A fluorescent target-guided Paal-Knorr reaction

Article abstract of DOI:10.1039/d0ra06962k

It has become increasingly apparent that high-diversity chemical reactions play a significant role in the discovery of bioactive small molecules. Here, we describe an expanse of this paradigm, combining a ‘target-guided synthesis’ concept with Paal-Knorr chemistry applied to the preparation of fluorescent ligands for human prostaglandin-endoperoxide synthase (COX-2).

Full text of DOI:10.1039/d0ra06962k

RSC Advances
View Article Online
View Journal | View Issue
PAPER
A fluorescent target-guided Paal–Knorr reaction†
a
a
b
*
Sachin B. Wagh,
Vladimir Maslivetc,
James J. La Clair
Cite this: RSC Adv., 2020, 10, 37035
a
*
and Alexander Kornienko
It has become increasingly apparent that high-diversity chemical reactions play a significant role in the
discovery of bioactive small molecules. Here, we describe an expanse of this paradigm, combining
a ‘target-guided synthesis’ concept with Paal–Knorr chemistry applied to the preparation of fluorescent
ligands for human prostaglandin-endoperoxide synthase (COX-2).
Received 12th August 2020
Accepted 30th September 2020
DOI: 10.1039/d0ra06962k
rsc.li/rsc-advances
Over the last decade, it has become clear that techniques such conditions. This reaction has been well documented through
as protein-templated fragment ligation (PTFL)1 or target-guided a variety of synthetic applications, such as the commercial
synthesis (TGS)2 demonstrate the potential of biological targets preparation of Lipitor.9 In addition, the Paal–Knorr reaction is
to not only participate in the structure–activity relationship well known biologically.10 It plays a role in human metabolism
(SAR) analyses, but also engage in the synthetic effort. Other and has been implicated in the neurotoxicity of n-hexane.10,11
approaches have shown the use of proteins and cells as vehicles
The mechanism of the Paal–Knorr reaction has been the
for lead isolation and selection,3 therein identifying the possi- subject of numerous investigations. The two main possibilities
bility for methods enabling medicinal chemical studies to be involve initial formation of enamine (a to b, Fig. 1) or hemi-
conducted directly in the presence of a protein target.
aminal (a to d Fig. 1). The calculation of potential energy
To date, reversible chemical reactions catalyzed by protein surfaces using density functional theory suggests that the
pockets referred to as PTFL, include syntheses of hemiacetals, hemiaminal cyclization is the preferred reaction pathway.12 This
acetals, imines, hydrazones, oximes, N,S-acetals, boronate is further corroborated by real-time monitoring of the reaction
esters, disuldes, olen cross-metathesis, and nitro-aldol between aniline and acetonylacetone using extractive electro-
additions.4 Unfortunately, many of these reactions result in spray ionization tandem mass spectrometry, where the dihy-
unstable ligands and oen require additional operations to droxy compound (d, Fig. 1) was found to be a key intermediate
improve stability, such as the reduction of imines to amines on the reaction path.13
with NaBH3CN. Such added steps are undesirable, as amines
The Paal–Knorr reaction requires a mild acid, which is not
could have different binding preferences than imine inter- surprising since each step in the reaction, namely initial attack
mediates.4c Irreversible processes, commonly referred to as TGS, of aniline 2 onto a carbonyl in 1 resulting in adduct a,
are primarily limited to addition of thiols to various electro-
philic molecules5a and the click reaction,5b resulting in the
formation of a thioether and triazole products, respectively.
Clearly, adaptation of diverse irreversible reactions to TGS is
required to enable the use of multifarious drug-friendly
moieties.6
Pyrroles are a fundamental motif found in natural products
and marketed drugs. It has been shown to deliver leads with
anti-cancer, anti-inammatory, anti-bacterial, anti-viral, anti-
malarial, anti-convulsant, anti-hypertensive, anti-psychotic,
anti-cholesterolimic and anti-nociceptive activities.7 While
many methods to synthesize pyrroles exist, the classical Paal–
Knorr reaction,8 involving the condensation of a primary amine
with a 1,4-dicarbonyl, stands out due to the mild reaction
aThe Department of Chemistry and Biochemistry, Texas State University, San Marcos
Fig. 1 Paal–Knorr synthesis of 1,2-diarylpyrroles 3 from diketone 1 and
aniline 2 can proceed through two possible mechanistic pathways.
