Synthetic molecules for disruption of the MYC protein-protein interface

Article abstract of DOI:10.1016/j.bmc.2018.07.019

MYC is a key transcriptional regulator involved in cellular proliferation and has established roles in transcriptional elongation and initiation, microRNA regulation, apoptosis, and pluripotency. Despite this prevalence, functional chemical probes of MYC

Full text of DOI:10.1016/j.bmc.2018.07.019

Accepted Manuscript
Synthetic Molecules for Disruption of the MYC Protein-Protein Interface
Nicholas T Jacob, Pedro O Miranda, Ryan J Shirey, Ritika Gautam, Bin Zhou,
M Elena de Orbe Izquierdo, Mark S Hixon, Jonathan R Hart, Lynn Ueno, Peter
K Vogt, Kim D Janda
PII:
S0968-0896(18)31031-9
https://doi.org/10.1016/j.bmc.2018.07.019
BMC 14456
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 June 2018
3 July 2018
10 July 2018
Please cite this article as: Jacob, N.T., Miranda, P.O., Shirey, R.J., Gautam, R., Zhou, B., de Orbe Izquierdo, M.E.,
Hixon, M.S., Hart, J.R., Ueno, L., Vogt, P.K., Janda, K.D., Synthetic Molecules for Disruption of the MYC Protein-
Protein Interface, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.07.019
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Synthetic Molecules for Disruption of the MYC Protein-Protein
Interface
Nicholas T Jacob, Pedro O Miranda†, Ryan J Shirey, Ritika Gautam, Bin Zhou, M Elena de Orbe
Izquierdo, Mark S Hixon∫, Jonathan R Hart, Lynn Ueno, Peter K Vogt, Kim D Janda*
Department of Chemistry, BCC-582, 10550 N Torrey Pines Road, The Scripps Research Institute, La
Jolla, CA 92037
ABSTRACT. MYC is a key transcriptional regulator involved in cellular proliferation and has
established roles in transcriptional elongation and initiation, microRNA regulation, apoptosis, and
pluripotency. Despite this prevalence, functional chemical probes of MYC function at the protein level
have been limited. Previously, we discovered 5a, that binds to MYC with potency and specificity,
downregulates the transcriptional activities of MYC and shows efficacy in vivo. However, this scaffold
posed intrinsic pharmacokinetic liabilities, namely, poor solubility that precluded biophysical
interrogation. Here, we developed a screening platform based on field-effect transistor analysis (Bio-
FET), surface plasmon resonance (SPR), and a microtumor formation assay to analyze a series of new
compounds aimed at improving these properties. This blind SAR campaign has produced a new lead
compound of significantly increased in vivo stability and solubility for a 40-fold increase in exposure.
This probe represents a significant advancement that will not only enable biophysical characterization of
this interaction and further SAR, but also contribute to advances in understanding of MYC biology.

INTRODUCTION
The v-myc myeleocytomatosis viral oncogene homolog (MYC) protein is an essential regulator of
cell-cycle progression occupying and apical space in the trasncriptome.1 Importantly, MYC has been
directly implicated in most human cancers and its role recognized as a hallmark of cancer initiation and
maintenance.2-6 In general, MYC expression has a strong correlation to dramatic effects on cellular
proliferation and function, but the molecular determinants responsible for these effects remain
controversial.7 Part of the difficulty in studying MYC is its frenetic mode of action: although having an
ephemeral existence, it is able to seemingly affect transcription in both a local and global manner.
Moreover, MYC exists as an intrinsically disordered protein (IDP), taking on structure only in the
presence of other basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factors of the MAX
network.8,9 This lack of structure and instability greatly impairs the ability to structurally or
biophysically characterize MYC interactions. In all, these attributes have worked to make MYC an
attractive, but elusive target in drug discovery.
Previously, we described an inhibitory scaffold of the MYC-MAX-DNA complex, KJ-Pyr-9, 5a.10
This compound exhibited potent inhibition of the transforming capabilities of an oncogenic ATG-MYC
virus in chicken embryo fibroblasts (CEF); importantly, it also demonstrated a higher specificity of
inhibition for MYC than other oncogenes. However, the scaffold presents intrinsic liabilities: namely, a
very modest solubility (~8µM), compounding the difficulty in characterizing this interaction, as well as
several undesirable moieties, namely the nitro group. To overcome these obstacles, we developed a
multifaceted screening platform combining in vitro biophysical characterization with in vivo methods.
RESULTS AND DISCUSSION

