), 7.58 (t, J = 7.9 Hz, 1H), 7.48 (t,
1
3
J = 7.6 Hz, 1H), 3.91 (s, 3H), 3.07 (q, J = 7.1 Hz, 6H), 1.15 (t, J = 7.3 Hz, 9H); C NMR (125 MHz,
DMSO-d ): δ 165.7, 165.0, 164.8, 142.0, 141.8, 139.0, 138.9, 135.8, 134.7, 134.1, 133.3, 132.1,
6
1
4
1
31.8, 129.2, 128.7, 128.1, 127.4, 127.1, 125.9, 125.8, 125.3, 124.2, 124.0, 123.0, 120.7, 52.4,
5.8, 8.6; FTIR (neat) νmax 3255, 3005, 2706, 1719, 1671, 1655, 1595, 1522, 1490, 1417, 1330,
–1
274, 1164, 1106, 1038, 1010, 920, 899, 869, 827, 789, 763, 724, 708, 661cm ; HRMS (ESI) [M-
–
–
H] calcd. for C H N O S , 579.1231; found, 579.1254. Estimated water solubility: 163.8 ꢀg/mL
3
2 23 2 7
(240 ꢀM).
4
-(4’-(4-Nitrobenzamido)biphenyl-4-ylcarboxamido)-1-naphthoic acid (11)
Preparation of compound 11 followed the general synthetic scheme of Scheme 1 via the
synthesis of specific intermediaries 23, 24, and 25 as summarized in Supporting Information
Scheme S3. The general procedure for hydrogenation as described below for 18 was followed
with 4-(4’-nitrobiphenyl-4-ylcarboxamido)-1-naphthoic acid (25) (60 mg, 0.145 mmol) to give
the free amine (58 mg) without further purification. The general procedure for the coupling
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reaction of 4-nitrobenzoic acid (16) (33 mg, 0.2 mmol) and the free amine gave the title
compound 11 as a yellow solid (54 mg, 71%) (>99.9% pure by HPLC analysis (UV spectra at 254
1
nm)). H NMR (400 MHz, DMSO-d ): δ 13.11 (br, 1H), 10.71 (s, 1H), 10.63 (s, 1H), 8.98 (d, J = 8.5
6
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
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59
60
Hz, 1H), 8.40 (d, J = 8.7 Hz, 2H), 8.27 - 8.15 (m, 6H), 7.96 (d, J = 8.6 Hz, 2H), 7.91 (d, J = 8.3 Hz,
2
H). 7.85 (d, J = 8.7 Hz, 2H), 7.80 (d, J = 7.9 Hz, 1H), 7.70 (t, J = 7.5 Hz, 1H), 7.64 (t, J = 7.6 Hz,
1
3
1H); C NMR (125 MHz, DMSO-d ): δ 165.9, 164.0, 149.2, 142.7, 140.5, 138.9, 136.8, 134.6,
6
1
32.8, 131.7, 129.3, 129.1, 128.8, 128.7, 128.6, 127.2, 126.9, 126.5, 126.2, 125.7, 124.9, 123.60,
123.56, 122.4, 120.8; FTIR (neat) νmax 3283, 1673, 1648, 1598, 1519, 1492, 1458, 1416, 1347,
–1
–
1
328, 1292, 1243, 1196, 1107, 913, 822, 764, 713, 684 cm ; HRMS (ESI) [M-H] calcd. for
–
C H N O , 530.1358; found, 530.1348. Estimated water solubility: 9.3 ꢀg/mL (17.6 ꢀM).
3
1 20 3 6
8
-(4’-Nitrobiphenyl-4-ylcarboxamido)naphthalene-1-sulfonic acid (14).
The general procedure for the coupling reaction as described below for 17 was followed with 8-
amino-1-naphthalenesulfonic acid (12) (1.50 g, 6.7 mmol) and 4’-nitro[1,1’-biphenyl]-4-
carboxylic acid (13) (1.63 g, 6.7 mmol) to give the triethylamine salt of 14 as a yellow solid (2.4
1
g, 66%). H NMR (500 MHz, DMSO-d ): δ 12.70 (s, 1H), 8.79 (br, 1H), 8.38 - 8.29 (m, 5H), 8.20 (d,
6
J = 7.6 Hz, 1H), 8.09 (d, J = 8.8 Hz, 2H), 8.02 (d, J = 8.1 Hz, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.83 (d, J =
7
.8 Hz, 1H), 7.59 (t, J = 7.9 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 3.08 (q, J = 7.2 Hz, 6H), 1.15 (t, J = 7.3
1
3
Hz, 9H); C NMR (125 MHz, DMSO-d ): δ 164.8, 147.0, 145.8, 141.8, 140.1, 135.9, 135.8, 133.2,
6
1
31.9, 128.9, 128.1, 127.4, 127.0, 126.1, 125.3, 124.2, 124.1, 124.0, 123.0, 45.8, 8.6; FTIR (neat)
νmax 3028, 2735, 1668, 1596, 1515, 1494, 1480, 1429, 1393, 1339, 1328, 1280, 1231, 1186,
–1
1
162, 1126, 1110, 1039, 1010, 924, 894, 869, 854, 843, 826, 788, 763, 740, 692 cm ; HRMS
+
+
(
ESI) [M+H] calcd. for C H N O S , 449.0802; found, 449.0781.
23 17 2 6
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8
-(4’-Aminobiphenyl-4-ylcarboxamido)naphthalene-1-sulfonic acid (15).
