Analogues of boswellic acids as inhibitors of pro-inflammatory cytokines TNF-α and IL-6

Article abstract of DOI:10.1016/j.bmcl.2015.11.035

A library of boswellic acid analogues were synthesized and tested for their anti-inflammatory potential on key inflammatory mediators, TNF-α and IL-6. The study led to the identification of lead compounds showing significant inhibition of the cytokines, TNF-α and IL-6 both in vitro and in vivo.

Full text of DOI:10.1016/j.bmcl.2015.11.035

Accepted Manuscript
Analogues of Boswellic acids as inhibitors of pro-inflammatory cytokines TNF-
α and IL-6
Simmi Sharma, Shilpa Gupta, Vidushi Khajuria, Asha Bhagat, Zabeer Ahmed,
Bhahwal Ali Shah
PII:
S0960-894X(15)30243-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.11.035
BMCL 23291
Reference:
Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
11 July 2015
26 October 2015
12 November 2015
Please cite this article as: Sharma, S., Gupta, S., Khajuria, V., Bhagat, A., Ahmed, Z., Shah, B.A., Analogues of
Boswellic acids as inhibitors of pro-inflammatory cytokines TNF-α and IL-6, Bioorganic & Medicinal Chemistry
Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.11.035
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Analogues of Boswellic acids as inhibitors of pro-inflammatory cytokines TNF-α and IL-6
Simmi Sharma,b,‡ Shilpa Gupta,a,c, ‡ Vidushi Khajuria,a,c Asha Bhagat,c Zabeer Ahmed*c and
Bhahwal Ali Shah*,a,b
b
c
aAcademy of Scientific and Innovative Research, Natural Product Microbes, Inflammation
Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-
Tawi, J&K 180001, India.
*Corresponding authors e-mail address: bashah@iiim.ac.in (BAS)
Keywords. β-Boswellic acid, anti-inflammatory, Cytokine, TNF-α, IL-6.
Abstract: A library of boswellic acid analogues were synthesized and tested for their anti-
inflammatory potential on key inflammatory mediators, TNF-α and IL-6. The study led to the
identification of lead compounds showing significant inhibition of the cytokines, TNF-α and IL-
6 both in vitro and in vivo.
Boswellic acids (BAs) are pentacyclic triterpenoids comprising essentially of β-boswellic
acid (BA, 1) as the main triterpenic acid along with 11-keto-β-boswellic acid (KBA, 2) and
corresponding acetates i.e., acetyl β-boswellic acid (ABA, 3) and acetyl 11-keto-β-boswellic acid
(AKBA, 4),1 derived from Boswellia serrata.2 Gum resin of B. serrata has been the major target
for the anti-inflammatory drug development in recent past3 and its has been widely used in
Ayurvedic preparations since antiquity for the treatment of inflammation4,5and arthritis.6,7,8,9,10,11
Also their activities against other inflammatory conditions like ulcerative colitis,12 chronic
colitis,13 and asthma14are well documented. Besides they have attracted considerable attention
for their activity against cancer and ability to induce apoptosis.15,16 They have been reported to
inhibit growth and induce apoptosis in brain tumors,17 malignant glioma cells,18 colon cancer
cells19 and leukemic cells.20
As pointed out in the press release of Nobel Prize Assembly, Barry J. Marshall and J. Robin
Warren, that many diseases in humans such as Crohn’s disease, ulcerative colitis, rheumatoid
arthritis and atherosclerosis are associated with chronic inflammation21,22 and because of the fact
1