With no structural characterization of the interaction between 5a and MYC, a blind SAR campaign
was undertaken primarily directed at accessing a more soluble scaffold to enable formal
characterization. A series of compounds with bioisosteric replacements of the undesirable moieties and
aimed at increased solubility were produced via the Kröhnke pyridine synthesis. Thus, a family of α,β-
unsaturated ketones 2 were synthesized using two pathways (Scheme 1): (a) Reaction of a methyl
ketone bearing variety of different five-membered heterocycles such as thiazole, imidazole and triazole
(b) an oxazole reaction with the corresponding acylchloride, which allowed for introduction of the
oxazole moiety. A second reaction between 2 and pyridinium salts 1 (obtained from the corresponding
phenacyl bromide derivative) using ammonium acetate at 100 ºC gave the corresponding 2,4,6-
trisubstituted pyridines 3. Compounds 5i-k were obtained at this point, while all others contained a p-
methylestergroup in the R3 position. The methylester group of 3 was hydrolyzed to obtain carboxylic
acid 4. The carboxylic acid was reacted with oxalyl chloride and N,N-dimethylformamide in order to
obtain the corresponding acyl chlorides, which were then reacted with different nucleophiles to form
compounds 5. In order to access truncated pyridine scaffolds, such as 7a and 7b, a palladium-catalyzed
Suzuki cross-coupling strategy was used as shown in Scheme 2. After the preparation of these
compounds, the next step was to examine their activity relative to 5a.
Scheme 1. Synthesis of pyridine scaffolds 5a-5o.

Scheme 2. Synthesis of pyridine scaffolds 7a and 7b.
Initially, compound solubilities were measured by dynamic light scattering (Table 1). In order to
screen new compounds for MYC binding, a field-effect transistor (Bio-FET) was used. Bio-FET offers
several advantages for characterizing this interaction11,12: 1. A much lower limit of detection as compared
with SPR or isothermal calorimetry. 2. Detection being conducted on a graphene surface, which
reduces the interaction of the disordered protein with the chip surface. 3. Site-specific functionalization,
which reduces the amount of noise caused from aggregates on the chip surface. The basic-helix-loop-
helix (bHLH) motif of MYC has been established a solvent-accessible region critical for MYC
function.10,13 A His-tagged bHLH construct was immobilized on the chip (see Materials and Methods)
and compounds screened for interaction. We were able to observe saturation of surface-bound MYC
across chips at 10 µM 5a in 3% DMSO in MES buffer to give a maximal response signal. This was used
as the control on each chip screened and gave a benchmark in terms of both solubility and affinity for
the protein (Figure 1a, Table 1). Compounds were screened by measuring the chip response at 10 µM
and normalized to the response found for 5a in that run. All compounds were also screened against a
monomeric form of MAX (mMAX), as well as MYC-MAX and MAX-MAX dimers (see Supplemental
Material). The lack of observed binding to mMAX, MAX-MAX and the unbound control surface, as
well as the general ability to regenerate the chip surface, indicates that there is a specificity in the
observed binding interactions.
The affinity of the MYC-MAX interaction is relatively weak (Kd = 176 nM14), making measurement
of the perturbation in this interaction difficult. However, positive results have been obtained measuring
the binding affinity for DNA using a stabilized MYC-MAX complex.10 Using surface plasmon
resonance (SPR), we established a characteristic binding response of stabilized MYC-MAX dimers to

immobilized E-BOX oligonucleotides on the chip surface. Heterodimers were stabilized through
disulfide formation through a C-terminal cystine residue introduced to the MYC bHLH and MAX
constructs. This stabilization was found necessary to prevent aggregation of MYC monomer on the chip
surface during runs. Incubation of dimer with treatment compound prior to flowing over the chip surface
showed a perturbation in the sensogram when compared to the vehicle control. Compounds were
incubated with dimer at 10 µM and their ability to prevent interaction with DNA measured (Figure 1b).
Importantly, compounds observed to have no interaction with MYC were also found to have no effect in
preventing dimer binding to DNA, and no interaction was observed between the DNA and compounds
alone.

Table 1: Activity summary.
ID
MW
Solubility
(µM)a
8
FET Req
(%)b
100
106
79
CEF IC50
(µM)c
1
385.4
365.4
382.4
299.3
379.4
380.4
423.5
407.4
366.4
387.5
390.4
379.4
443.5
380.4
393.4
322.4
246.3
366.4
381.4
393.4
5a
5b
5c
10
3
64
30
250
8
0
>100
40
5d
5e
41
32
55
40
5f
125
15
97
5
5g
5h
5i
154
72
2.5
2
5
< 1
18
188
92
>100
2
5j
5k
5l
25
3.2
76
NT
10
32
5m
5n
5o
7a
7b
10a
10b
10c
500
2
0
>100
>100
10d
0
32
0
10
0
NT
7
8
66
< 1
25
0
>100
>100
0
a – Measured by DLS in MES (pH 6.0) with < 0.1 %
DMSO. b – Relative to 5a Req. c – Measured in CEF
transfected with ATG-MYC. d – inhibited all cell growth.
NT – not tested.

