Synthesis and biological evaluation of quinic acid derivatives as anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2009.07.096

Quinic acid (QA) esters found in hot water extracts of Uncaria tomentosa (a.k.a. cat's claw) exert anti-inflammatory activity through mechanisms involving inhibition of the pro-inflammatory transcription factor nuclear factor kappa B (NF-κB). Herein, we d

Full text of DOI:10.1016/j.bmcl.2009.07.096

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5458–5460
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of quinic acid derivatives
as anti-inflammatory agents
,
*
Kui Zeng, Karin Emmons Thompson, Charles R. Yates , Duane D. Miller
Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN 38163, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Quinic acid (QA) esters found in hot water extracts of Uncaria tomentosa (a.k.a. cat’s claw) exert anti-
Received 15 May 2009
Revised 16 July 2009
Accepted 20 July 2009
Available online 23 July 2009
inflammatory activity through mechanisms involving inhibition of the pro-inflammatory transcription
factor nuclear factor kappa B (NF-
QA derivatives. Inhibition of NF- B was assessed using A549 (Type II alveolar epithelial-like) cells that
stably express a secreted alkaline phosphatase (SEAP) reporter driven by an NF- B response element.
A549-NF- B cells were stimulated with TNF- (10 ng/mL) in the presence or absence of QA derivative
for 18 hours followed by measurement of SEAP activity. Amide substitution at the carboxylic acid posi-
tion yielded potent inhibitors of NF- B. A variety of modifications to the amide substitution were toler-
ated with the N-propyl amide derivative being the most potent. Further examination of the SAR
demonstrated that acetylation of the hydroxyl groups reduced NF- B inhibitory activity. QA amide deriv-
atives lacked anti-oxidant activity and were found to be neither anti-proliferative nor cytotoxic at con-
centrations up to 100 M. In conclusion, we have discovered a novel series of non-toxic QA amides
that potently inhibit NF- B, despite their lack of anti-oxidant activity. Mechanistic studies and pre-clin-
ical efficacy studies in various inflammatory animal models are on-going.
jB). Herein, we describe the synthesis and biological testing of novel
j
j
j
a
Keywords:
NFkB
Quinic acid derivatives
Anti-inflammatory
Synthesis
j
j
l
j
Ó 2009 Elsevier Ltd. All rights reserved.
The woody plant Uncaria is widely dispersed in tropical regions,
including Southeast Asia, Africa, and South America. Uncaria genus
plants have provided numerous structurally diverse medicinal nat-
ural products (e.g., alkaloids, terpenes, quinovic acid glycosides,
flavonoids, and coumarins).1 The species with the most compounds
identified (ꢀ50; 15 of which are reported as novel) is the Peruvian
Uncaria tomentosa commonly known as Uno de Gato or cat’s claw.
Most commercial cat’s claw preparations are based on oxindole
alkaloid content as alkaloids represent the most abundant class
of compounds found in Uncaria.2
Hot water cat’s claw extracts (e.g., C-Med 100) have very low
alkaloid content (<0.5%) and yet retain significant immune enhanc-
ing activity. For example, the extract significantly accelerated
recovery from doxorubicin-induced leukopenia in rats.3 Moreover,
elevated leukocyte numbers were noted in humans, mice, and rats
receiving repeat doses of the extract.4,5 Enhanced leukocyte counts
correlated with prolonged lymphocyte survival, thus providing a
potential mechanistic basis for the immune enhancing properties
of the extract.5 Prolonged cell survival has been linked to enhanced
DNA repair.4,6,7 Recently, quinic acid (QA) esters have been identi-
fied as biologically active components in the extract.8
The mechanism by which QA and its esters exert their anti-
inflammatory activity is unclear, but appears to be related to inhi-
bition of the pro-inflammatory transcription factor nuclear factor
kappa B (NF-
tate (PMA) and ionomycin-induced NF-
cells at concentrations that neither induced cell death nor inhibited
proliferation.9 Moreover, QA prevented degradation of the NF-
inhibitor protein I B- without affecting levels of the phosphory-
lated I B-
protein.9 Base hydrolysis of the extract dramatically re-
jB). For example, QA inhibited phorbol myristate ace-
jB activation in Jurkat T
j
B
j
a
j
a
duces biological activity. For example, the lactone ester of QA (QAL)
inhibits proliferation of mitogen-stimulated mouse lymphocytes,
whereas QA does not affect proliferation.9 Further, base hydrolysis
of the extract dramatically reduces its anti-proliferative effect
against HL-60 and human mononuclear cells.8 Together, these data
suggest the ester derivatives of QA found in the extract comprise a
significant fraction of the biological activity.