Current experimental and theoretical results favor the hemiaminal (a to
78666, USA. E-mail: a_k76@txstate.edu
bXenobe Research Institute, P. O. Box 3052, San Diego, USA. E-mail: i@xenobe.org
† Electronic supplementary information (ESI) available: Experimental procedures
e via d) over the enamine (a to c via b) cyclization.
and copies of spectral data. See DOI: 10.1039/d0ra06962k
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 37035–37039 | 37035

View Article Online
RSC Advances
Paper
cyclization of the latter to give d, and subsequent dehydrations for 1a or 2a) in PBS pH 7.2 without inducing undesired reactivity
leading ultimately to pyrroles 3, could be accelerated by an acid such as acetal formation (MeOH, EtOH) or oxidation (DMSO).
catalyst. It is also tempting to propose that the Paal–Knorr
We then prepared pyrrole 3aa synthetically (Fig. 2a) and
reaction should proceed efficiently in presence of protein con- found that it displayed blue uorescence with lmax of 435 ꢀ
taining hydrogen bond donor residues provided that the 10 nm (Fig. 2b). Using 3aa as a guide, we developed a uores-
substituents present in diketone 1 or aniline 2 and ultimately in cent screen to evaluate reactions containing 250 mM diketones
pyrrole 3 could be accommodated at the binding site (see TOC 1a–m with 250 mM anilines 2a–m in PBS at pH 7.2 containing
graphic and Fig. S1†).
5% CH3CN. At this concentration, we screened the levels of
Moreover, when starting materials, intermediates and COX-2 required to generate a uorescent response. Fluores-
pyrrole products 3 are sterically and electronically compatible cence spectra were collected with lex at 260 nm and measuring
with the protein pocket, binding can bring reactants into close the uorescence from lem at 280–600 nm from each well,
proximity thus promoting the transformation. This proposal is compared to wells without COX-2. At concentrations above
consistent with previous reports suggesting that the Paal–Knorr
reaction could be accelerated in the presence of proteins.14
To reduce this proposal to practice, we adopted the trans-
formation of aryl diketones 1a–m and anilines 2a–m to afford
a panel of pyrroles 3aa–mm as the focus of our study (Fig. 2a).
We envisioned that this reaction would be catalyzed at the active
site of COX-2, an important anti-inammatory drug target.15
This choice was based on the prior work describing diary-
lpyrroles as COX-2 selective inhibitors,16 and the presence of
multiple hydrogen-bond donor residues at its active site (H75,
Q178, Y355, Y385, R499). It should also be noted that this
enzyme has been previously reported to catalyze an “in situ”
click reaction, resulting in the formation of triazole products.17
This Paal–Knorr target-guided strategy is schematically illus-
trated in Fig. S1.†
We began by applying the Stetter reaction16 utilizing methyl
vinyl ketone to prepare 1,4-diketones 1a–m from aldehydes 4a–
m (Fig. 2a). Here, we found that use of 20 mol% of thiazolium
catalyst provides moderate to high yields of the diketone
products in a highly reproducible fashion. Using diketone 1a
and aniline 2a as a model, we then established screening to
produce 3aa in aqueous media. This began by developing
conditions where 1a and 2a were sufficiently soluble in aqueous
media. Using NMR methods, we found that 5% CH3CN in PBS
pH 7.2 provided sufficient solubility (at 250 mM concentration
Fig. 3 Discovery of fluorescent ligands to COX-2. (a–d) Primary
screen. Ketones 1a–m (250 mM) were screened in an array against
anilines 2a–m (250 mM). The entire array was run in parallel for 2 h at
0 ꢁC then 12 h at rt in presence (0.1 mM COX-2) or absence of COX-2.
(a) Heat maps depicting difference in fluorescence between the
reactions in the presence or absence of COX-2. Blue indicates a gain in
fluorescence and green a loss in fluorescence. (b) Selected spectra
used to generate the fluorescent intensity change heat maps. (c) In
addition to intensity changes, different wavelengths of fluorescence
were also observed from the spectra collected in the presence or
absence of COX-2. Wavelengths are given from <310 nm (light purple)
to >350 nM (dark purple). White indicates no fluorescence. (d) Selected
spectra used to generate the spectral shift heat maps. We observed
a change in the intensity and fluorescence maxima across the 169
reactions (13 ꢂ 13 reaction matrix). (e–g) Selectivity screen. A subset of
the primary screen was subjected to selectivity analyses. Using iden-
tical conditions, we compared the fluorescence obtained in the
presence of (e) 0.1 mM COX-2 to that generated in the presence of (f)
0.1 mM COX-1 and (g) 0.1 mM human serum albumin (HSA) in order to
identify COX-2 selective products relative to COX-1 and general
protein binders using HSA. Structures of all products in (e–g) are
provided in Fig. S2.† Structures with the largest response (D > 20) in (a)
are provided in Fig. S4.†
Fig. 2 Approach (a) A two-step process was used to prepare dike-
tones 1a–m from aldehydes 4a–m using the Stetter reaction followed
by Paal–Knorr condensation with anilines 2a–m to afford pyrroles
3aa–mm. (b) Fluorescence of 3aa was visible upon excitation at 260–
290 nm.