A
250
200
150
100
50
0
i
j
l
5
f
a
c
e
k
a
0
b
d
g
h
5
5
5
5
m
5
5
5
5
5
5
5
5
1
Treatment (10 µM)
B
150
100
50
0
i
j
l
5
f
a
c
e
k
a
a
0
b
d
g
h
o
b
7
5
5
5
m
5
5
5
5
7
5
5
5
5
5
5
1
Treatment
C
100
10
1
0.1
i
f
a
c
e
k
a
0
b
g
h
5
5
5
m
5
5
5
5
5
5
5
1
Treatment
Figure 1: Activity screening of test compounds. a) Relative binding to MYC compared with 5a. b)
Inhibition of MYC-MAX binding to DNA measured by SPR. c) IC50 of optimized compounds in CEF
assay. Error bars are SEM, n = 3.
The ultimate assessment of anti-MYC activity was done through a well-established focus assay
examining the ability of an ATG-MYC expressing vector to establish microtumors in CEF.15
Compelling compounds from binding studies were examined for inhibition of microtumor formation in
this assay (Figure 1c). Several derivatives were found to inhibit microtumor formation with similar

efficacy to 5a (Table 1). Overall, the CEF and FET data correlated well (Supplemental Figure 1), apart
from 5j, which was of much poorer solubility than 5a. This was opposed to the SPR data, which did not
correlate well with either the FET or CEF data. We reason that this discrepancy is due to the covalent
linkage of the MYC-MAX dimers, which alters the affinity necessary to perturb the complex greatly.
Additionally, the MYC-MAX-DNA complex is far more stable than the heterodimer itself (Fieber,
JMB, 2001), and thus shifts the equilibrium of MYC-MAX heterodimerization. We also note that while
the FET and CEF data correlate very well, there is a discrepancy between the affinity for MYC and the
potency in cells. This is a common pharmacodynamic phenomenon when targeting protein-protein
interactions. MYC is pleiotropic, the MYC-MAX complex coordinates and controls the expression of
thousands of other genes, and thus the precise molecular determinants responsible for any observed
phenotype related to a change in MYC concentration are unknown. The intervening factors will be
illuminated by further studies of MYC biology with an effective inhibitor.
The results of these experiments provide the beginnings of a structure-activity map for MYC
inhibition. In general, the whole framework of 5a appears necessary as compounds 7a and 7b lost all
activity when entire substituents of the central pyridine were eliminated. This may indicate the “Y-
shape” of the scaffold is a necessary pharmacophore. Critically, the most prominent liability, the p-nitro
moiety in R1, was found dispensable, able to be effectively replaced with a cyano (5b) or thiazole
heterocycle (5g). This also assuaged concerns that inhibition proceeds through a covalent mechanism.
By contrast, it was found there was little plasticity in the chemical space of the furan substituent at the
R2 position. Elimination of the furan ablated activity (7a) and replacement with another heterocycle
resulted in a loss or significant attenuation of activity (5d, 5f, and 5o). In general, there are concerns that
furan functionality is prone to oxidation and the formation of reactive metabolites,16 however, it was not
known the degree to which this moiety presented a pharmacokinetic liability in these particular
scaffolds. We reasoned that the π-conjugation throughout the scaffold would impart increased stability
to the furan from oxidation. To test this, we examined the stability of several compounds incubated with
rat liver microsomes (Figure 2). We found that while the nitro group of 5a was readily eliminated, the

furan was exceedingly recalcitrant to oxidation in these conditions. Importantly, the mass of oxidized
furan ion was not detected.
Ablation of the p-benzylamide lost all activity (7b), but complete replacement of the p-benzylamide in
5j was the only compound found to have significantly greater binding to MYC than 5a, indicating a
pliability. However, the solubility was reduced and it was not found to have any activity in the CEF
assay. We reason that the lack of aqueous solubility even at low doses completely precludes cell
penetration. In a similar vein, carboxylic acids of 4 were also found inactive in both the Bio-FET and
CEF assays. However, it was found there was significant plasticity in the space around the amide
substituent. The use of a N-methylsulfonyl benzamide in 5m had a modest improvement in solubility,
but it was found less
5a
100
5g
7a
50
0
0
10
20
30
40
50
Time (min)
Figure 2: Oxidation susceptibility by rat liver microsomes by mass analysis. Compounds were
incubated with microsomes at 37 ºC for indicated time. Recovery calculated from AUC of extracted ion.
effective in binding MYC and had a 10-fold loss of potency compared to 5a. Similar results were
obtained with 5k, containing a tetrazole in place of the amide, indicating an advanced plasticity in this
region.
Importantly, it was also found that changes to the core heterocycle were tolerated. Diversity was
introduced into the core heterocycle with the formation of a pyrimidine ring in an attempt to optimize
solubility. An α,β-unsaturated ketone 2i was refluxed with furancarboximidine 8 in the presence of
excess sodium methoxide in ethanol to produce carboxylic acid 9, which was converted to the

