Diversity through phosphine catalysis identifies octahydro-1,6- naphthyridin-4-ones as activators of endothelium-driven immunity

Article abstract of DOI:10.1073/pnas.1015254108

The endothelium plays a critical role in promoting inflammation in cardiovascular disease and other chronic inflammatory conditions, and many small-molecule screens have sought to identify agents that prevent endothelial cell activation. Conversely, an au

Full text of DOI:10.1073/pnas.1015254108

Diversity through phosphine catalysis identifies
octahydro-1,6-naphthyridin-4-ones as activators
of endothelium-driven immunity
Daniel Cruza,1, Zhiming Wangb,1,2, Jon Kibbiea, Robert Modlinc, and Ohyun Kwonb,3
aDepartment of Medicine, Division of Cardiology, A2-237 Center for Health Sciences, University of California, Los Angeles, CA 90095-1679; bDepartment
c
of Chemistry and Biochemistry, University of California, 607 Charles E. Young Drive East, Los Angeles, CA 90095-1569; and Department of Medicine,
Division of Dermatology, University of California, 10833 Le Conte Avenue, 52-121 Center for Health Sciences Building, Los Angeles, CA 90095
Edited by Stuart L. Schreiber, Broad Institute, Cambridge, MA, and approved January 31, 2011 (received for review December 6, 2010)
The endothelium plays a critical role in promoting inflammation in
cardiovascular disease and other chronic inflammatory conditions,
and many small-molecule screens have sought to identify agents
that prevent endothelial cell activation. Conversely, an augmented
immune response can be protective against microbial pathogens
and in cancer immunotherapy. Yet, small-molecule screens to iden-
tify agents that induce endothelial cell activation have not been
reported. In this regard, a bioassay was developed that identifies
activated endothelium by its capacity to trigger macrophage in-
flammatory protein 1 beta from primary monocytes. Subsequently,
a 642-compound library of 39 distinctive scaffolds generated by a
diversity-oriented synthesis based on the nucleophilic phosphine
catalysis was screened for small molecules that activated the
endothelium. Among the active compounds identified, the major
classes were synthesized through the sequence of phosphine-
catalyzed annulation, Tebbe reaction, Diels–Alder reaction, and in
some cases, hydrolysis. Ninety-six analogs of one particular class of
compounds, octahydro-1,6-naphthyridin-4-ones, were efficiently
prepared by a solid-phase split-and-pool technique and by solution
phase analog synthesis. Structure-function analysis combined
with transcriptional profiling of active and inactive octahydro-1,
6-naphthyridin-4-one analogs identified inflammatory gene net-
works induced exclusively by the active compound. The identifica-
tion of a family of chemical probes that augment innate immunity
through endothelial cell activation provides a framework for un-
derstanding gene networks involved in endothelial inflammation
as well as the development of novel endothelium-driven immu-
notherapeutic agents.
one-step ring-forming reactions were carried out using either
commercially available or otherwise readily available imines,
maleimides, aldehydes, aziridines, and electron-deficient olefins,
resulting in scaffolds s1–s8 (11–20). For electron-deficient acet-
ylenes, a one-step double-Michael reaction with readily available
dinucleophiles was effected by a diphenylphosphinopropane
catalyst, providing heterocyclic frameworks s9–s20 (21, 22).
The α,β-unsaturated ester functionality in tetrahydropyridine
s1, dihydropyrrole s2, and bicyclic succinimide s3 was further
utilized in a highly diastereoselective Michael addition of thiols
to produce piperidine s21, pyrrolidine s22, and bicyclic succini-
mide s23 (23). The carbonyl group of the α,β-unsaturated ester
was also methylenated using Tebbe reagent to provide alkoxy
dienes, which upon exposure to dienophiles (imines, maleimides,
triazolinediones, tetracyano ethylene, and benzoquinones) un-
derwent diastereoselective Diels–Alder reaction and produced
multicyclic compounds s24–s35 (24). A stereoselective hydrolysis
of the enol ether group in the Diels–Alder adducts further pro-
duced multicyclic ketones s36–s39. The library of 642 compounds
of 39 distinctive scaffolds was tested in several bioassays and re-
sulted in the identification of geranylgeranyltransferase type I
(GGTase I) inhibitors (25, 26), RabGGTase inhibitors (23),
and antimigratory compounds (24). These outcomes powerfully
demonstrate the premise behind DOS—the more structural
diversity in the screening collection, the higher the probability
of discovering small-molecule biomodulators. Therefore, the
nucleophilic phosphine catalysis-based synthesis resulted in a
compound library with rich structural diversity, well poised to
probe critical biological questions.