37036 | RSC Adv., 2020, 10, 37035–37039
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
30 nM, a consistent trend was obtained over 3 repetitions and from 300 nm to >350 nm as illustrated by maxima for 1e + 2b, 1c
0.1 mM COX-2 was set as the concentration for screening. Next, + 2b and 1j + 2b at lem ¼ 340, 325, and 318 nm, respectively
we evaluated the optimal reaction times. Time course uores- (Fig. 3d).
cent monitoring indicated that this process was ideally con-
Next, we challenged the Paal–Knorr approach to generate
ducted by starting the reaction at 0 ꢁC for 2 h and then warming uorescent pyrroles that would demonstrate selectivity for COX-
ꢁ
ꢁ
it up to 23 C over 1 h and incubating for $10 h at 23 C.
2, the predominating cyclooxygenase at sites of inammation,
Over 2 repetitions, we screened a 13 ꢂ 13 array of 1a–m and not COX-1, constitutively expressed in the gastrointestinal
against 2a–m (Fig. 3) using 250 mM 1a–m, 250 mM 2a–m, 0.1 mM tract.18 For therapeutic use, the identication of fully-selective
COX-2 in PBS pH 7.2 containing 5% CH3CN. Spectra for each COX-2 inhibitors would reduce the effects on gastric mucosal
reaction were compared against the corresponding reactions in prostaglandin synthesis observed with non-specic COX-2 tar-
the absence of COX-2 and the difference between these spectra geting candidates.19 While phylogenetically more primitive and
were determined by subtracting the spectrum of the reaction very similar to COX-1,15 COX-2 is distinguished by a smaller
without COX-2 from that with COX-2 (Fig. 3a). As shown in pocket within its active site. This pocket has played a key role in
Fig. 3b, the presence of COX-2 either caused an increase (1j + 2f) the selectivity of agents such as rofecoxib (Vioxx) or celecoxib
or reduction in uorescence (1j + 2g). This data was effectively (Celebrex).20
displayed using a heat map based on the maximal change in the
Using the data in Fig. 3a–d, we selected 16 reactions and
level of uorescence (Fig. 3a). Interesting, the difference prepared a 4 ꢂ 4 reaction array based on the highest differential
between the reactions with and without COX-2 also led to in activity (dark green to dark blue, Fig. 3a) during our primary
a change in wavelength (Fig. 3c). These shis were observed COX-2 screen. We found only modest changes in uorescence
in the presence of 0.1 mM COX-1 (Fig. 3f) or 0.1 mM HSA (Fig. 3g)
when compared to 0.1 mM COX-2 (Fig. 3e). This selectivity was
clearly evident in reactions conducted with 1j (right column,
Fig. 4 Validation studies. (a) Comparison of the reaction of 1j + 2d in
the absence (green) or presence (blue) of COX-2 against 3jd in the
absence (red) or presence (purple) of COX-2 (b) comparison of the
reaction of 1j + 2e in the absence (green) or presence (blue) of COX-2
against 3je in the absence (red) or presence (purple) of COX-2. (c)
Fig. 5 Microscale NMR evaluation. (a) 1H NMR trace indicating the
Comparison of the reaction of 1j + 2f in the absence (green) or
presence (blue) of COX-2 against 3jf in the absence (red) or presence
formation of 3jd from the reaction of 250 mM 1j with 1250 mM 2d in the
(purple) of COX-2. (d) Comparison of the reaction of 1c + 2d in the
presence of 0.1 mM COX-2. (b) Comparable spectral data from the
absence (green) or presence (blue) of COX-2 against 3cd in the
absence (red) or presence (purple) of COX-2. Reactions were con-
ducted using 250 mM 1c or 250 mM 1j and 250 mM 2d–f using same
conditions as in Fig. 3. Spectra were collected from reactions with 0.1
mM COX-2 or without COX-2. Spectra of the products were collected
using 25 mM 3cd or 25 mM 3jd–jf with 0.2 mM COX-2 or without COX-
2. A two-fold increase in COX-2 was used with 3jd–jf to highlight the
spectral changes.
