Dipyridodiazepinone derivatives; synthesis and anti HIV-1 activity

Article abstract of DOI:10.3762/bjoc.5.36

Ten dipyridodiazepinone derivatives were synthesized and evaluated for their anti HIV-1 reverse transcriptase activity against wildtype and mutant type enzymes, K103N and Y181C. Two of them were found to be promising inhibitors for HIV-1 RT.

Full text of DOI:10.3762/bjoc.5.36

Dipyridodiazepinone derivatives; synthesis and anti
HIV-1 activity
Nisachon Khunnawutmanotham1, Nitirat Chimnoi1, Arunee Thitithanyanont2,
Patchreenart Saparpakorn3, Kiattawee Choowongkomon4,
Pornpan Pungpo5, Supa Hannongbua3 and Supanna Techasakul*,1,3
Full Research Paper
Open Access
Address:
Beilstein Journal of Organic Chemistry 2009, 5, No. 36.
1Chulabhorn Research Institute, Vibhavadee-Rangsit Highway,
Bangkok 10210, Thailand, 2Department of Microbiology, Faculty of
Science, Mahidol University, Bangkok 10400, Thailand, 3Department
of Chemistry, Faculty of Science, Kasetsart University, Bangkok
10903, Thailand, 4Department of Biochemistry, Faculty of Science,
Kasetsart University, Bangkok 10903, Thailand and 5Department of
Chemistry, Faculty of Science, Ubonratchathani University,
Ubonratchathani 34190, Thailand
doi:10.3762/bjoc.5.36
Received: 09 April 2009
Accepted: 03 July 2009
Published: 22 July 2009
Editor-in-Chief: J. Clayden
© 2009 Khunnawutmanotham et al; licensee Beilstein-Institut.
License and terms: see end of document.
Email:
Supanna Techasakul* - supanna@cri.or.th
* Corresponding author
Keywords:
AIDS; anti HIV-1 RT; dipyridodiazepinone; nevirapine; synthesis
Abstract
Ten dipyridodiazepinone derivatives were synthesized and evaluated for their anti HIV-1 reverse transcriptase activity against wild-
type and mutant type enzymes, K103N and Y181C. Two of them were found to be promising inhibitors for HIV-1 RT.
Introduction
Dipyridodiazepinone nevirapine (1) [1] (Figure 1) is a potent 8-arylthiomethyl moiety, when compared with 9 [4] (Figure 2)
non-nucleoside inhibitor of human immunodeficiency virus as reference, are effective inhibitors of this mutant enzyme.
type 1 reverse transcriptase (HIV-1 RT) and is approved as a Some dipyridodiazepinone derivatives containing an N-methyl-
therapeutic agent for the treatment of AIDS. In the clinic,
nevirapine monotherapy results in relatively rapid drug resist-
ance due to mutation of the RT enzyme. To develop a second-
generation inhibitor with improved activity against the mutant
RT enzyme, many efforts have been focused on the synthesis of
dipyridodiazepinone derivatives [2-8]. On the basis of
molecular modeling analysis on the wild-type (WT) and Y181C
HIV-1 RT, it was found that the dipyridodiazepinone derivat-
Figure 1: Structure of nevirapine (1).
ives containing an unsubstituted lactam nitrogen and a 2-chloro-
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Beilstein Journal of Organic Chemistry 2009, 5, No. 36.
(Scheme 1) [9]. However, by using the same procedure to
prepare aminopyridinecarboxamide 15b (R = CH3), only poor
yields of 15b were obtained. Therefore, 15b (R = CH3) was
prepared by formation of pyridinecarboxamide 17, obtained
from 12 and 16 [10,11], followed by the displacement of 2′
chloro by the ethylamino group.
Afterwards, the aminopyridinecarboxamide intermediates 15
were treated as previously reported [4] to give 8-arylthiomethyl-
dipyridodiazepinones, as shown in Scheme 2.