solid-phase library synthesis ∣ immune activator ∣ medicinal chemistry ∣
nucleophilic phosphine-catalyzed annulations ∣
structure-activity relationship analysis
In order to most efficiently interrogate compound libraries, high-
throughput screening systems to detect compounds with prespeci-
fied molecular targets have been developed. However, with few
exceptions, a limitation of current screens is that they do not
integrate the interactions that occur between heterogeneous cell
types involved in disease pathogenesis (27, 28). We aimed to estab-
lish a biological platform amenable to high-throughput screening,
but with sufficient complexity to identify molecular probes that
he synthesis and use of bioactive small molecules to gain
T
insight into biological systems is a major facet of modern
chemical biology (1) and several hypotheses regarding the most
effective ways to increase the probability of discovering chemical
probes of desired activity have been put forth (2–4). In this
context, combinatorial chemistry emerged as the fastest way to
generate a large number of candidate compounds (5, 6). How-
ever, early practices of large library synthesis and screening un-
veiled that the large number alone is not sufficient for increased
hit rates and the structural diversity within the library may be
important. The idea of generating a structurally diverse collection
of compounds within a streamlined sequence of reactions has
been elegantly formulated in the algorithms of diversity-oriented
synthesis (DOS) (7–10). In this vein, a series of nucleophilic phos-
phine catalysis reactions has been developed, resulting in the
production of 20 distinctive carbo- and heterocyclic scaffolds
(Scheme 1, s1–s20). The goal in developing previously unde-
scribed reactions was to provide previously unexampled hetero-
cyclic frameworks that deviate from the relatively limited pool of
molecular motifs used by pharmaceutical companies. For allenes,
Author contributions: D.C., Z.W., and O.K. designed research; D.C., Z.W., and J.K.
performed research; D.C., Z.W., R.M., and O.K. analyzed data; and D.C., Z.W., and O.K.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: Crystallographic data for 1a and 1a′ have been deposited in the
Cambridge Structural Database, Cambridge Crystallographic Data Centre, Cambridge
CB2 1EZ, United Kingdom, http://www.ccdc.cam.ac.uk/ (CSD reference nos. 767112 and
802608, respectively).
1D.C. and Z.W. contributed equally to this work.
2Present address: School of Chemical Engineering, Changzhou University, Changzhou
213164, Jiangsu, People’s Republic of China.
3To whom correspondence should be addressed. E-mail: ohyun@chem.ucla.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1015254108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1015254108
PNAS ∣ April 26, 2011 ∣ vol. 108 ∣ no. 17 ∣ 6769–6774

the production of macrophage inflammatory protein 1 alpha
(MIP1α) from primary human monocytes (42). Interestingly,
neither EC nor monocytes treated with IFNγ produced this
chemokine; coculture between the activated EC and monocytes
was required. Therefore we hypothesized that compounds that
“activate” the endothelium could be identified indirectly—
endothelium encountering activating compounds would gain
the capacity to trigger chemokine production by monocytes. To
test this hypothesis, we designed an EC-monocyte coculture
system amenable to high-throughput compound screening
(Fig. S1A). In this platform, MIP1β production, rather than
MIP1α or IL8, proved to be the most reliable marker of EC
activation by IFNγ (Fig. S1B). Subsequently, EC were treated
with DMSO (vehicle control), IFNγ (EC-activation control),
and a collection of 642 compounds (final concentration, 10 μM)
(Fig. S1C). Of the 642 compounds, 37 (5.8%) promoted MIP1β
production (Fig. 1A). By contrast, IFNγ-treated EC inhibited
IL-8 production from monocytes; likewise, every EC-activating
compound also decreased IL8 production (Fig. S2A). MIP1α
was not induced by IFN-treated EC; however, most compounds
that induced MIP1β also induced MIP1α production (Fig. S2B).