reaction of 250 mM 1j with 1250 mM 2e in the presence of 0.1 mM COX-
2. (c) Comparable spectral data from the reaction of 250 mM 1j with
1250 mM 2f in the presence of 0.1 mM COX-2. (d) Comparable spectral
data from the reaction of 250 mM 1c with 1250 mM 2d in the presence
of 0.1 mM COX-2. These reactions were run at 10-fold increased scale
compared to Fig. 3 (2 mL) to ensure accurate yield calculations. Five-
fold excess of the aniline component was used in these reactions to
encourage turnover. Additional spectra are provided in the ESI.†
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 37035–37039 | 37037

View Article Online
RSC Advances
Paper
Fig. 3e–g). Here, the 3,5-dibromo-substitution in diketone 1j suggesting that the reaction occurred within a pocket of COX-2
resulted in a positive gain in uorescence when presented to 2e (Fig. 6). To further explore this selectivity, we applied our NMR
and 2f in the presence of COX-2 and a loss in uorescence when methods to evaluate reactions that did not show a uorescent
presented to 2d or 2g.
response. As shown in Fig. 5d, we did not observe a reaction
We then evaluated if pyrrole formation would occur in the between 1c and 2d in the presence of COX-2, suggesting that
absence of COX-2 under the same set of conditions as used in a lack of uorescent response suggests no Paal–Knorr reactivity.
the screens (Fig. 3). We scaled up the reactions of 1j with 2d, 2e,
Interestingly, the uorescence quench appears to arise from
and 2f, so that we could chemically monitor them with TLC and the fact that binding to COX-2 requires a conformational
1H NMR analyses (Fig. S5†). The reactions were conducted using regulation leading to a reduction of conjugated states when
250 mM of diketone and 250 mM aniline in 0.4 mL of PBS buffer bound. This suggestion is in part supported by the X-ray crystal
containing 5% CH3CN at 23 ꢁC. NMR and uorescent moni- structure of celecoxib bound to COX-2 (Fig. 6a).20 In order to
toring (every 2.5 h for 15 h) indicated no conversion or uo- explain why 3je formed and 3cd did not, we evaluated how these
rescence characteristic from pyrroles 3 during this period.
pyrroles occupy the celecoxib pocket in COX-2. We found that
We then prepared samples of 3cd, 3jd, 3je, and 3jf synthet- 3je (Fig. 6b) can dock within the same bi-aryl pocket as cele-
ically (Fig. 2a) and compared the effect of COX-2 on their uo- coxib. Pyrrole 3cd (Fig. 6c), which only contains methyl groups
rescence relative to that observed in the reactions in Fig. 3. As in these positions, would present additional steric require-
shown in Fig. 4, we observed a decrease in uorescence from ments as well as lack the electronegativity required for inter-
reactions of 1j with 2d in the presence of COX-2, while the actions with COX-2.
reactions between 1j and 2e or 2f, resulted in a gain in uo-
Overall, we have shown that the Paal–Knorr reaction can be
rescence, when compared to reactions in absence of COX-2. In induced in the presence of COX-2 and utilized the combination
contrast, the incubation of synthetically prepared 3cd and 3jd–jf of uorescence screening and capillary NMR validation. Our
all resulted in a loss in uorescence upon binding to COX-2. work demonstrates a clear pipeline to implement this proce-
This uorescence quench was observed aer adding COX-2 to dure for the development of uorescent probes to a targeted
3cd, 3jd, 3je, or 3jf. This indicates that while the levels of uo- protein (COX-2). Efforts are now underway to translate these
rescence will increase over the course of a reaction due to the materials into uorescent probes for cellular applications as
formation of 3jd, 3je, 3jf or 3cd in the presence of COX-2, well as explore their pharmacological potential.
product binding to COX-2 would mask the uorescent response.
Next, we turned to NMR for validation. As shown in Fig. 5a–c,
Conflicts of interest
we were able to observe the production of 3jd, 3je or 3jf in the
presence of COX-2 by 1H NMR with a 35 mL sample in a 1.7 mm
There are no conicts to declare.
capillary NMR tube. Using integration to compare relative
concentrations of diketone 1j to pyrroles 3jd–jf, we determined
that conversions for 3jd, 3je or 3jf were 22%, 31%, and 5%, Acknowledgements
respectively. This corresponded to a turnover of 528, 740, 120
This work was supported by funding from the NIH under grant
1R21GM131717-01.