corresponding amide via the same strategy of forming acylchloride and reacting it with NH4OH to give
pyrimidine 10 (Scheme 3). 10a has an activity profile comparable to 5a. Further diversification of the
scaffold using a pyrimidine core presents an opportunity to produce more soluble derivatives.
Scheme 3. Pyrimidine synthesis.
Overall, 5g is ideal in that it is readily accessed, has greatly increased solubility, lacks the labile nitro
group, and demonstrates similar binding and efficacy to 5a. The affinity of 5g is 12.5 ± 4.1 nM,
comparable to the affinity measured for 5a of 6.5 ± 2.6 nM (Supplementary Figure 4). A 10 mg/kg i.v.
PK study was conducted in mouse to determine if these favored properties translated into improved PK.
Non-compartmental analysis of the mouse PK followed by allometric scaling to the rat afforded a
comparison to the rat PK study conducted previously10 on 5a and is shown in Table 2. This relatively
simple substitution has significantly increased the mean residence time from 1 hour to 4.8 producing a
30-fold improvement in exposure (AUC) (Supplementary Figure 6). Replacement of the nitro group,
along with the increased solubility, stability and concomitant increase in PK, make 5g a significant
improvement on this scaffold, with little cost to ligand efficiency (LE). The LE of 5a and 5g was found
to be 0.40 and 0.36, respectively (Supplementary Table 1). This demonstrates that there is sufficient
plasticity in the chemical space discovered through 5a to improve the physiochemical properties without
compromising efficacy.

Table 2. PK comparison of 5a and 5g.
5aa
385.3
1.6
5ab
385.3
1.0
5gc
423.5
4.8
MW (g/mol)
Half-life (h)
AUC (ng*h/mL)
MRT (h)
258
1.7
1,450
0.94
6.44
115
44,000
4.3
Vss (L)
6.44
65
0.97
3.8
CI (mL/min/kg)
a
b
– values determined in Sprague Dawley rat – values are
allometrically scaled from those determined in rat.10 – values
c
determined in C57Bl6 mice.
CONCLUSION
Through analysis of a blind SAR campaign, we developed a new series of compounds that retain the
efficacy of 5a, but with improved in vivo stability and solubility. We hypothesize there is a goldilocks
effect, whereby the hydrophobicity imparted by the conjugation of the scaffold is intrinsically necessary
for binding to MYC, but this hydrophobicity negatively affects the pharmacokinetic properties of such a
molecule. Thus, the slight activity compromise of 5g for the sake of solubility may represent an
optimally balanced scaffold for targeting MYC. The necessary potency of a MYC inhibitor for in vivo
efficacy remains to be delineated. 5g provides new opportunities assessing the balance between
retaining target level potency while optimizing drug-like properties. This information will help to open
access to thorough biophysical characterization of this inhibition interaction to allow for a structure-
based SAR campaign to be undertaken.
EXPERIMENTAL SECTION
All solvents and chemicals were acquired from Thermo-Fisher or Millipore Sigma unless otherwise
specified. Solvent was anhydrous unless otherwise specified. Purification of compounds was carried out

by prep TLC on 1mm PLC silica gel 60 F254 plates and by flash chromatography on a Teledyne ISCO
Combiflash Rf + Lumen. Compounds and intermediates were characterized by NMR on a Bruker DPX-
400 or NEO-500 instrument in indicated solvent, and high-resolution mass-spectrometry on an Agilent
ESI-TOF. Purity of > 95% was determined by HPLC analysis on an Agilent 1260 Infinity (see
Supplement). Compounds were screened for known PAINS compounds through 2 database searches
(ZINC Patterns, FAF-Drugs4).
General Procedure for compounds 5a-o. Chalcone 2 (1g, 4 mmol.), and NH4OAc (9.2 g, 120 mmol)
were dissolved in a mixture of acetic acid (20 mL) and DMF (30 mL). To it, pyridinium salt 1 (1.2 g. 4
mmol) was added and the reaction mixture was heated at 100 ºC overnight. Solvent was evaporated
under vacuum and the remaining brown oil was dissolved in DCM (100 mL) and solid NaHCO3 was
added until the gas release ceased. The organic phase was dried over MgSO4 and evaporated under
reduced pressure. The product 3 is crashed out using Et2O-MeOH as a brown solid. This material was
carried on without further purification. LiOH (2.5 g, 104 mmol) was added to a solution of 3 in
THF:H2O (9:1) and stirred overnight. The reaction mixture was filtered through a pad of silica gel and
evaporated to dryness to afford carboxylic acid 4. 4 (3g, 8.2 mmol.) was dissolved in dry DCM (100
mL) and oxalyl chloride (0.7 mL, 8.2 mmol) followed by 1 drop of DMF. The reaction was stirred at
room temperature overnight, then the solvent was removed in vacuo and the remaining solid was
redissolved in dry DCM (100 mL). The solution was poured onto a NH4OH solution (50 mL) and the
reaction mixture was stirred for 30 min. The organic layer was separated, dried and evaporated under
vacuo to afford a brown oil which was purified to obtain pyridine 5.
5b: 1HNMR (600 MHz, DMSO-d6):  = 8.54 (d, J = 12 Hz, 2H), 8.35 (s, 1H), 8.12 (m, 3H), 8.08 (d, J
13
= 6 Hz, 2H), 8.04 (d, J = 12 Hz, 2H), 7.94 (s, 1H), 7.50 (brs, 1H), 7.42 (s, 1H), 6.75 (s, 1H); CNMR
(150 MHz, DMSO-d6):  = 167.23, 154.81, 152.78, 149.39, 148.74, 144.66, 142.48, 139.50, 134.99,
132.70, 128.25, 127.77, 127.19, 118.83, 117.28, 115.59, 112.48, 111.77, 110.14; HRMS (ESI-TOF) :
m/z calcd for C24H17N3O2: 366.1237 (M + H)+; found: 366.1238.