QA is utilized by gastrointestinal bacteria as a carbon source
for aromatic acid synthesis. Consequently, only a small fraction
of QA is absorbed after oral administration of either QA or chlor-
ogenic acid (CGA), which is a caffeic acid (CA)-containing ester of
QA.10 Our group has focused on the discovery and development
of stable QA derivatives. Toward this end, we have identified
water soluble amide analogs of QA (1,3,4,5-tetrahydroxy-1-cyclo-
hexanecarboxylic acid) which possess potent anti-inflammatory
activity.
* Corresponding author. Tel.: +1 901 448 6026; fax: +1 901 448 3446.
E-mail address: dmiller@utmem.edu (D.D. Miller).
These authors contributed to the paper equally.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.096

K. Zeng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5458–5460
5459
Synthesis of the QA analogs was carried out using chemistry
previously described.11–13 The synthesis of the lactone 2 was car-
ried out using PTSA in refluxing benzene and DMF according to
the procedure of Neelu Kaila et al. with minor revision.13,14 The lac-
tone was then allowed to react with N-propyl amine with acetic
acid at 85 °C and the resulting amide 3 was purified using flash
chromatography.11,15 The amides 4–11, and 13 were formed by
using different amines with lactone 2 in a manner similar to what
was described for compound 3. QA, 1, was treated with acetic
anhydride /pyridine to give compound 12.13 Each compound was
characterized with Mass Spectroscopy, NMR, and elemental
analysis.
The general synthesis of QA analogs is shown in Scheme 1.
As previously described, human alveolar Type II-like epithelial
cells (A549 cells) stably transfected with the secreted alkaline
phosphatase (SEAP) gene containing the response element for
NF-j
B, were used to screen for anti-inflammatory activity.16
Briefly, cells (6 ꢁ 104 cells/well) were plated overnight followed
by treatment with 10 ng/mL human recombinant TNF-
a (Bio-
Figure 1. NF-
j
B inhibition by QA and analogs using A549 cells stably transfected
with a secreted alkaline phosphatase (SEAP) reporter. NF-
18 hours after addition of TNF- (10 ng/mL) and either QA or synthesized
derivatives (3–13; 1 M). Dexamethasone (Dex; 1 M) was used as a positive
control. Data are presented as percent (%) inhibition relative to control (ctl; TNF-
jB activity was measured
source, Camarillo, CA) and QA analogs (1
measured 18 h later in supernatant samples (50
Great EscAPeTM chemiluminescence kit (Clontech, Mountain View,
CA) and a microplate luminometer (Packard HT microplate read-
l
M). SEAP activity was
a
lL) using the
l
l
a
a
alone) and represent mean % inhibition standard deviation (n = 3). Blank: media
only.
er). Cells were lysed for protein quantification (Pierce BCA Protein
Assay Kit, Microplate Procedure) and SEAP activity was normalized
to the total protein content. Inhibitory potency (IC50) was deter-
mined from dose–response curves (n = 3 separate experiments).
The results of QA derivative anti-inflammatory high throughput
screening are presented in Figure 1. The N-propyl amide derivative
Next, we determined the anti-oxidant potential of QA deriva-
tives using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical
scavenging method.17 The dietary polyphenol CGA, an ester of QA
and CA, possesses potent anti-oxidant activity, which is attributed
to the presence of CA. We thus compared the anti-oxidant poten-
tial of CGA, CA, QA and 3 to determine if the anti-inflammatory
activity of QA and 3 was attributable to anti-oxidant activity.
Ascorbic acid 6-palmitate was included as a structurally-distinct
molecule with described anti-oxidant activity. As expected, Figure
2 demonstrates the anti-oxidant activity of CGA, CA, and ascorbic
acid. However, QA and 3 have no such anti-oxidant activity. The
fact that QA does not possess anti-oxidant activity is consistent
(3) was found to have the greatest extent of NF-jB inhibition fol-
lowing derivative screening. We initially focused on substitution
at the carboxylic acid position, as previous work has shown that
QA ester derivatives have greater biological activity compared to
QA.8,9 The carboxylic acid substituent appears critical to the inter-
action of QA derivatives with E- and P-selectin, key mediators of
leukocyte endothelial cell interactions during inflammation.13
Interestingly, our most potent QA analog 3 demonstrates anti-
inflammatory activity despite the fact that it lacks a carboxylic acid
functional group. Further examination of the SAR demonstrated
that acetylation of the hydroxyl groups, which yields compound
12, leads to reduced NF-jB inhibitory activity. A variety of modifi-
cations to the amide substitution of 3 were tolerated, for example,
9, 10 and 13, a N-isopropyl amide, but no substitution was better
than the N-propyl amide. The NF-jB inhibitory potency (IC50) of
with a previous report.18 Therefore, the NF-
jB inhibitory activity
of QA derivatives is attributed to mechanisms unrelated to anti-
oxidant activity.