for 3jd, 3je or 3jf, respectively, indicating that, while slow, this
templated reaction did turnover. Additional studies indicated
that celecoxib blocked this reaction (Fig. S6†), further
Notes and references
1 (a) O. Seitz, J. Pept. Sci., 2019, 25, e3198; (b) M. Jaegle,
E. L. Wong, C. Tauber, E. Nawrotzky, C. Arkona and
J. Rademann, Angew. Chem., Int. Ed., 2017, 56, 7358.
2 X. Hu and R. Manetsch, Chem. Soc. Rev., 2010, 39, 1316.
3 P. Singh, K. Madhaiyan, M.-D. Duong-Thi, B. W. Dymock and
S. Ohlson, SLAS Discovery, 2017, 22, 440.
4 (a) L. Zhang, J. Dong, X. Xu and Q. Liu, Chem. Rev., 2016, 116,
287; (b) E. Burda and J. Rademann, Nat. Commun., 2014, 5, 1;
Fig. 6 Structural evaluation. (a) X-ray crystal structure (PDB ID 3LN1)
(c) M. F. Schmidt, A. El-Dahshan, S. Keller and J. Rademann,
depicting celecoxib (cyan) binding pocket in COX-2. The aromatic
Angew. Chem., Int. Ed., 2009, 48, 6346.
rings of celecoxib undergo deconjugative rotation, so that the aryl
5 (a) E. Oueis, F. Nachon, C. Sabot and P.-Y. Renard, Chem.
torsional angles21 are 41 ¼ ꢃ30ꢁ, 42 ¼ ꢃ44ꢁ. (b) Image depicting 3je
Commun., 2014, 50, 2043; (b) E. Oueis, C. Sabot and
P.-Y. Renard, Chem. Commun., 2015, 51, 12158.
6 J. Ohkanda, Chem. Rec., 2013, 13, 561.
7 V. Bhardwaj, D. Gumber, V. Abbot, S. Dhiman and
P. Sharma, RSC Adv., 2015, 5, 15233.
(yellow) docked within the celecoxib binding pocket of COX-2 shown
in (a). Here, the 3,5-dibromoaryl ring in 3je could mimic the two S]O
bonds within the sulphonamide in celecoxib (proximal to R499 and
Q178). Energy minimized conformations of 3je impose torsion angles
of 41 ¼ ꢃ48ꢁ, 42 ¼ ꢃ57ꢁ indicating that conformation access within the
biaryl motifs of 3je was comparable to that in celecoxib. (c) Image
depicting 3cd (yellow) docked within the celecoxib-binding pocket of
COX-2. Expanded renderings in Fig. S3.†
8 D. R. Dasari, J. J. La Clair and A. Kornienko, Chembiochem,
2017, 18, 1792.
37038 | RSC Adv., 2020, 10, 37035–37039
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
9 Y. V. Novozhilov, M. V. Dorogov, M. V. Blumina,
A. V. Smirnov and M. Krasavin, Chem. Cent. J., 2015, 9, 7.
J. L. Currie, K. Seibert and P. C. Isakson, J. Med. Chem.,
1997, 40, 1619.
10 A. Kornienko and J. J. La Clair, Nat. Prod. Rep., 2017, 34, 17 A. Bhardwaj, J. Kaur, M. Wuest and F. Wuest, Nat. Commun.,
1051. 2017, 8, 1.
11 J. W. Werner-Allen, J. F. DuMond, R. L. Levine and A. Bax, 18 J. A. Mitchell, N. S. Kirkby, B. Ahmetaj-Shala, P. Armstrong,
Angew. Chem., Int. Ed., 2016, 55, 7374.
12 B. Mothana and R. J. Boyd, J. Mol. Struct.: THEOCHEM, 2007,
811, 97.
13 X. Zhang, M. Pei, D. Wu, S. Yang and Z. Le, Sci. Rep., 2019, 9,
19279.
M. Crescente, P. Ferreira, M. E. L. Pires, R. K. Vaja and
T. D. Warner, Pharmacol. Ther., 2020, 5, 107624.
19 S. A. Schug, B. Parsons, C. Li and F. Xia, J. Pain Res., 2017, 10,
2451.
20 B. J. Orlando and M. G. Malkowski, Acta Crystallogr., Sect. F:
Struct. Biol. Commun., 2016, 72, 772.
14 N. Salehi and B. B. F. Mirjalili, Iran. J. Catal., 2019, 9, 185.
´
´
15 M. D. Ferrer, C. Busquets-Cortes, X. Capo, S. Tejada, J. A. Tur, 21 T. Egawa, K. Shinashi, T. Ueda, E. J. Ocola, W.-Y. Chiang and
A. Pons and A. Sureda, Curr. Med. Chem., 2019, 26, 3225.
16 I. K. Khanna, R. M. Weier, Y. Yu, P. W. Collins,
J. M. Miyashiro, C. M. Koboldt, A. W. Veenhuizen,
J. Laane, J. Phys. Chem. A, 2014, 118, 1103.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 37035–37039 | 37039