1
5c: HNMR (600 MHz, DMSO-d6):  = 8.54 (m, 3H), 8.47 (d, J = 12 Hz, 1H), 8.16 (brs, 1H), 8.15
(d, J = 6 Hz, 2H), 8.10-8.07 (m, 5H), 8.00 (d, J = 6 Hz, 1H), 7.51 (brs, 1H); 13CNMR (150 MHz,
DMSO-d6):  = 167.98, 167.20, 155.01, 151.43, 149.35, 144.47, 141.78, 139.12, 135.21, 132.87,
128.35, 127.75, 127.27, 123.37, 119.94, 118.76, 116.21, 112.08; HRMS (ESI-TOF) : m/z calcd for
C22H14N4OS: 383.0961 (M + H)+; found: 383.0960.
1
5d: HNMR (600 MHz, DMSO-d6):  = 8.58 (s, 1H), 8.56 (d, J = 6 Hz, 2H), 8.44 (s, 1H), 8.41 (s,
1H), 8.16 (brs,1H), 8.15 (d, J = 6 Hz, 2H), 8.09 (d, J = 6 Hz, 2H), 8.06 (d, J = 6 Hz, 2H), 7.56 (s, 1H),
7.51 (s, 1H); 13CNMR (150 MHz, DMSO-d6):  = 167.19, 159.87, 155.32, 149.11, 146.41, 142.04,
141.50, 138.94, 135.23, 132.80, 128.99, 128.35, 127.90, 127.28, 120.01, 119.00, 118.75, 112.03; HRMS
(ESI-TOF) : m/z calcd for C22H14N4O2: 367.1189 (M + H)+; found: 367.1183.
5e: 1HNMR (600 MHz, DMSO-d6):  = 8.51 (d, J = 6 Hz, 2H), 8.45 (d, J = 6Hz, 1H), 8.41 (d, J = 1.5
Hz, 1H), 8.15 (brs, 1H), 8.09 (d, J = 3Hz, 4H), 8.03 (d, J = 6 Hz, 2H),7.96 (d, J = 12 Hz, 2H), 7.50 (brs,
13
1H), 7.42 (s, 1H), 7.11 (d, J = 1 Hz, 1H), 4.23 (s, 3H); CNMR (150 MHz, DMSO-d6):  = 167.25,
154.25, 151.18, 148.53, 143.60, 142.73, 139.54, 135.02, 132.83, 132.38, 128.36, 128.00, 127.09,
125.79, 119.09, 118.81 117.64, 111.75, 36.47; HRMS (ESI-TOF) : m/z calcd for C23H17N5O: 380.1506
(M + H)+; found: 380.1504.
1
5f: HNMR (600 MHz, DMSO-d6):  = 8.60 (s, 1H), 8.53 (d, J = 6 Hz, 2H), 8.50 (s, 1H), 8.36 (s,
1H), 8.20 (d, J = 6 Hz, 2H), 8.16 (brs, 1H), 8.09 (d, J = 12 Hz, 2H), 8.05 (d, J = 12 Hz, 2H), 7.53 (brs,
1H), 4.50 (s, 3H); 13CNMR (150 MHz, DMSO-d6):  = 167.18, 155.05, 149.18, 147.61, 142.36, 139.00,
135.62, 135.17, 134.48, 132.85, 128.20, 127.91, 127.39, 120.20, 119.50, 118.75, 118.45, 112.0, 37.96;
HRMS (ESI-TOF) : m/z calcd for C22H16N6O: 381.1458 (M + H)+; found: 381.1457.
1
5g: HNMR (600 MHz, DMSO-d6):  = 8.46 (d, J = 12 Hz, 2H), 8.28 (s, 1H), 8.16 (brs, 1H), 8.12
(m, 3H), 8.08 (d, J = 12 Hz, 2H), 8.00 (d, J = 3 Hz, 1H), 7.94 (s, 1H), 7.86 (s, d, J = 3 Hz, 1H), 7.51
(brs, 1H), 7.40 (s, 1H), 6.75 (s, 1H); 13CNMR (150 MHz, DMSO-d6):  = 167.29, 166.58, 155.80,
153.00, 149.26, 148.55, 144.50, 144.08, 139.78, 139.67, 134.90, 133.78, 128.77, 127.77, 127.13,