Scheme 1. Diversity-oriented synthesis based on nucleophilic phosphine
catalysis. For clarity, detailed designation of stereochemistry and substituents
are removed. For the detailed structural information, see the following
references: For scaffold s1, refs. 11 and 12; s2, refs. 13 and 14; s4, ref. 15; s5,
ref. 16; s6, refs. 17 and 18; s7, ref. 19; s8, ref. 20; s9–s12, ref. 21; s13–s20,
ref. 22; s21–s23, ref. 23; s24–s39, ref. 24.
resulted in (i) endothelial cell activation and (ii) subsequent
endothelial cell (EC)-triggered induction of innate immune
responses. Activation of the endothelium occurs when proinflam-
matory cytokines such as interferon gamma (IFNγ) induce the pro-
ductionofchemokinesand expressionofcelladhesionmoleculeson
theendothelialsurface. Consequently, leukocyteshometoactivated
endothelium and transmigration ensues. Such activation of the
endothelium is a physiologic and necessary process, desirable for
eradicating infections and some cancers (29–31).
Despite the homeostatic benefits of endothelial cell activation
in host defense, such activation also plays a central role in
the pathogenesis of many chronic inflammatory disorders (32);
this has resulted in chemical screens predominantly focused on
the identification of compounds that decrease endothelial cell in-
flammation (33–37). In stark contrast, far less effort has been put
forth to understand the underlying networks by which compounds
may activate or inflame the endothelium. Yet, such insights may
have important implications for understanding salutary and
maladaptive aspects of inflammation at the vessel wall. Further-
more, given that an increasing number of drugs have been
removed from the market as a result of cardiovascular complica-
tions (38–41), understanding how small molecules may promote
inflammation in the vasculature is an important question. Herein,
we report how a DOS library approach, applied to a unique
biological platform, has led to the identification of a previously
undescribed class of chemical probes that trigger human EC-
driven activation of innate immunity, a biological process integral
to both host defense and disease pathogenesis.
Fig. 1. Identification of small molecules that activate human endothelial
cells. (A) IFNγ (10 ng∕mL), DMSO controls (n ¼ 60 total replicates), and 642
compounds (10 μM) were tested for their ability to promote MIP1β produc-
tion. The library contained 37 compounds capable of inducing MIP1β at least
to the level of IFNγ (red dashed line). * represents five distinct scaffolds
with activity, whereas ** represents the two naphtyridine families. (B) Struc-
tures of five octahydronaphthyridinones selected for further study. (C) Con-
firmation of a subset of active compounds: Coculture is required for MIP1β
production. Data represents mean ꢀ SEM of triplicate wells from one of
two comparable experiments. # p values for MIP1β: (a) 105A3 ¼ 0.008,
(b) A8 ¼ 0.003, (c) A10 ¼ 0.026, (d) A11 ¼ 0.007, and (e) B2 ¼ 0.046. ##
p values for MIP1α: (a) 105A3 ¼ 0.042, (b) A8 ¼ 0.016, (c) A10 ¼ 0.009,
(d) A11 ¼ 0.041, and (e) B2 ¼ 0.13. (D) Activated endothelium triggers MIP1β
production from CD14+ monocytes. Percent positive MIP1β cells and mean
fluorescence intensity (MFI) of MIP1βþ cells are shown.
Results
Identification of Small Molecules That Activate Endothelium. Activa-
tion of the endothelium is intricately linked to innate immunity.
For example, ECs activated by IFNγ were shown to trigger
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Intriguingly, most EC-activating compounds (35 out of 37;
Fig. S3) possess the scaffolds derived from the sequence of phos-
phine-catalyzed annulation, Tebbe methylenation, Diels–Alder
reaction, and sometimes hydrolysis (scaffolds s24–s27, s31, s32,
and s36 in Scheme 1). As reported earlier (24), tetrahydropyri-
dine s1 and pyrrolidine s2 were formed through the phopshine-
catalyzed [4 þ 2] and [3 þ 2] annulation reaction between alleno-
ates and imines (11–14) and converted into the corresponding
ethoxy dienes via Tebbe reaction. The subsequent Diels–Alder
reaction of the dienes with maleimides, N-phenyl triazolinedione,
and N-sulfonamido arylimines provided multicyclic enol ethers
s24, s25/s26, s27, s31, s32, each of which provided 9, 5, 5, 3,
and 8 hits out of 10, 12, 5, 9, and 10 compounds, respectively,
in confirmatory experiments (Fig. S4). The enol ether adducts
s24, s25/s26, and s31 were stereoselectively hydrolyzed into the
corresponding ketones s36, s37/s38, and s39. Although ketones
s37/s38 and s39 did not provide any EC-activating compounds,
all 10 of the octahydro-1,6-naphthyridin-4-ones were active.