QA esters potently inhibit proliferation of mitogen-stimulated
mouse lymphocytes without increasing cytotoxicity.9 We thus
determined the anti-proliferative potential of QA and 3 against
A549 cells using the MTS assay.19 Neither QA nor 3 exhibited cyto-
our most active analog 3 was determined as 2.83 1.76 lM.
toxic activity toward A549 cells at concentrations up to 100 lM.
O
HO
O
OH
O
HO
HO
1
NH
R
a
b
O
OH
HO
OH
HO
HO
OH
OH
OH
1
1
3: R = -C H , 13: R
=
3
7
1
2
1
4: R = -C
H
12 25
1
AcOH
°
5: R
6: R
=
=
-C
H
16 33
(Ac)2O
1
N
H
85
C
1
7: R
8: R
=
O
OH
O
AcO
HO
1
=
N
H
N
OAc
AcO
O
1
OH
9: R
=
HO
OAc
OH
O
2
1
NH
12
10: R
=
11
Scheme 1. Reagents and conditions: (a) PTSA, DMF, C6H6, Dean-Stark, reflux, 78%; (b) R1–NH2, AcOH, oil bath 85 °C.

5460
K. Zeng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5458–5460
10. Adamson, R. H.; Bridges, J. W.; Evans, M. E.; Williams, R. T. Biochem. J. 1970, 116,
437.
11. Boyd, S. A.; Fung, A. K.; Baker, W. R.; Mantei, R. A.; Stein, H. H.; Cohen, J.;
Barlow, J. L.; Klinghofer, V.; Wessale, J. L.; Verburg, K. M., et al J. Med. Chem.
1994, 37, 2991.
12. Girard, C.; Dourlat, J.; Savarin, A.; Surcin, C.; Leue, S.; Escriou, V.; Largeau, C.;
Herscovici, J.; Scherman, D. Bioorg. Med. Chem. Lett. 2005, 15, 3224.
13. Kaila, N.; Somers, W. S.; Thomas, B. E.; Thakker, P.; Janz, K.; DeBernardo, S.;
Tam, S.; Moore, W. J.; Yang, R.; Wrona, W.; Bedard, P. W.; Crommie, D.; Keith, J.
C., Jr.; Tsao, D. H.; Alvarez, J. C.; Ni, H.; Marchese, E.; Patton, J. T.; Magnani, J. L.;
Camphausen, R. T. J. Med. Chem. 2005, 48, 4346.
14. Lactone. 1,3,4-Trihydroxy-6-oxa-bicyclo[3.2.1]octan-7-one (2). In
a 200 mL
round-bottom flask fitted with a stirring bar, reflux condenser, Dean-Stark trap,
and argon inlet, 5.0 g of QA (26.0 mmol) was placed and 10 mL of dry DMF was
added via syringe and the slurry stirred at room temperature. Next, benzene
60 mL and PTSA 0.5 g were added, and the slurry was heated to reflux for 26 h.
TLC was used to confirm reaction completion. A 1:1 mixture of EtOAc and
heptane (100 mL) was added to the cooled reaction mixture. The mixture was
stirred for 1 h at room temperature and filtered. The collected solid was again
stirred with a 1:1 mixture of EtOAc and heptane (100 mL) for 1 h at room
temperature and filtered. Tituration was repeated once more with
a 1:1
mixture of EtOAc and heptane (100 mL) and the precipitate was purified by
flash column chromatography with EtOAc to give 3.5 g of Lactone 2 (78% yield).
