Fluorinated betulinic acid derivatives and evaluation of their anti-HIV activity

Article abstract of DOI:10.1016/j.bmcl.2015.11.029

Several fluorinated derivatives of the anti-HIV maturation agent bevirimat (1) were synthesized and evaluated for anti-HIV replication activity. The modified positions were the C-2, C-3, C-28, and C-30 positions, either directly on the betulinic acid (2) skeleton or in the attached side chains. Compound 18, which has a trifluoromethyl group added to C-30 of its isopropenyl group, exhibited similar potency to 1 against HIV-1_NL4-3. In total, our current studies support our prior conclusion that C-30 allylic modification is unlikely to be a pharmacophore for anti-HIV activity, but could be a meaningful route to manipulate other properties of 2-related compounds.

Full text of DOI:10.1016/j.bmcl.2015.11.029

Bioorganic & Medicinal Chemistry Letters 26 (2016) 68–71
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fluorinated betulinic acid derivatives and evaluation of their
anti-HIV activity
Jizhen Li a,b, Masuo Goto , Xiaoming Yang , Susan L. Morris-Natschke , Li Huang , Chin-Ho Chen ,
Kuo-Hsiung Lee
a
b
b
b
c
c
b,d,
⇑
Department of Organic Chemistry, College of Chemistry, Jilin University, 2519 Jiefang Road, Changchun 130023, China
Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, United States
Surgical Science, Department of Surgery, Duke University Medical Center, Durham, NC 27710, United States
b
c
d
Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Several fluorinated derivatives of the anti-HIV maturation agent bevirimat (1) were synthesized and eval-
uated for anti-HIV replication activity. The modified positions were the C-2, C-3, C-28, and C-30 positions,
either directly on the betulinic acid (2) skeleton or in the attached side chains. Compound 18, which has a
trifluoromethyl group added to C-30 of its isopropenyl group, exhibited similar potency to 1 against HIV-
Received 29 September 2015
Revised 6 November 2015
Accepted 9 November 2015
Available online 11 November 2015
1
NL4-3. In total, our current studies support our prior conclusion that C-30 allylic modification is unlikely
to be a pharmacophore for anti-HIV activity, but could be a meaningful route to manipulate other prop-
erties of 2-related compounds.
Keywords:
Fluorinated betulinic acid derivatives
Bevirimat
Ó 2015 Elsevier Ltd. All rights reserved.
Anti-HIV activity
HIV-1 infection affects more than 30 million people worldwide,
and its treatment remains a serious problem due to the emergence
of drug-resistant HIV strains and deleterious side effects. Thus, the
discovery and development of new drug candidates with novel anti
HIV mechanisms remain important to solve the problems of this
disease.
preclinical drug candidate can often be regarded as an important
strategy in drug design.
Fluorinated drugs and drug candidates based on natural prod-
ucts are present in many therapeutic classes. Fluorine has unique
physical properties, such as strong electronegativity, small atomic
size, and low polarizability of the CAF bond, and can mimic either a
2
–7
Bevirimat (DSB, 1 in Fig. 1), a triterpene natural product deriva-
tive, represents a promising class of anti-HIV agents with a novel
mechanism. It inhibits HIV-1 maturation by blocking the cleavage
of p25 to functional p24, resulting in the production of noninfec-
tious HIV-1 particles. In recent years, efforts to improve the
anti-HIV activity of 1 have focused on modifications at the C-3
and C-28 positions, particularly, insertion of side chains containing
various functional groups, such as anhydrides, amino acids, and
hydrogen or hydroxy group.
A recent literature review exten-
, intrinsic
sively discussed fluorine’s effects on conformation, pK
a
potency, membrane permeability, metabolic pathways, and phar-
macokinetic properties, concluding that its incorporation into a
molecule can be significantly important in medicinal chemistry
8
and the design of valuable future drugs. Thus, the introduction
of one or more fluorine atoms into the structure of 1 could dramat-
ically influence the lipophilicity, conformational flexibility, meta-
bolic stability, or other properties, which could be useful in
modulating the biological activity or pharmaceutical profile of 1.
With this consideration in mind, herein, we report the synthesis
of fluorinated derivatives of 1 and evaluation of their anti-HIV
activities.
Readily modifiable positions of the betulinic acid (BA, 2) scaf-
fold include the C-2, C-3, C-28, and C-19 positions. We introduced
fluorine atoms either directly at C-2 or C-3 as shown in Scheme 1
or indirectly in the C-29, or C-3 and C-28 side chains of 2 as shown
in Schemes 2 and 3 (see Supplementary data for the detailed
synthetic procedures). Therefore, we could evaluate the effects of
fluorine atoms in several different positions.
1
N-heterocycles. Correspondingly, success was achieved by the
synthesis of new compounds with better EC50 data than 1 itself.
However, improving the anti-HIV potency is not necessarily the
sole driving force for modification of 1. For instance, structural
changes can help define pharmacophores related to biological pro-
file as well as potency. Also, the possibility of creating a new lead
compound with action at a different HIV process/enzyme is always
significant and meaningful in the field of anti-HIV research.
Consequently, alteration of the activity or biological profile of a
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2015.11.029
960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
0