Aminopyridinecarboxamides 15 were regioselectively bromin-
ated to produce bromo compounds 18. The diazepinone ring
was formed by treatment with sodium hexamethyldisilazane in
pyridine to yield tricyclic compounds 19. Coupling of 19 with
vinyltributyltin in the presence of tetrakis(triphenylphosphine)
Figure 2: Structures of dipyridodiazepinone derivatives with promising
anti-HIV activity.
ated of lactam also exhibited good potency against the WT palladium(0) provided 8-vinyl compounds 20 which underwent
enzyme. The 8-amino derivative of nevirapine and its hydro- ozonolysis to produce aldehydes 21 in good yields. The reduc-
chloride salt also provided interesting potency. The first two tion of 21 with NaBH4 produced alcohols 22, which were
compounds, 2 and 3, were synthesized and their virustatic and converted to the corresponding chlorides 23 through treatment
virucidal activities against HIV-1 subtype E were reported with thionyl chloride in dichloromethane. The reaction of 23a
previously [9]. As part of our continuing efforts directed with thiophenolate, 3-methoxythiophenolate, and 3-fluoro-
towards the development of potential HIV-1 RT inhibitors, we thiophenolate in N,N-dimethylformamide yielded 2, 3, and 4,
have extended the synthesis of dipyridodiazepinone derivatives respectively, whilst the reaction of 23b with thiophenolate and
(Figure 2) and evaluation of their activity against wild-type RT 3-methoxythiophenolate yielded 5 and 6, respectively. Methyla-
and K103N and Y181C mutant RT enzymes.
tion of the lactam nitrogen of 5 and 6 with methyl iodide
provided 7 and 8. Compound 9 was also prepared via methyla-
tion of 2, which was used as the reference compound.
Results and Discussion
Synthesis of compounds 2–9
Compounds 2–9 were synthesized via efficient routes as shown Synthesis of compounds 10 and 11
in Scheme 1 and Scheme 2. The aminopyridinecarboxamide Compounds 10 and 11 were synthesized as shown in Scheme 3.
15a (R = H) was prepared from 2-(ethylamino)-3-pyridine- The starting 3-amino-2-cyclopropylamino-4-methylpyridine
carboxylic acid (13) and 3-amino-2,6-dichloropyridine (14) (27) was prepared from commercially available 2-hydroxy-4-
methyl-3-nitropyridine (24) through a sequence involving treat-
ment with POCl3, followed by chloro displacement from the
resulting 2-chloro compound with the aminocyclopropyl group,
and finally reducing the nitro to the amino group. 2-Chloro-5-
nitronicotinic acid (30) was prepared by nitration of commer-
cially available 2-hydroxynicotinic acid (28) followed by treat-
ment with POCl3. Then amine 27 and the acid 30 underwent
coupling to produce carboxamide 31. Diazepinone ring closure
was performed by heating 31 in hexamethyldisilazane. After-
wards, the nitro group was reduced to produce the hydro-
chloride salt 10. Treatment of 10 with 50% aqueous NaOH
yielded its corresponding free amino compound 11.
Biological testing against HIV-1 reverse tran-
scriptase
Scheme 1: Reagents and conditions: (a) EtNH2, 120 °C, 4 h, 99% (b)
The results from the biological testing of all compounds
i) (COCl)2, benzene, DMF, rt, 1 h; ii) amine 14 or 16, dioxane, cyclo-
hexane, pyridine, rt, 16 h, 80% (c) EtNH2, dioxane, 100 °C, 20 min,
47%.
synthesized, compared with nevirapine (1) and 9, against the
wild-type RT together with K103N and Y181C mutant RT are
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Beilstein Journal of Organic Chemistry 2009, 5, No. 36.
Scheme 2: Reagents and conditions: (a) Br2, HOAc, KOAc, rt, 1 h; (b) NaHMDS, pyridine, 90 °C, 1 h; (c) CH2=CH–SnBu3, Pd(PPh3)4, DMF, 90 °C, 1
h; (d) O3, CH2Cl2/MeOH, −78 °C then PPh3, rt, 1 h; (e) NaBH4, THF, H2O, rt, 0.5 h; (f) SOCl2, CH2Cl2, Et3N, rt; (g) NaH, ArSH, DMF, rt, 1 h; (h) NaH,
DMF, 50 °C, 0.5 h then MeI, rt, 0.5 h.