The octahydro-1,6-naphthyridine framework was particularly
interesting in that it exhibited the highest hit rate, and the so-
lid-phase split-and-pool synthesis of the octahydro-1,6-naphthyr-
idin-4-one analogs should be possible by using resin-bound
allenoates (vide infra).
With the anticipation of the analog synthesis, five octahy-
dro-1,6-naphthyridin-4-ones were selected for further validation
(Fig. 1B). Direct treatment of independent EC or peripheral
blood mononuclear cell (PBMC) with these activating com-
pounds did not result in significant production of MIP1β or
MIP1α; as with IFNγ, coculture of activated EC and PBMC was
required for chemokine induction (Fig. 1C). Intracellular flow
cytometry of cocultures revealed that when PBMCs are added
to ECs activated either by IFNγ or an octahydro-1,6-naphthyri-
din-4-one 105A10, the CD14+ monocytes, rather than ECs or
lymphocytes, are the dominant producers of MIP1β (Fig. 1D).
This underscores the finding that a screen testing isolated en-
dothelial cells or monocytes would have missed this particular
class of EC activators. Therefore, like IFNγ, this unique family of
compounds mediates EC-triggered induction of innate immune
activation.
phosphine-catalyzed [4 þ 2] annulation of resin-bound allenoate
4 with N-tosylbenzaldimine (25). The resin-bound tetrahydropyr-
idine 5 was treated with Tebbe reagent and anhydrous pyridine in
THF and converted into the dienol ether 6. To test the efficiency
of the Tebbe reaction, the enol ether 6 was cleaved off the resin
using TFA (2.5%) in dichloromethane (DCM). Because there are
very few successful examples of Tebbe reaction in the solid phase
(43, 44), we were pleased to find the methylenation of the poly-
mer-supported α,β-unsaturated enoate 5 proceeded smoothly in
good yield; enone 60 was obtained in 53% overall yield over four
steps and excellent purity (>95%, 1H NMR). The subsequent
endo-selective Diels–Alder reaction with N-tosylbenzaldimine
in toluene at 80 °C gave octahydro-1,6-naphthyridine 7, which
was hydrolyzed off the resin using 2.5% TFA in DCM to provide
octahydro-1,6-naphthyridin-4-ones 1a and 1a′ in 38% overall
yield with high diastereoselectivity (diastereoisomeric ratio ¼
97∶3) after chromatographic purification. The structures of both
compounds 1a and 1a′ were unequivocally established through
X-ray crystallography. X-ray crystallography revealed that the tet-
rahydropyridine (11) and the octahydronaphthyridine (24) fea-
tured antirelationships between their C7-phenyl and N6-tosyl
groups. Interestingly, the imine dienophile approached the diene
6 from the opposite face of the N6-tosyl group. It is also note-
worthy that the hydrolysis of the enol ether produced octahydro-
naphthyridinone 1a and 1a′ featuring a cis-fused [4.4.0] bicyclic
framework.
Having successfully established the solid-phase reaction condi-
tions, next we prepared the N-sulfonylimine building blocks.
According to the result of phosphine-catalyzed [4 þ 2] annulation
of resin-bound allenoates from our previous work (25), we chose
10 N sulfonylimines, which provided excellent reaction yields
(87–108%) and purities (80–99%) (Fig. S5). It is noteworthy that
the strategy of our combinatorial library construction is very effi-
cient in terms of building blocks because the N sulfonylimines
were used in both phosphine-catalyzed [4 þ 2] annulation and
the Diels–Alder reaction. With the building blocks in hand,
the library synthesis commenced. The individual lanterns were
tagged with colored spindles and cogs to encode the imine build-
ing blocks of the [4 þ 2] annulation used for each lantern.
Because the Diels–Alder reaction was the last split step of the
synthesis, tagging for the imine building blocks of the Diels–Alder
reaction was not necessary. By using the tagging and split-and-
pool combinatorial techniques, 2-methyl-2,3-butadienoic acid
and 10 N-sulfonylimine building blocks resulted in the prepara-
tion of 100 (1 × 10 × 10) octahydro-1,6-naphthyridin-4-one ana-
logs. The overall yields are up to 39% in five steps and the
purities of the final products are up to 99% after the prep HPLC
purification.