J. Li et al. / Bioorg. Med. Chem. Lett. 26 (2016) 68–71
69
3
0
with NaBH
anhydride.
4
, and finally, esterification with 2,2-dimethylsuccinic
2
0
2
9
1
9
2
H
H
Thirdly, fluorine atoms were introduced at various positions in
the side chains on the BA C-3 and C-28 positions as shown in
Scheme 3. Compounds 21 and 22, in which the C-28 side chain ter-
minates in a trifluoromethylphenyl and difluorocyclohexyl group,
respectively, were synthesized easily via esterification of 3 with
8
H
CO2H
1
H
CO2H
2
3
O
H
H
HO2C
O
HO
H
H
3
-(trifluoromethyl)phenethyl alcohol or 4,4-difluorocyclohexane
1
(Bevirimat, DSB)
2 (Betulinic acid, BA)
methanol, respectively. Fluorine atoms were incorporated in the
C-3 side chain by esterifying 2 directly with tetrafluorosuccinic
or hexafluoroglutaric anhydride to provide 23 and 24, respectively.
All of the synthesized new fluorinated compounds (8, 9, 11, 13,
Figure 1.
Firstly, as shown in Scheme 1, the CO
3) was protected with CH I to obtain the ester 4. Then, compound
was treated with lithium diisopropylamide (LDA) and N-fluo-
robenzenesulfonimide (NFSI) at À78 °C, resulting in the introduc-
tion of one fluorine atom at the C-2 -position (5) in 43% yield.
Similarly, if the CO H group was esterified with 2-cyclopropyl
ethanol, one fluorine atom was also introduced easily from the
-face at the C-2 position at À78 °C. In addition, when 4 was trea-
ted with CF Si(CH , a CF functional group was inserted at the C-3
position (12). Furthermore, application of classical organic syn-
thetic methods including reduction by NaBH , esterification with
,2-dimethylsuccinic anhydride, or removal of a methyl ester with
2
H group of betulinic acid
1
8, 21–24) were evaluated for anti HIV activity by determining
9
(
4
3
inhibition of NL4-3 virus replication in MT4 lymphocytes. Table 1
shows the anti-HIV results for the tested compounds, with the lead
compound 1 as a positive control.
With one exception, the fluorinated compounds were much less
active than the non-fluorinated lead compound 1. Five compounds
a
2
(8, 9, 13, 23, and 24) did not show anti-HIV potency at the highest
a
concentration tested. Indeed, the inactivity of 9, which differs
structurally from 1 only by a fluorine rather than hydrogen at C-
2
anti HIV activity. The importance of the dimethylsuccinyl C-3 ester
group to the anti-HIV activity was confirmed by the inactivity of
the fluorinated esters 23 and 24. Two compounds with fluorine
in the C-28 ester side chain showed only weak anti-HIV activity
with (4,4-difluorocyclohexyl)methyl (22) resulting in greater
potency than trifluoromethylphenethyl (21). However, compounds
3
3
)
3
3
, might suggest that a proton at C-2 could play a critical role for
4
2
LiI, the desired fluorinated products 8, 9, 11, and 13 were obtained.
Secondly, the fluorinated modification of the isopropenyl group
on C-19 is shown as Scheme 2. Betulin (14) was protected as its
diacetate 15. Then, a CF
5% yield by treating 15 with S-(trifluoromethyl)diarylsulfonium
salt using copper thiophenecarboxylate (CuTC) as catalyst to pro-
duce the fluorinated key intermediate 16. The desired product 18
was obtained in a four-step sequence, deprotection of 16 with
3
group was added at the C-30 position in
2
1
and 18, which has a trifluoromethyl group added to C-30 of the
isopropenyl group of 1, exhibited similar potency against HIV-
NL4-3. Based on the data, this position was the only one among
1
those tested to actually tolerate the addition of a fluorine atom.
This finding was not unexpected, because prior studies had shown
2
N NaOH in THF/EtOH, oxidization with CrO
/H
3 2
SO
4
, reduction
H
H
H
CO2CH3
H
CO2CH3
F3C
O
H
F3C
HO
d
H
O
H
38%
H
HO2C
13
1
2
f
83%
H
O
H
H
H
H
H
CO2CH3
O
H
H
CO2CH3
F
H
CO2H
a
g
H
O
H
H
b
H
H
75%
90%
O
1
0
O
43%
H
O
H
H
5
H
3
4
c
81%
b, c, d 58%
H
H
O
H
CO2H
CO2CH3
H
F
F
F
e
58%
O
H
H
O
H
HO
HO
7
H
6
HO2C
O
H
H
11
d
50%
d
54%
H
H
H
CO2H
H
CO2CH3
F
F
O
O
H
H
HO2C
O
HO2C
O
H
9
H
8
Scheme 1. Reagents and conditions: (a) CH
20 °C; (e) LiI, DMF, 140 °C; (f) CF Si(CH
3
I, K
, Bu
2
CO
3
, DMF, 40 °C; (b) LDA, NFSI, THF, À78 °C–rt; (c) NaBH
4
, EtOH/THF, rt; (d) 2,2-dimethylsuccinic anhydride, DMAP, pyridine,
N,CH Cl , rt.
+
À
1
3
3
)
3
4
N F , THF, rt; then 6 M HCl; (g) (CO)
Cl
2 2
2
, CH Cl
2
, rt; then 2-cyclopropylethanol, Et
3
2
2