Scheme 3: Reagents and conditions: (a) POCl3, 150 °C, 6 h, 85%; (b) cyclopropylamine, xylene, 105 °C, 4 h, 99%; (c) SnCl2·2H2O, conc. HCl,
CH3COOH, rt, 3 h, 83%; (d) 69% HNO3, conc. H2SO4, 50 °C, 7 h, 79%; (e) POCl3, reflux, 4 h, 78%; (f) i) 30, (COCl)2, benzene, DMF, rt, 1 h; ii) 27,
THF, DIPEA, rt, 5 h, 53%; (g) HMDS, 110 °C, 24 h, 90%; (h) SnCl2·2H2O, conc. HCl, CH3COOH, rt, 3h, 73%; (i) 50% aq. NaOH, rt, 1 h, 90%.
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Beilstein Journal of Organic Chemistry 2009, 5, No. 36.
shown in Table 1. It was found that compounds 2–8 exhibited
higher inhibitory activity against WT-RT and both mutant RTs
compared to nevirapine. Interestingly, 5 and 6 were found to be
about four times more potent against WT-RT than 9, and they
provided comparable activity against K103N mutant RT. Incor-
poration of a methyl group at the R1 position and the free N of
the amide seems to be responsible for this higher activity.
However, 9 showed better potency against the Y181C mutant
RT compared to the other two compounds. Compound 5,
without methoxy substituent, was found to be slightly more
potent than 6 except for Y181C mutant RT. Additional
N-methyl groups in 7 and 8 led to diminished activity relative to
that of 5 and 6. 10 and 11, 8-amino analogues of nevirapine,
were found to be ineffective inhibitors.
Table 1: Inhibitory activity of the synthesized compounds against HIV-
1 reverse transcriptase.
Compounds
IC50a (μM)
K103N
WT
Y181C
nevirapine (1) 1.070 ± 0.60b 27.10 ± 5.20 228.5 ± 24.84
2
3
4
5
0.427 ± 0.31 6.23 ± 2.48
1.50 ± 0.34
0.757 ± 0.15 19.40 ± 2.80 2.90 ± 0.19
0.183 ± 0.10 13.90 ± 1.23 0.459 ± 0.29
0.0186 ±
0.002
0.224 ± 0.14 0.269 ± 0.08
6
7
8
9
0.0229 ± 0.01 0.428 ± 0.39 0.0593 ± 0.07
0.124 ± 0.03 4.37 ± 0.66
0.0828 ± 0.03 4.59 ± 2.23
0.507 ± 0.36
0.118 ± 0.06
0.0858 ±
0.00001
0.39 ± 0.23
0.00463 ±
0.0009
10
11
17.40 ± 2.11 62.10 ± 5.14 126.0 ± 28.03
6.05 ± 1.60 97.0 ± 16.8 61.0 ± 6.9
aIC50 is the concentration of inhibitor required for 50% inhibition of
reverse transcriptase. bStandard errors obtained from duplicate experi-
ments.
Molecular docking
To understand the binding mode of the new potent derivatives
5, 6 and 9 were docked into the HIV-1 RT binding site by using
the default parameters of the GOLD v3.2 program. The wild
type HIV-1 RT structure (pdb code 1klm) was taken from the
protein data bank. Additionally, two HIV-1 RT mutants, K103N
and Y181C, were used and analyzed by mutating positions 103
and 181 of the wild-type structure through the use of the Sybyl
7.2 program. The docked conformations of 5, 6, 9 and
nevirapine are shown in Figure 3, and their GoldScores are
presented in Table 2. In the wild-type and K103N binding sites,
the docked orientations of 5, 6 and 9 are similar to that of
nevirapine. In the Y181C binding site, except for 5, the orienta-
tions of the others were similar to nevirapine orientation. The
Figure 3: Docked orientations of nevirapine (green), 9 (yellow), 5, and
6 (atom type color – carbon: grey, chloride: green, hydrogen: white,
nitrogen: blue, oxygen: red and sulfur: yellow) in WT (a), K103N (b),
and Y181C (c) binding pockets.