The general procedure for hydrogenation as described below for 18 was followed with 8-(4’-
nitrobiphenyl-4-ylcarboxamido)naphthalene-1-sulfonic acid (14) (2.8 g, 5.1 mmol) to give the
0
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8
9
0
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4
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6
7
8
9
0
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8
9
0
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7
8
9
0
1
triethylamine salt of 15 as a white solid (2.3 g, 86%). H NMR (500 MHz, DMSO-d ): δ 12.56 (s,
6
1
H), 8.80 (br, 1H), 8.31 (dd, J = 1.1, 6.1 Hz, 1H) , 8.25 - 8.12 (m, 3H), 8.00 (d, J = 7.2 Hz, 1H), 7.80
(d, J = 7.3 Hz, 1H), 7.67 (d, J = 8.3 Hz, 2H), 7.57 (t, J = 7.8 Hz, 1H), 7.49 (d, J = 7.0 Hz, 2H), 7.47 (t,
J = 7.7 Hz, 1H), 6.68 (d, J = 8.4 Hz, 2H), 5.33 (s, 2H), 3.05 (q, J = 7.2 Hz, 6H), 1.14 (t, J = 7.3 Hz,
1
3
9H); C NMR (125 MHz, DMSO-d ): δ 165.2, 148.9, 143.1, 141.8, 135.7, 133.4, 132.5, 131.8,
6
1
28.6, 127.4, 127.3, 126.3, 125.7, 125.3, 124.6, 124.1, 123.9, 123.0, 114.2, 45.7, 8.6; FTIR (neat)
νmax 3431, 3338, 3227, 3006, 2712, 1648, 1602, 1531, 1492, 1474, 1429, 1397, 1331, 1284,
–1
1
227, 1196, 1184, 1161, 1061, 1036, 1008, 921, 891, 823, 788, 761, 725, 704, 663 cm ; HRMS
+
+
(ESI) [M+H] calcd. for C H N O S , 419.1060; found, 419.1058.
23 19 2 4
8
-(4-nitrobenzamido)naphthalene-1-sulfonic acid (17) and general procedure for the coupling
reaction.
For the synthesis of 17 and as a general procedure of coupling a modified version of the
74
coupling reaction from reference was used. Under an argon atmosphere, trimethylamine
(1.74 mL, 12.5 mmol) was added dropwise to a mixture of 4-nitrobenzoic acid 16 (1.09 g, 6.5
mmol), O-(6-chlorobenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate
HCTU (2.7 g, 6.5 mmol) and DMF (10 mL) at 0°C and the resulting reaction mixture was stirred
for 1 h at the same temperature. Subsequently, 8-amino-1-naphthalenesulfonic acid 12 (1.12 g,
5
.0 mmol) was added at the same temperature. The resulting reaction mixture was allowed to
stir overnight at room temperature (RT). Diethyl ether (50 mL) was added to the reaction
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Journal of Medicinal Chemistry
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mixture, and a yellow precipitate formed. This precipitate was collected by filtration and
washed with diethyl ether (3 x 10 mL) to afford the triethylamine salt of 17 as a yellow solid (2.0
1
g, 65%). H NMR (500 MHz, DMSO-d ): δ 12.83 (s, 1H), 8.82 (br, 1H), 8.39 (d, J = 9.0 Hz, 2H), 8.36
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(d, J = 8.9 Hz, 2H), 8.30 (dd, J = 1.2, 7.2 Hz, 1H), 8.17 (d, J = 7.6 Hz, 1H), 8.03 (dd, J = 0.9, 8.1 Hz,
1
H), 7.86 (d, J = 8.1 Hz, 1H), 7.60 (t, J = 7.8, 1H), 7.49 (t, J = 7.4 Hz, 1H), 3.08 (q, J = 7.2 Hz, 6H),
1
3
1.15 (t, J = 7.3 Hz, 9H); C NMR (125 MHz, DMSO-d ): δ 163.9, 148.9, 141.6, 141.5, 135.7, 132.7,
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1
31.9, 129.4, 127.5, 126.5, 125.3, 124.3, 124.1, 123.3, 122.9, 45.8, 8.6; FTIR (neat) νmax 3244,
3013, 2714, 1675, 1602, 1519, 1489, 1454, 1432, 1391, 1347, 1337, 1277, 1241, 1198, 1164,
–1
+
+
1
128, 1039, 1010, 916, 880, 853, 824, 782, 763, 713 cm ; HRMS [M+H] calcd. for C H N O S ,
17 13 2 6
373.0489; found, 373.0479.
8
-(4-aminobenzamido)naphthalene-1-sulfonic acid (18) and general procedure for the
hydrogenation reaction.
For the synthesis of 18 and as a general procedure of coupling a modified version of the
75
hydrogenation reaction from reference was used. A mixture of 8-(4-
nitrobenzamido)naphthalene-1-sulfonic acid (17) (0.82 g, 1.73 mmol) and 10% Pd on carbon (27
mg) in a solvent mixture of EtOH (2.0 mL) and DMF (1.0 mL) was hydrogenated (H balloon) at
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0°C for 3.5 h. The reaction mixture was filtered via a short pad of Celite®, concentrated in
vacuo, and recrystallized from MeOH to afford the triethylamine salt of 18 as a white solid (0.61
1
g, 80%). H NMR (500 MHz, DMSO-d ): δ 12.22 (s, 1H), 8.86 (br, 1H), 8.28 (d, J = 6.1 Hz, 1H), 8.14
6
(d, J = 6.7 Hz, 1H), 7.96 (d, J = 7.4 Hz, 1H), 7.92 (d, J = 8.6 Hz, 2H), 7.73 (d, J = 7.3 Hz, 1H), 7.51 (t,
J = 7.8 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 6.64 (d, J = 8.5 Hz, 2H), 3.38 (br, 2H), 3.06 (q, J = 7.1 Hz,
1
3
6H), 1.15 (t, J = 7.2 Hz, 9H); C NMR (125 MHz, DMSO-d ): δ 165.3, 150.5, 141.9, 135.8, 133.9,
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