Synthesis of Naphthyridinone Analogs. The discovery of promising
chemical probes capable of promoting a robust innate immune
response through the activation of the endothelium warranted
the development of efficient and rapid synthesis of analogs,
allowing for determination of structure-activity relationship
(SAR). We envisioned a short, modular synthetic route using
SynPhase lanterns as the solid support (Scheme 2). Establishment
of the solid-phase synthesis began with the coupling of the Wang
resin 3 with 2-methyl-2,3-butadienoic acid (2) and the subsequent
Ts
Ph
N
O
O
86 h for loading
134 h reaction
Ts
O
N
+
OH
COOH
O
4 days washing
Ref. 25
1 day for washing
Ref. 25
Ph
3
5
2
4
SynPhase lanterns
Wang resin
Tebbe/Py, THF
6 days reaction
2 days washing
Ph
O
O
H
Ts
H
H
N
Ts
Ts
Ts
N
N
2.5% TFA
Ph
2.5% TFA
CH2Cl2
Ts
Ph
Ts
Ph
N
+
O
N
O
N
O
Ph
N
Ph Ph
N
toluene
heat
Ph
CH2Cl2
H
Ph
N
Ts
1a, major
Ts
1a', minor
6
6'
4 steps, 53%
6 days reaction
3 days washing
7
Ts
5 steps, 38% overall yield, dr = 97:3
Scheme 2. Solid-phase synthesis of octahydro-1,6-naphthyridin-4-ones.
Cruz et al.
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At the same time, additional octahydro-1,6-naphthyridine
analogs with different functional groups were synthesized in
the solution phase (Scheme 3) for the further SAR analysis. The
octahydro-1,6-naphthyridin-4-one analogs 11a–11ab with differ-
ent substituents, Ar, R, Ar′, and R′, were synthesized according
to procedures reported previously (Scheme 3A) (24). For the
preparation of an octahydronaphthyridinone without the C7
substituent, a previously undescribed sequence of reactions was
designed (Scheme 3B). The commercially available ethyl 4-
piperidone-3-carboxylate hydrochloride (12) was protected with
p-toluenesulfonyl (tosyl, TS) group and the ketone was chemose-
lectively reduced with sodium borohydride (NaBH4). Methane-
sulfonyl(mesyl)ation of the resulting alcohol and β-elimination
of the mesylate provided tetrahydropyridine 14. The Tebbe
methylenation, Diels–Alder reaction with N-tosylbenzaldimine,
and acid hydrolysis gave the desired product H6. To probe the
importance of the C4-carbonyl group, the corresponding alcohol
and its ethyl ether were also prepared (Scheme 3C). The diiso-
butylaluminium hydride reduction of compound 105A10 gave the
alcohol 2B6, which in turn was O-alkylated to the ethyl ether 2B7.
Naphthyridinone without the N1 substituent was also prepared
(Scheme 3D). To prepare tetrahydronaphthyridine G9, octahy-
dronaphthyridine 15 was subjected to the Fukuyama et al.’s
denosylation condition (45) followed by morpholinesulfonyl
chloride and triethylamine for aromatization (46).
A
B
C
Fig. 2. EC-activating effects of 96 naphthyridinones. (A) Data represent
percent induction relative to IFNγ for each of the 96 analogs analyzed over
two experiments. (B) Structures of selected active (H6, 2B6, 2B7, 105A3, D6,
A10, D10, D11, E3, C2, C8, and C9) and inactive octahydro-1,6-naphthyridin-4-
one analogs (H11, 2B2, D5, D9, E2, E8, F7, F10, and G9). (C) Validation of
seven (active and inactive) analogs. Data represent the mean ꢀ SEM from
two independent experiments performed in triplicate wells (n ¼ 6 wells per
condition), * p value < 0.05, ** p value < 0.03, *** p value < 0.003.