boswellic acids are known anti-inflammatory agent, we envisaged revisiting certain derivatives
of boswellic acids synthesized by us, which in past have shown anti-cancer activity.23,24 Thus, in
our continuous effort to develop bioactive leads based on the natural products25 including BAs,26
we wish to report acyl analogues of BAs including their epimers as well as 4-amino analogues as
inhibitors of pro-inflammatory cytokines through in vitro and in vivo expression of TNF-α, and
IL-6.
Our efforts initiated with the synthesis of 3-acyl derivatives of BAs and their epimers by
following previously reported procedure.23 Treatment of BA with corresponding anhydrides i.e.,
acetic, propanoic, butyric and hexanoic anhydrides in presence of dimethyl amino pyridine
(DMAP) as a catalyst in dry DCM produce 3-O-α-acetyl-β-boswellic acid (3), 3-O-α-propionyl-
β-boswellic acid (5) and 3-O-α-butyryl-β-boswellic acid (7) and 3-O-α-hexanoyl-β-boswellic
acid (9)respectively in almost quantitative yields. Likewise3-O-α-acetyl-11-keto-β-boswellic
acid (4), 3-O-α-propionyl-11-keto-β-boswellic acid (6), 3-O-α-butyryl-11-keto-β-boswellic acid
(8) and 3-O-α- hexanoyl-11-keto-β-boswellic acid (10) were also prepared in similar yields
(95%) (Scheme-1).
Scheme-1. Reagents and conditions: (i) R2O, DMAP, DCM (95%).
For the synthesis of 3-epi BAs, BA/KBA in DMF was treated with K2CO3. After stirring it for 30
minutes, benzyl bromide was added at 0ºC and further kept it on stirring for 12 h to afford
benzyl-3-α-hydroxyurs-12-en-24-oate (11) in 95% yield. The ester was oxidized with PCC in
DCM to convert it to corresponding 3-keto derivatives i.e.,benzyl-3-oxours-12-en-24-oate (12) in
85% yield. The 3-keto derivative was then reduced by NaBH4 in methanol to produce benzyl-3-
β-hydroxyurs-12-en-24-oate (13) having (S) or β-configuration at C-3. The debenzylation of
2

ester was brought about by hydrogenation using Pd/C as catalyst in EtOH: EtOAc (1:1) to afford
3-epi-β-boswellic acid (14) in 90% yields. To obtain 3-epi-11-keto-β-boswellic acid (15) half
portion of 14 was oxidized with CrO3 in 80% yields
Scheme-2. Reagents and conditions: (i) K2CO3, BnBr, DMF (95%) (ii) PCC, DCM, rt (80%) (iii)
NaBH4, MeOH, rt (90%) (iv) H2, Pd/C, EtOH: EtOAc (1:1) (v) R2O, DMAP, DCM (95%) (vi)
CrO3, AcOH.
The 3-O-acyl analogues of 3-epi-β-boswellic acid (14) and 3-epi-11-keto-β-boswellic acid (15)
were similarly prepared using alkyl anhydrides in dry DCM using DMAP as a catalyst to obtain
3-O-β-acetyl-boswellic acid (16), 3-O-β-butyryl-β-boswellic acid (18) and 3-O-β-hexanoyl-β-
boswellic acid (20) respectively. Similarly, 3-O-β-acetyl-11-keto-β-boswellic acid (17), 3-O-β-
butyryl-11-keto-β-boswellic acid (19) and 3-O-hexanoyl-11-β-keto-boswellic acid (21) were
prepared in almost quantitative yields (Scheme-2).
3