7
0
J. Li et al. / Bioorg. Med. Chem. Lett. 26 (2016) 68–71
F3C
H
H
H
H
OH
OAc
i
OAc
j,k
4%
H
H
H
h
H
54%
AcO
H
2
5%
5
HO
AcO
H
H
H
1
4
15
16
F3C
F3C
H
H
H
H
CO2H
H
CO2H
c,d
48%
O
H
HO2C
O
O
H
17
H
18
Scheme 2. Reagents and conditions: (h) Ac
2
O, pyridine, CH
2
Cl
2
, rt; (i) S-(trifluoromethyl)diarylsulfonium salt, CuTc, 2,4,6-collidine, DMA, 40 °C; (j) 2 N NaOH, THF/EtOH, rt;
(
k) CrO
3
/H
2
SO
4
, acetone, 0–5 °C.
H
H
O
c,d
O
H
H
H
46%
O
O
O
H
H
l
O
O
CF3
HO2C
O
CF3
H
H
19
H
21
H
55%
F
H
F
CO2H
H
H
F
F
H
O
m
O
H
O
H
47%
O
O
c,d
49%
3
O
H
H
HO2C
O
H
20
22
H
H
H
H
CO2H
H
CO2H
H
CO2H
F
F
O
F
F
O
o
n
H
H
H
HO2C
O
32%
HO
30%
HO2C
O
H
H
H
F
F F
F
24
2
F
F
23
Scheme 3. Reagents and conditions: (l) (CO)
2
Cl
2 2
, CH Cl
2
, rt; then 3-(trifluoromethyl)phenethyl alcohol, Et
3
2
N, CH Cl
2
, rt; (m) (CO)
2 2 2 2
Cl , CH Cl , rt; then 4,4-difluorocyclohexane
3
methanol, Et N, CH
2
Cl
2
, rt; (n) tetrafluorosuccinic anhydride, CH
2
Cl , 0 °C–rt; (o) hexafluoroglutaric anhydride, CH
2
2
2
Cl , 0 °C–rt.
that C-30 allylic modification is unlikely to be a pharmacophore for
anti-HIV activity, but could be a meaningful route to manipulate
other properties of 2-related compounds.
Table 1
1
2
Anti-HIV data for fluorinated target compounds
Compd
EC50
(
l
M) NL4–3
# of tests
CC50 (lM) MT4
Acknowledgment
8
9
>16
>17
1.1 ± 0.28
>6
0.097 ± 0.025
3.0 ± 0.49
0.31 ± 0.067
>6
4
4
3
3
3
3
3
1
1
4
>16
>17
>6
>6
>6
>5
>6
>6
>6
This work was supported by the National Institute of Allergy
and Infectious Diseases (NIAID) Grant AI33066 (K.H.L.).
1
1
1
2
2
2
2
1
1
3
8
1
2
3
4
Supplementary data
>6
Supplementary data (general methods, synthetic procedures,
biological assay) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2015.11.029.
(Bevirmat, DSB)
0.075 ± 0.0155
>17
Note: For compounds 8 and 9, the highest concentration tested was 17
mL). For the remaining compounds 11, 13, 18, and 21–24, the highest concentration
tested was 6 M (4 g/mL).
lM (10 lg/
l
l
References and notes
1.
See: (a) Sun, I.-C.; Chen, C. H.; Kashiwada, Y.; Wu, J. H.; Wang, H. K.; Lee, K. H. J.
Med. Chem. 2002, 45, 4271; (b) Yu, D.; Sakurai, Y.; Chen, C. H.; Chang, F. R.;
Huang, L.; Kashiwada, Y.; Lee, K. H. J. Med. Chem. 2006, 49, 5462; (c) Dang, Z. D.;
Lai, W. H.; Qian, K.; Lee, K. H.; Chen, C. H.; Huang, L. J. Med. Chem. 2009, 52,
that certain changes in the isopropenyl group (e.g., saturation of
the 20(29) double bond,10 thioether, or oxyether substitution
11
12
at C-30) did not appreciably impact the anti-HIV-1 potency of
7887; (d) Qian, K.; Kuo, R. Y.; Chen, C. H.; Huang, L.; Morris-Natschke, S. L.; Lee,
derivatives of 2. Thus, our studies confirm the previous conclusion
K. H. J. Med. Chem. 2010, 53, 3133; (e) Qian, K.; Bori, I. D.; Chen, C. H.; Huang, L.;