126.48, 120.88, 116.62, 114.92, 112.43, 109.89; HRMS (ESI-TOF) : m/z calcd for C25H17N3O2S:
424.1114 (M + H)+; found: 424.1116.
5h: 1HNMR (600 MHz, DMSO-d6):  = 8.52 (s, 1H), 8.45 (d, J = 12 Hz, 2H), 8.27 (s, 1H), 8.15 (brs,
1H), 8.12 (m, 3H), 8.04 (s, 1H), 7.93 (m, 3H), 7.94 (s, 1H), 7.84 (s, 1H), 7.50 (brs, 1H), 7.39 (s, 1H),
6.74 (s, 1H); 13CNMR (150 MHz, DMSO-d6):  = 167.28, 155.89, 153.03, 152.14, 150.21, 149.21,
148.51, 144.48, 139.81, 138.18, 134.88, 128.25, 128.17, 127.68, 127.13, 124.35, 122.81, 116.45,
114.76, 112.42, 109.84; HRMS (ESI-TOF) : m/z calcd for C25H17N3O3: 408.1343 (M + H)+; found:
408.1350.
1
5i: HNMR (600 MHz, CDCl3):  = 8.28 (d, J = 12 Hz, 2H), 7.96 (s, 1H), 7.85 (s, 1H), 7.82 (d, J =
12 Hz, 2H), 7.78 (d, J = 6 Hz, 2H), 7.61 (s, 1H), 7.58-7.52 (m, 3H), 7.27 (s, 1H), 6.62 (s, 1H); 13CNMR
(150 MHz, CDCl3):  = 154.94, 153.20, 150.02, 149.62, 143.12, 142.98, 137.76, 132.07, 128.92,
128.75, 127.18, 126.63, 118.43, 116.77, 115.67, 112.07, 111.76, 109.01; HRMS (ESI-TOF) : m/z calcd
for C22H14N2O: 323.1179 (M + H)+; found: 323.1182.
1
5j: HNMR (600 MHz, DMSO-d6):  = 8.37 (d, J = 12 Hz, 2H), 8.31 (s, 1H), 8.15 (s, 1H), 8.12 (s,
1H), 8.11 (d, J = 12 Hz, 2H), 8.05 (d, J = 6 Hz, 1H), 8.00 (d, J = 6Hz, 1H), 7.94 (s, 1H), 7.86 (s, 1H),
7.37 (s, 1H), 6.74 (s, 1H); 13CNMR (150 MHz, DMSO-d6):  = 166.48, 164.37, 156.18, 152.55, 149.47,
144.83, 144.51, 144.09, 141.89, 139.04, 133.99, 127.67, 126.58, 123.05, 120.95, 114.96, 113.28,
112.53, 110.20; HRMS (ESI-TOF) : m/z calcd for C21H13N3OS2: 388.0573 (M + H)+; found: 388.0574.
5k: 1HNMR (600 MHz, DMSO-d6):  = 8.54 (d, J = 12 Hz, 2H), 8.34 (s, 1H), 8.18 (d, J = 12Hz, 2H),
8.11 (s, 1H), 8.07 (d, J = 12 Hz, 2H), 8.03 (d, J = 12 Hz, 3H), 7.94 (s, 1H), 7.41 (s, 1H), 6.74 (s, 1H);
13CNMR (150 MHz, DMSO-d6):  = 160.11, 154.69, 152.94, 149.45, 149.31, 144.53, 142.68, 135.00,
134.00, 132.68, 127.74, 127.26, 126.28, 118.86, 116.79, 115.05, 112.42, 111.64, 109.90; HRMS (ESI-
TOF) : m/z calcd for C23H14N6O: 391.1302 (M + H)+; found: 391.1315.
Scheme for synthesis of compounds 5m-5o
Carboxylic acid 4a (1g, 2.7 mmol.) was dissolved in dry DCM (100 mL) and oxalyl chloride (0.5 mL,
5 mmol) followed by DMF (1 drop) were added. The reaction was stirred at room temperature