15. Amide. 3. In a 50 mL round-bottom flask fitted with a stirring bar, reflux
Figure 2. Reaction kinetics of DPPH for determining antioxidant activity. Caffeic
acid (CA), chlorogenic acid (CGA), ascorbic acid (AA), QA and Compound 3 were
prepared in 50% acetone. The conventional colorimetric DPPHꢂscavenging capacity
was determined by UV absorption measured at 515 nm. CA, CGA and AA showed
strong anti-oxidant activity, while QA and 3 had no anti-oxidant activity.
condenser, 0.2 g lactone
2 (1.1 mmol), and propylamine (0.8 mL, 0.6 g,
10.1 mmol) were combined, then glacial acetic acid (0.2 mL, 0.20 g,
3.4 mmol) was added. The solution was warmed to 85 °C in an oil bath for
30 min, at which time TLC (EtOAc) indicated complete consumption of the
starting lactone. The reaction mixture was purified by flash column with
CHCl3:MeOH:NH4OH (100:10:1). The amide 3 was isolated as a white solid
206.0 mg (mp: 132–133 °C, 80% yield).
Thus, it appears that inhibition of NF-jB by QA and 3 is related not
to anti-proliferative or cytotoxic activity, but rather to a yet to be
16. Dirks, N. L.; Li, S.; Huth, B.; Hochhaus, G.; Yates, C. R.; Meibohm, B. Pharmazie
2008, 63, 893.
17. DPPH. DPPHꢂ and all standard antioxidant compounds were dissolved in 50%
determined mechanism.
In conclusion, we have synthesized novel QA analogs that po-
tently inhibit NF-jB activity in TNF-a-stimulated human alveolar
Type II-like epithelial cells (A549). We have demonstrated that
the QA analogs presented in this work do not exert their activity
via anti-oxidant or cytotoxic mechanisms. Mechanistic studies
and pre-clinical efficacy studies of our lead molecule 3 in various
inflammatory animal models are on-going.
acetone. The DPPHꢂ stock solution at
a concentration of 0.625 mM was
prepared monthly and kept at 4 °C in dark. The 0.208 mM fresh DPPHꢂ working
solution was made daily by further diluting the stock solution in 50% acetone
for each test. Stock solutions of CA, CGA, AA and compound 3 were prepared in
50% acetone at concentrations of 10 mM, respectively, and stored at 4 °C. A
series of working solutions were made by appropriate dilutions of the above
standard phenolic acid stock solutions with 50% acetone.Conventional
colorimetric analysis. The conventional colorimetric DPPHꢂscavenging
capacity assay was performed according to a previously described laboratory
protocol. Briefly, an aliquot of 500
50% acetone was added to 500
l
L of different concentrations of sample in
l
L of 0.208 mM DPPHꢂ solution. The initial
Acknowledgements
concentration was 0.104 mM for DPPHꢂ in all reaction mixtures. Each mixture
was vortexed for a few seconds and test immediately. The absorbance (A) of
each reaction mixture at 515 nm was measured against a blank of 50% acetone
using a UV–isible spectrometer. The level of DPPHÅ remaining for each reaction
time was calculated as:
This research was supported by funds from the Department of
Pharmaceutical Sciences, College of Pharmacy, University of Ten-
nessee Health Science Center, and the Van Vleet Endowed Profes-
sorship (D.D.M.). This technology is patent-pending and is owned
by the University of Tennessee Research Foundation.
%DPPHÅ remaining ¼ ðAsample-t=AcontrolÞ ꢁ 100:
18. Kono, Y.; Shibata, H.; Kodama, Y.; Sawa, Y. Biochem. J. 1995, 312, 947.
19. MTS. Cytotoxicity Studies in Cultured human lung adenocarcinoma A549SN
cells. A549SN cells were suspended in culture medium at
lL were plated into 96-well flat-
bottom plates. Following incubation for 24 h at 37 °C, drugs, vehicles and
a
density of
10 ꢁ 104 cells/mL. Then 1 ꢁ 104 cells in 200
References and notes
controls consisting only of medium and cells were dispensed in 200
lL
volumes in duplicate into the appropriate wells, and incubated for 18 h. QA
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derivatives were tested at concentrations ranging from 0.01 to 100 lM.Cell
viability was assessed by the MTS-CellTiter 96Ò aqueous non-radioactive cell
proliferation assay (Promega, Madison, WI, USA) according to the
manufacturer’s protocol after 2 h of culture. The number of living cells in the
culture is directly proportional to the absorbance at 490 nm by a formazan
product bioreduced from MTS by living cells. Absorbance was measured using
a
DTX 880 multimode detector (Beckman Coulter, Fullerton, CA). The
absorbance of media-only well was subtracted from reading of control or
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