As reported earlier,24 to synthesize 4-amino analogues of BAs, ABA (3)/AKBA (4) was
subjected to Curtius reaction involving firstly refluxing of 3/4 with SOCl2 in dry benzene to
prepare corresponding acid chlorides. The excess of SOCl2 was distilled out under reduced
pressure followed by in situ reaction with NaN3 in dry acetone to produce corresponding
isocyanates, 3--acetoxy-4-isocyanato-24-norurs-12-ene (22) and 3--acetoxy-4-isocyanato-11-
oxo-24-norurs-12-ene (23) in 90% yield. Then TFA: H2O (2:1, 10 ml) was added and the
reaction mixture was kept on stirring at room temperature for 3 h. After that, solution was
neutralized and the product (24) and (25) was extracted with DCM and was then purified by
column chromatography (Scheme-3).
Scheme-3. Reagents and conditions: (i) SOCl2, benzene, reflux (ii) NaN3, acetone, reflux (90%)
(iii) TFA: H2O (2:1) stirring, rt (80%).
The release of pro-inflammatory cytokines is an important mechanism by which the immune
cells regulate the inflammatory responses and contribute to various inflammatory and
autoimmune disorders. All the synthesized compounds were analyzed for their ability to decrease
lipopolysaccharide (LPS) induced tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)
production in human peripheral blood mononuclear cells (PBMCs).27 Co-incubation of boswellic
acid analogues with LPS at 5 h showed a considerable inhibition of TNF-α and IL-6 at 10 µM,
when compared to LPS-induced control cells and rolipram standard (Table 1).
Table.1 In vitro TNF-α and IL-6 expression
Compound
Conc. (µM)
% TNF-α inhibition
44.5±3.7
% IL-6 inhibition
48.6±3.0
10
10
1
2
56.3±9.4
59.6±1.2
4

10
43.83±1.9
ND
3
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
55.03±1.47
40±3.36
38.9±0.89
41.4±1.13
34.1±2.5
53.93±1.2
43.5±2.05
36.6±1.6
34.83±1.5
22.5±2.6
50.5±1.9
13.8±1.4
42.3±1.7
49.6±1.7
51.1±0.8
44.7±1.1
39.5±2.2
52.3±1.8
53.43±1.4
64.03±2.8
ND
4
5
6
7
8
9
10
14
15
16
17
18
19
20
21
22
23
72.33±4.36
18.28±2.19
41.37±6.96
53.47±9.06
57.93±2.16
39.6±2.61
68.93±4.47
44.17±2.8
42.53±1.84
44.67±1.47
40.63±2.92
40.57±2.44
68.33±5.16
78.93±1.44
34.6±2.54
69.8±1.5
63.3±1.2
58.4±1.3
26.2±1.73
24
25
Rolipram
Compounds having % inhibition >50 are considered as active compounds.28,29 Results are summarized in table for
both % of cytokine inhibition and expressed as the mean ± SEM.
From the results given in Table 1, it is clear that at 10 µM concentrations, the analogues 4, 9, 15,
16, 18, 24 and 25 displayed maximum inhibitory effects of 55.03, 72.33, 53.47, 57.93, 68.93,
68.33 and 78.93% respectively, wherein the compounds 9, 16 18, 24 and 25 showed TNF-α
inhibition better than parent compounds. Among all these compounds 9 and 25 presented
significant level of TNF-α inhibition of over 70%. Similarly compounds 4, 9, 16, 19, 20, 21, 23,
24 and 25 suppressed extracellular IL-6 expression level to 53.93, 50.5, 51.1, 52.3, 53.43, 64.03,
69.8, 63.3 and 58.4% respectively at the dose of 10 µM, wherein the compounds 21, 23, 24 were
found to be more potent than parent compounds. The compounds showing more than 50%
inhibition of TNF-α and IL-6 expressions are considered to have potent TNF-α and IL-6
inhibitory activity respectively. In structure activity relationship studies, acyl analogues of 11-K-
β -BAs with short carbon chain resulted in enhancement of TNF-α activity while higher acyl
homologues displayed better TNF-α inhibition in case of β-BAs. Also the epimerisation at 3-OH
position resulted in enhancement of TNF-α inhibition in both β-BAs & 11-K-β–BAs. In case of
IL-6 supression higher acyl homologues gave better results than lower acyl homologues. Also
5