overnight. Then, the solvent was removed in vacuo and the remaining solid was redissolved in dry DCM
(100 mL). The solution was poured onto a solution of (a) methylamine and DIPEA (1:1) (25 mL); (b)
methanesulfonamide (300 mg, 3.15 mmol) and DIPEA (10 mL) or (c) hydrazine and DIPEA (1:1) (25
mL) and the reaction mixture was stirred for 30 min. Then, the organic layer was washed with water,
separated, dried and evaporated under vacuo to afford 5l, 5m and 5n respectively. The products were
purified by prep TLC using DCM/MeOH (85:15) as eluent.
5l: 1HNMR (600 MHz, DMSO-d6):  = 8.63 (m, 2H), 8.53 (d, J = 12 Hz, 2H), 8.35 (s, 1H), 8.14 (d, J
13
= 12 Hz, 2H), 8.11 (s, 1H), 8.04 (m, 3H), 7.94 (s, 1H), 7.42 (s, 1H), 6.74 (s, 1H), 2.84 (s, 3H); CNMR
(150 MHz, DMSO-d6):  = 165.92, 154.82, 152.78, 149.39, 148.70, 144.66, 142.48, 139.29, 135.19,
132.70, 127.83, 127.77, 127.24, 118.83, 117.25, 115.55, 112.48, 111.77, 110.13; HRMS (ESI-TOF) :
m/z calcd for C24H17N3O2: 380.1393 (M + H)+; found: 380.1394.
5m: 1HNMR (600 MHz, DMSO-d6):  = 8.54 (d, J = 6 Hz, 2H), 8.32 (s, 1H), 8.12 (d, J = 6 Hz, 2H),
8.08 (s, 1H), 8.01 (m, 4H), 7.94 (brs, 1H), 7.93 (s, 1H), 7.40 (s, 1H), 6.74 (m, 1H), 2.89 (s, 3H);
13CNMR (150 MHz, DMSO-d6):  = 169.75, 154.73, 152.86, 149.32, 144.57, 142.58, 140.75, 137.93,
132.68, 129.74, 129.00, 127.76, 126.34, 118.85, 117.10, 115.40, 112.43, 111.68, 109.98, 43.13 ; HRMS
(ESI-TOF) : m/z calcd for C24H17N3O4S: 444.1012 (M + H)+; found: 444.1017.
5n: 1HNMR (600 MHz, DMSO-d6):  = 9.96 (s, 1H), 8.54 (d, J = 12 Hz, 2H), 8.35 (s, 1H), 8.14 (d, J
= 6 Hz, 2H), 8.11 (s, 1H), 8.03 (d, J = 12 Hz, 3H), 7.94 (s, 1H), 7.41 (s, 1H), 6.75 (s, 1H), 4.57 (s, 2H);
13CNMR (150 MHz, DMSO-d6):  = 165.11, 154.81, 152.77, 149.39, 148.69, 144.66, 142.48, 139.34,
134.01, 132.70, 127.76, 127.27, 118.82, 117.24, 115.55, 112.47, 111.77, 110.13 ; HRMS (ESI-TOF) :
m/z calcd for C23H16N4O2: 381.1346 (M + H)+; found: 381.1358.
Synthesis of 7a
To a solution of 4-bromo-2-(4-cyanophenyl)pyridine (200 mg, 0.77 mmol.) in dry DME (3 mL) was
added 4-aminocarbonyl phenyl boronic acid (152 mg, 0.80 mmol.) and water (1 mL). Then, K2CO3 (320
mg, 2.3 mmol.) and Pd(PPh3)4 (36 mg, 0.03 mmol.) were added and the reaction heated at 85 ºC
overnight. The reaction mixture was evaporated, adsorbed over silica gel and purified by column