Structure-Activity Relationship. With the synthesis of analogs from
the solid phase and the solution phase completed (Fig. S6), the
products were dissolved in DMSO and analyzed for EC activa-
tion. Because the original 10 naphthyridinones were all EC-acti-
vating, the hope was that the SAR studies would reveal structural
elements that obliterate EC-activating effects and thereby
pinpoint the crucial motifs for the biological activity. Indeed,
the focused library of 96 analogs produced compounds with
distinct capacities for EC activation (Fig. 2A), indicating some
essential structural features for the biological activity of the
octahydro-1,6-naphthyridin-4-ones. In particular, the aryl groups
on the N1- and N6-arylsulfonly groups were indispensible;
both N1-mesyl (Ms) and N6-mesyl naphthyridinone compounds
(H11 and 2B2, Fig. 2B) lost their activity. Based on the fact that
the N1-mesylated naphthyridinone was inactive, it was not sur-
prising to find that the derivative G9 with the pyridine ring
was inactive as well. On the other hand, removal of the C7-aryl
group (analog H6) did not affect the naphthyridinone’s EC acti-
vation. As we discovered in the initial screening, both enol ether
(s24 in Scheme 1) and ketone octahydronaphthyridines (s36 in
Scheme 1) were active, indicating that ketone group is not essen-
tial for the activity. Although we speculated that the tetrasubsti-
tuted enol ether functionality is intact under physiological
conditions based on the fact that ketones (s37–s39) of EC-
activating enol ethers (s25, s26, and s31) were not active, we
wanted to confirm that the naphthyridinones are by no means
acting as covalent modifiers through Schiff base formation. In-
deed, alcohol 2B6 and its ethyl ether 2B7 were active, confirming
that the ketone group was less essential for naphthyridinone’s
EC-activating effect. The solid-phase split-and-pool synthesis
provided a large number of active and inactive analogs that con-
tain various substituents around the benzene rings of N1 and N6
arylsulfonyl as well as C2- and C7-aryl groups. One subtle yet
powerful observation was that naphthyridinones D5, D9, E8,
F7, and F10 (except analog E2) bearing N1 benzenesulfonyl
and C2-p-methoxyphenyl group did not exhibit EC activation,
whereas various naphthyridinones with the same N6 and C7 sub-
stituents retained their activity (Fig. 2B). Based on its structure,
analog E2, which appeared to have some activity in the initial test
(Fig. 2A), was predicted to behave more like the inactive analogs;
indeed, confirmatory studies revealed it was relatively inactive
(Fig. 2C). Although some synergistic substituent effects obscured
a clear-cut SAR, useful patterns emerged (Fig. 3). The predictive
power of the SAR data illustrates the advantage of having a
diverse and significant number of analogs, which was possible
only because of the short and efficient synthetic route based on
the solid-phase nucleophilic phosphine catalysis.
A
B
Based on the disparate biological activities of D10 and E2, we
predicted that these two structurally similar molecules would
induce distinct gene networks involved in EC activation. To test
this idea, we performed transcriptome profiling in EC treated
with active (D10) and inactive (E2) analogs. In comparison to
vehicle control, the EC-activating analog D10 induced 201 gene
probes (fold change >1.25, p value < 0.05), whereas the inactive
analog E2 activated only 49 gene probes (Fig. 4A). Remarkably,
despite their structural similarity, the two analogs induced
distinct sets of genes: Only two genes were shared in common
between the two analogs. More specifically, genes involved
C
D
Scheme 3. Solution-phase synthesis of octahydro-1,6-naphthyridin-4-ones.
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Discussion
The endothelium is perpetually exposed to systemic chemical and
mechanical stressors, such as infection, hypertension, smoking,
and diabetes-cardiovascular risk factors known to cause endothe-
lial inflammation (49, 50). This is intricately linked with the
induction of chemokines and adhesion molecules by the endothe-
lium, which culminates in transmigration of circulating mono-
cytes, a process that may be protective in infection and some
cancers, but detrimental in chronic inflammatory diseases, such
as atherosclerosis. We screened a library of 642 carbo- and het-
erocycles of 39 distinctive scaffolds assembled through DOS
based on the nucleophilic phosphine catalysis for their ability to
activate human endothelial cells. Seven distinctive scaffolds of 35
compounds were identified by their capacity to bestow upon
the endothelium the ability to trigger MIP1α and MIP1β produc-
tion from previously quiescent monocytes. Taking advantage of
the exceedingly simple assembly strategy for one specific scaf-
fold, octahydro-1,6-naphthyridin-4-one, 96 analogs were pre-
pared through solid-phase split-and-pool synthesis and solution
phase medicinal chemistry. The diverse library revealed structur-
al features indispensible for activation of the endothelium, simul-
taneously serving as tools to dissect molecular networks necessary
to mediate EC-triggered activation of innate immune responses.