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Beilstein Journal of Organic Chemistry 2009, 5, No. 36.
Tyr188 in the Y181C binding pocket were formed as compared
with the WT binding pocket. For 6, H-bond interaction between
a hydrogen atom of the methoxy group of 6 and an oxygen atom
of backbone Lys104 revealed a longer bond length (3.58 Å) in
the Y181C binding pocket as compared to the WT binding
pocket (2.14 Å).
Table 2: GoldScores of nevirapine, 5, 6 and 9 in wild-type (WT),
K103N and Y181C HIV-1 RT.
Compounds
GoldScores
K103N
WT
Y181C
nevirapine 58.13 (±
0.27)a
58.66 (± 0.39) 56.92 (± 0.04)
Conclusion
5
6
9
72.07 (± 1.60) 79.23 (± 1.42) 66.44 (± 0.63)
79.19 (± 0.83) 83.22 (± 0.85) 71.92 (± 1.69)
73.66 (± 2.36) 76.27 (± 1.00) 68.84 (± 1.09)
The remarkable anti HIV-1 activity of dipyridodiazepinone
derivatives, particularly compounds 5 and 6, was presented in
this study. A preliminary SAR study showed that the methyl
group at the R1 position and the free N of amide are crucial for
potent activity. This is possibly because of the strong interac-
tion with the amino acid residue in the RT enzyme. The
aIn parenthesis is the standard error of the GoldScore from the triplicate
of docking calculations.
GoldScores of 5, 6 and 9 were higher than those of nevirapine secondary test of these two compounds was regarded to be valu-
by 17.61–24.56, 9.52–15.00 and 15.33–21.06 in the WT, able for future investigation.
K103N, and Y181C binding pockets, respectively. In the wild-
type binding pocket, the H-bond interaction with the backbone
Supporting Information
oxygen atom of Lys103 was found to contain 5, 6 and 9 but no
nevirapine was present. Compounds 5, 6 and 9 also formed an
Supporting information provides details about the chemical
H-bond interaction with the backbone nitrogen atom of Val106.
methods, analytical data and biological testing.
Since there is a methyl group at the R1 position of 5 and 6, their
Supporting Information File 1
Experimental part.
docked conformations were slightly shifted below the binding
pocket as compared to the docked conformation of 9. This shift
caused the formation of a stronger H-bond interaction of 5 and
6 with Lys101, Val179, Tyr188 and Val189 compared to 9. The
methyl group at R1 position of 5 and 6 also formed stronger
H–π interaction with Trp229. Moreover, the methoxy
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-5-36-S1.doc]
substituent of 6 revealed a strong attractive interaction with Acknowledgments
Lys104, but the movement of ring C in 6 caused a steric interac- We would like to thank the Thailand Research Fund
tion with the side chain of Lys102.
(DBG4780009 for S.T., RTA5080005 for S.H., DBG5180022
for P.P., and MRG5080267 for P.S.), National Synchrotron
In the case of the docked conformations of 5, 6 and 9 in the Research Center for K.C., and the Ministry of Education for
K103N binding pocket, the H-bond interactions with the back- their financial support. We also gratefully acknowledge the help
bone atom of Asn103 were still detected, but their H-bond inter- of Dr. Carrie Dykes of the University of Rochester, New York
actions with Val106 were lost. Furthermore, the docked for the mutant clones, K103N and Y181C; and Dr. Hiroaki
conformation of 5 showed stronger H-bond interaction with Mitsuya of the HIV and AIDS Malignancy Branch, USA for the
Lys101 compared to 6 and 9. The adjustment of the ethyl group WT clone.
also formed an important H-bond interaction with the oxygen
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