J. Li et al. / Bioorg. Med. Chem. Lett. 26 (2016) 68–71
71
Lee, K. H. J. Med. Chem. 2012, 55, 8128; (f) Dang, Z.; Ho, P.; Zhu, L.; Qian, K.; Lee,
K. H.; Huang, L.; Chen, C. H. J. Med. Chem. 2013, 56, 2029.
Nakano, T.; Makino, M.; Morizawa, Y.; Matsumura, Y. Angew. Chem., Int. Ed.
Engl. 1996, 35, 1019.
O’Hagan, D.; Harper, D. B. J. Fluorine Chem. 1999, 100, 127.
Murphy, C. D.; Schaffrath, C.; O’Hagan, D. Chemosphere 2003, 52, 455.
Swinson, J. Halocarbon Products Corp, Pharmaceuticals, 2005.
Isanbor, C.; O’Hagan, D. J. Fluorine Chem. 2006, 127, 303.
Wang, J.; Sanchez-Rosello, M.; Acena, J. L.; del Pozo, C.; Sorochinsky, A. E.;
Fustero, S.; Soloshonok, V. A.; Liu, H. Chem. Rev. 2014, 114, 2432.
Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.; Meanwell, N. A. J. Med.
Chem. 2015. http://dx.doi.org/10.1021/acs.jmedchem.5b00258.
9. Asada, Y.; Sukemori, A.; Watanabe, T.; Malla, K. J.; Yoshikawa, T.; Li, W.; Kuang,
X.; Koike, K.; Chen, C. H.; Akiyama, T.; Qian, K.; Nakagawa-Goto, K.; Morris-
Natschke, S. L.; Lu, Y.; Lee, K. H. J. Nat. Prod. 2013, 76, 852.
10. Kashiwada, Y.; Hashimoto, F.; Cosentino, L. M.; Chen, C. H.; Lee, K. H. J. Med.
Chem. 1996, 39, 1016.
11. Evers, M.; Poujade, C.; Soler, F.; Ribeill, Y.; James, C.; Lelievre, Y.; Gueguen, J. C.;
Reisdorf, D.; Morize, J.; Pauwels, R.; De Clercq, E.; Henin, Y.; Bousseau, A.;
Mayaux, J. F.; Le Pecq, J. B.; Derett, N. J. Med. Chem. 1996, 39, 1056.
12. Qian, K.; Yu, D.; Chen, C. H.; Huang, L.; Morris-Natschke, S. L.; Nitz, T. J.;
Salzwdedl, K.; Reddick, M.; Allaway, G. P.; Lee, K. H. J. Med. Chem. 2009, 52,
3248.
2
.
3.
4.
5.
6.
7.
8
.