1
chromatography using EtOAc as eluent to afford 7a as a white solid (160 mg). HNMR (600 MHz,
DMSO-d6):  = 8.82 (d, J = 6 Hz, 1H), 8.45 (m, 3H), 8.13 (brs, 1H), 8.06 (m, 4H), 7.84 (d, J = 6 Hz,
1H), 7.49 (brs, 1H); 13CNMR (150 MHz, DMSO-d6):  = 167.25, 154.98, 150.54, 147.61, 142.84,
139.54, 134.89, 132.69, 128.25, 127.66, 127.09, 121.28, 118.83, 111.60; HRMS (ESI-TOF) : m/z calcd
for C19H13N3O: 300.1131 (M + H)+; found: 300.1132.
Synthesis of 7b
To a solution of 2-bromo-6-(furan-2-yl) pyridine (280 mg, 1.25 mmol.) in dry DME (3 mL) was
added 4-cyanophenyl boronic acid (220 mg, 1.3 mmol.) and water (1 mL). Then, K2CO3 (520 mg, 3.75
mmol.) and Pd(PPh3)4 (60 mg, 0.05 mmol.) were added and the reaction heated at 85 ºC overnight. The
reaction mixture was evaporated, adsorbed over silica gel and purified by column chromatography using
Hex/EtOAc (8:2) as eluent to afford 7b as a white solid (180 mg). 1HNMR (600 MHz, CDCl3):  = 8.23
(d, J = 6 Hz, 2H), 7.86 (t, J = 6 Hz, 1H), 7.80 (m, 1H), 7.73 (d, J = 6 Hz, 1H), 7.66 (d, J = 6 Hz, 1H),
7.59 (s, 1H), 7.22 (s, 1H), 6.60 (m, 1H); 13CNMR (150 MHz, CDCl3):  = 154.24, 153.10, 149.15,
143.11, 142.84, 137.26, 132.05, 127.04, 118.42, 117.52, 111.69, 108.78; HRMS (ESI-TOF) : m/z calcd
for C16H10N2O: 247.0866 (M + H)+; found: 247.0866.
General Procedure for compounds 10a-c. To a solution of furancarboximidine 8 (250 mg, 1.72
mmol) in IPA (20 mL) was added Na (45 mg, 2 mmol) and the reaction was refluxed for 2 hrs.
Afterwards, 2 (500 mg, 1.72 mmol) was added and the mixture was refluxed overnight. Then, it was
evaporated to dryness under vacuum, dissolved in DCM:MeOH (8:2) and filtered through a pad of silica
gel. The filtrate was evaporated to dryness to give a red oil. Carboxylic acid 9 was precipitated using
EtOAc:Hex (1:1) as a yellow solid (55%). This material was carried on without further purification. 9
(200 mg, 0.55 mmol) was dissolved in DCM (20 mL) and oxalyl chloride (0.2 mL, 36 mmol) followed
by 1 drop of DMF. The reaction was stirred at room temperature overnight. Solvent was removed in
vacuo and the remaining solid was redissolved in DCM (50 mL). The solution was poured onto a
NH4OH solution (50 mL) and the reaction mixture was stirred for 30 min. The organic layer was

separated, dried and evaporated under vacuo to afford a brown oil which was purified to obtain
pyrimidine 10.
1
10a: HNMR (600 MHz, DMSO-d6):  = 8.66 (s, 1H), 8.65 (d, J = 2 Hz, 2H), 8.55 (d, J = 12 Hz,
2H), 8.18 (s, 1H), 8.10 (m, 4H), 8.03 (s, 1H), 7.60 (s, 1H), 7.55 (s, 1H), 6.79 (s, 1H); 13CNMR (150
MHz, DMSO-d6):  = 167.24, 163.91, 162.60, 157.33, 151.72, 146.12, 140.41, 138.39, 136.65, 132.89,
128.20, 128.04, 127.35, 118.55, 114.35, 113.49, 112.61, 111.30; HRMS (ESI-TOF) : m/z calcd for
C22H14N4O2: 367.1189 (M + H)+; found: 367.1202.
10b: 1HNMR (400 MHz, DMSO-d6):  = 8.35 (d, 2H), 8.22 (m, 1H), 8.10 (d, 2H), 7.82 (bs, 2H), 7.90
(m, 4H), 7.80 (d, 2H), 7.50 (m, 3H); HRMS (ESI-TOF) : m/z calcd for C22H16N4O: 377.1402 (M + H)+;
found: 377.1482.
10c: 1HNMR (400 MHz, DMSO-d6):  = 8.66 (s, 1H), 8.65 (d, J = 2 Hz, 2H), 8.55 (d, J = 12 Hz, 2H),
8.18 (s, 1H), 8.10 (m, 4H), 8.03 (s, 1H), 7.60 (s, 1H), 7.55 (s, 1H), 6.79 (s, 1H); HRMS (ESI-TOF) : m/z
calcd for C22H16N4O: 382.1555 (M + H)+; found: 382.1482.
ASSOCIATED CONTENT
Supporting Information.
Supplementary figures, biological protocols, sensogram data, compound characterizations, and
expanded synthetic procedures (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
*E-mail: kdjanda@scripps.edu
Present Addresses

† Instituto de Productos Naturales y Agrobiología, CSIC, Avda. Franscisco Sánchez 3, 38206 La
Laguna, Tenerife, Spain. ∫ Mark S. Hixon Consulting LLC, 11273 Spitfire Road, San Diego, CA
92126.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Notes
This is manuscript number 29684 from the Scripps Research Institute.
ACKNOWLEDGMENT
This work was supported by the Skaggs Institute for Chemical Biology and the National
Institutes of Health (CTSA TL1TR001113 to N.T.J), and the Seventh Framework Program of the
European Union (REA Grant Agreement No. 623155 to P.O.M.).
ABBREVIATIONS
FET, field-effect transistor; SPR, surface plasmon resonance; bHLH, basic-helix-loop-helix;
DLS, dynamic light scattering; TLC, thin layer chromatography.
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