the compounds 24 & 25 where the amino group was introduced in place of acid group, resulted
in enhancement of anti-inflammatory potential. Further to confirm that the suppressive effect of
the active compounds from in vitro assay is not because of cytotoxicity, the compounds were
tested for their cell viability against normal breast epithelial cell line fR2 at 10µM concentration
for 48 h. Among these compounds 4, 9, 15, 16, 23, 24 & 25 were found to be non-toxic and
compound 21, 18 & 19 with very low toxicity on normal cell line fR2.
Table2. In vitro cell viability of lead compounds against fR2
Compd
1
2
4
9
15 16 18 19 20 21 23 24 25 control
% viability (50µM) 73 86 100 89 100 99 83 80 97 85 91 100 97 100
These compounds were further chosen for in vivo evaluation. The objective of in vivo
study was to determine if the lead compounds identified from in vitro studies were able to
replicate in animal models. The compounds were evaluated in PBMC cells because this
macrophage model produces high concentrations of IL-6 and TNF-α in culture upon activation
with LPS,30 and reduced the need for additional animals required to obtain and use primary
cultures. Cytokine levels were also evaluated in mice using LPS as a stimulant. Levels of IL-6
and TNF-α were not detectable in untreated cells and animals which served as control in vitro
and in vivo respectively. In vivo results of the compounds listed in table.2.
Table 3. Effect of lead compounds on In vivo expression of TNF-α and IL-6
Compound
Conc. (mg /kg)
% TNF-α inhibition % IL-6 inhibition
10
10
10
50.8±7.6
58.7±5.4
70.7±0.6
53.0±3.9
61.6±9.4
54.3±0.8
1
2
4
9
10
10
10
10
10
10
10
10
10
10
42.5±2.2
71.3±.3.7
ND
58.7±2.2
37.8±7.4
59.3±9.7
30.7±6.3
26.4±4.8
39.5±13.3
53.3±5.9
57.5±1.6
28.9±8.6
19.5
51.6±4.5
48.5±9.9
41.6±4.7
44.1±5.7
40.2±8.7
41.4±8.9
39.1±4.1
15
16
18
19
20
21
23
24
25
6

10
48.1±5.7
36.9±3.2
Rolipram
Compounds having % inhibition >50 are considered as active compounds.31,32 Results are summarized in table for
both % of cytokine inhibition and expressed as the mean ± SEM.
Among eleven analogous of BAs taken for in vivo assay, five compounds 4, 15, 18, 20 and 25
displayed more than 50% TNF-α inhibition that is 70.7, 71.3, 58.7, 59.3 and 53.3% respectively
wherein compounds 4 and 15 showed TNF-α inhibition more than parent compounds. Also the
compounds 4, 9 and 18 suppress IL-6 at level 54.3, 57.5 and 51.6% respectively, more than
standard rolipram. Parent compound 2 found to be most potent inhibitor of IL-6 and its epimer
that is compound 15 was found to be the strongest inhibitor of TNF-α. In vivo results shows
more potency in acid analogues as compared with amino analogues for both cytokines. Our
results revealed that in both in vitro and in vivo experiments TNF-α inhibition is more as
compared to IL-6.
In summary a series of 3-acyl analogues including their epimers and 4-amino analogues of BAs
was synthesized and evaluated for their activity against pro-inflammatory cytokines TNF-α and
IL-6 in LPS stimulated macrophage model. The in vitro studies showed seven analogues viz., 4,
9, 15, 16, 18, 24 and 25 having significant TNF-α inhibition activity, and eight compounds viz.,
4, 9, 16, 19, 20, 21, 23, 24 and 25 showing IL-6 inhibitory activity. Among these, compound 25
is most potent inhibitor of TNF-α & compound 23 is most potent inhibitor of IL-6. The in vivo
studies showed that compound 15 and parent compound 2 as lead inhibitor of TNF-α and IL-6
respectively. Thus, BAs analogues with interesting structural profiles present an important
platform for further development into the products of therapeutic significance.
Supporting Information:
Experimental procedures and compound characterization data.
Author Contributions
‡These authors contributed equally.
Acknowledgment
We thank Department of Science and Technology (OC/07/2013) and CSIR (BSC-0108), India
for financial assistance. S.S. thanks CSIR for fellowship. IIIM communication no.1737.
7

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Graphical Abstract
Boswellic acids analogues were synthesized and evaluated for their anti-inflamattory potential.
One of the lead compounds was found to be an inhibitor of TNF-α and IL-6 in both in vivo and
in vitro experiments