MIP-1 chemokines are known to play a key role in the chemo-
taxis of lymphocytes and monocytes, as well as protective roles in
HIV pathogenesis (51) and cancer immunotherapy (52). There-
fore, the identification of small molecules with potent capacity to
activate the innate immune response through the endothelium
raises the possibility of developing compounds for clinical scenar-
ios in which augmented immunity may be desirable. Nevertheless,
inflammation in the vessel wall can lead to endothelial dysfunc-
tion and myocardial infarction (49, 50). We demonstrate that
structurally similar compounds can have disparate effects on
triggering EC-mediated induction of innate immune responses.
Therefore, among otherwise equal drug candidates, it may be
preferable to select agents that do not have “off-target” EC
activation, which may then mediate undesirable innate immune
activation in the vascular wall. Indeed, the increasing number of
Fig. 3. SAR analysis of octahydro-1,6-naphthyridin-4-ones.
in the regulation of immune processes were more frequently
induced by D10 than E2 (Fig. 4B). The induced genes were
additionally analyzed by ingenuity pathway analysis; in total,
32 canonical pathways were engaged by the active analog, and
10 of the top 20 canonical pathways are implicated in inflamma-
tion, including atherosclerosis signaling (p < 0.03), IL8 signaling
(p < 0.006), and high mobility group box1 signaling (p < 0.004).
By contrast, E2 did not activate any networks related to immune
regulation. Of particular interest, E selectin was found to be un-
iquely induced by the active, but not by the inactive analog. This
adhesion molecule plays a critical role in leukocyte rolling and
firm adhesion to activated endothelium and is implicated in
chronic inflammatory diseases (47, 48). To confirm this finding
at the protein level, EC were treated with active or inactive ana-
logs and E-selectin expression was measured by flow cytometric
analysis. In comparison to vehicle control, only the active analog
D10 markedly enhanced E-selectin expression levels (Fig. 4C).
Therefore, despite subtle structural changes, these two naphthyr-
idinones display distinct effects on EC activation and disparate
gene expression profiles. Together, this work demonstrates how
a DOS approach can be combined with screening platforms of
adequate complexity, along with transcriptome profiling, to
provide a framework for understanding complex biological
processes, such as EC-triggered innate immunity.
B
D10
E2
-
D10
E2
-
A
COL3A1
SELE
IRAK2
ZFP36
RAG1
CXCR4
CXCR4
DUSP3
VCAM1
CXCR4
DLG1
ATP7A
DUSP3
INHBA
GEM
MYO1E
JUN
ITPKB
SQSTM1
BCL2L11
KITLG
DUSP3
HMOX1
BCL2L11
IRS2
SQSTM1
Fig. 4. Gene expression profiling of endothelial cells
DMSO
D10
E2
isotype
treated with active and inactive analogs. (A) Microarray
analysis comparing gene expression induction in EC
treated with active analog (D10, 10 μM) and inactive
analog (E2, 10 μM). (B) Subset of genes from A with
known involvement in inflammation: relative expres-
sion by active and inactive analogs. (C) E-selectin induc-
tion by naphthyridinone analogs. Percentages for
intercellular adhesion molecule (ICAM) and ICAM/E-
selectin expressing cells are displayed.
53
36
79
12
10
1
79
7
C
E selectin
Cruz et al.
PNAS
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April 26, 2011
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vol. 108
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drugs withdrawn from the market or failed in clinical trials due
to cardiovascular complications (38–41) indicates the need for
biological platforms that identify endothelial cell activation.
This work demonstrates that the integration of extremely
efficient DOS strategy and a biological screening platform of
adequate complexity can lead to deep insights into structure–
function relationships during small-molecule testing and develop-
ment. We identified a unique group of small molecules, octahy-
dro-1,6-naphthyridin-4-ones, that activate the endothelium, which
in turn trigger monocyte activation. Together, the work provides
a unique conceptual framework for dissecting critical regulatory
networks involved in EC activation by small molecules, as well as
the possibility of augmenting innate immunity through endothe-
lium-triggered immune responses.
Materials and Methods
Synthesis. Detailed synthetic procedures and characterization of the
compounds are available in SI Appendix.
Biology. Information regarding cells, reagents, assays, flow cytometry, and
microarray experiments is provided in SI Appendix.
ACKNOWLEDGMENTS. This research was supported by the US National
Institutes of Health (Grants R01GM071779 and P41GM081282 to O.K.; Grant
K088HL092290 to D.C.), and the Harold Amos Medical Faculty Development
Program (to D.C.).
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