MINISTRY OF EDUCATION
AND TRAINING
VIETNAM ACADEMY
OF SCIENCES AND TECHNOLOGY
GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY
-----------------------------
LE VAN HAI
SYNTHESIS AND EVALUATION OF ANTICANCER ACTIVITY
OF TUBULYSIN DERIVATIVES
Major: Organic Chemistry
Code: 9.44.01.14
SUMMARY OF CHEMISTRY DOCTORAL THESIS
HA NOI, 2020
The thesis was completed at:
Institute of Chemistry
Vietnam Academy of Science and Technology
Supervisor:
1. Asso. Prof. Dr. Tran Van Loc
Institute of Chemistry-Vietnam Academy of Science and Technology
2. Dr. Tran Van Chien
Institute of Chemistry-Vietnam Academy of Science and Technology
Reviewer 1:
Reviewer 2:
Reviewer 3:
The thesis will be presented in front of the doctoral thesis council at:
Graduate University of Science and Technology - Vietnam Academy of
Science and Technology- 18 Hoang Quoc Viet Road, Cau Giay, Hanoi.
At time ........, ……………………
The thesis can be found at:
- Library of Academy of Science and Technology
- National Library of Vietnam
OPENING
1. The essential of the thesis
The natural compounds have considered infinite supplies of
bioactive substances for research and medical application. The
abundance and diversity of the substance-frames had highly bioactive.
Especially, the mechanism of action of each layer of these frames
attracted of researchers so far. For the purpose of research on drug
development, compounds from microbial sources are often achieved by
total synthesis or will be modified in chemical structure to create large
quantities of products as well as new derivatives.
Tubulysins are the tetrapeptide class (figure 1.8) isolated from two
myxobacteria strains Angiococcus disciformis An d48 and Archangium
gephyra Ar 315. Studies have shown that tubulysins are the best class of
anticytokines known to nowaday. The cancer cell inhibitory of
tubulysins are show a wide range of human cancer cell lines such as
ovarian cancer, breast cancer, prostate cancer, colon, lung and blood
cancer.
In vitro and in vivo studies have shown that tubulysins inhibit the
growth of cancer cells higher than anticancer drugs in use such as
vinblastine, epothilone or taxol by approximately 20- to 1000 fold.
Figure 1.8. Chemical formula of the tubulysins
From the point of view of pharmaceutists, natural tubulysins are a
leading class for research and development of new anticancer drugs.
However, their content in bacteria are very low, which were not enough
for intensive research. Thus, the total synthesis of tubulysins and their
derivatives are very necessary and scientific significance.
1
2. Aim of the thesis
Synthesis of several derivatives and tubulysin analogues with the
replacement of the amino acid methylpipecolic (Mep) at the N-terminal,
replacing N,O-acetyl group by methyl group, and researching the role of
tubuphenylalanine (Tup) group at the C-terminal .
Evaluation of cytotoxic activity on a number of cancer cell lines in
order to further clarify the correlation between the activity and the
structure of the tubulysins, and to find new compounds with remarkable
activity.
3. The main research content of the thesis
+ Overview of the myxobacteria. Overview of tubulysin: Biological
activity, active-structure correlation.
+ Overview of synthesis of ɤ-amino acid tubuphenylalanine (Tup) and
tubuvaline (Tuv).
+ Synthesize Tuv
+ Synthesize Tup
+ Synthesize dipeptides.
+ Synthesize tripeptides.
+ Synthesize tetrapeptides (derivatives and analogues of tubulysin)
+ Evaluation of cytotoxic activity of tetrapeptides
Layout of the Thesis
The thesis includes 133 pages: Opening (2 pages), Chapter 1:
Overview (28 pages), Chapter 2: Research Methods and Experimental
(37 pages), Chapter 3: Results and discussions (55 pages), conclusions
(1 page), The list of published related to thesis (1 page).
The reference contains of thesis have 96 documents, which were
updated until the year 2020.
The appendix 81 pages have composed the spectrum of synthetic
substances.
2
CHAPTER 1. OVERVIEW
1.1. Myxobacteria, substances and biological activity
1.2. Microtube in drug research
1.3. Tubulysin: Isolation, determination of structure, biosynthesis
and biological activity.
Figure 1.10. Chemical formulas of natural tubulysins
3
1.4. Synthesis of ɤ-amino acids of tubulysin
1.5. The structure and biological activity correlation of the
tubulysins
CHAPTER 2. RESEARCH METHODOLOGY AND
EXPERIMENTAL
2.1. Research methodology
2.1.1. Organic synthetic methods
2.1.2. Determination of organic compounds structure
2.1.3. Evaluation of cytotoxic activity
2.2. Experimental
2.2.1. Chemicals and solvents
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2.2.2. The general scheme of synthesis of tetrapeptides
2.2.3. Synthesis of tubuphenylalanine
2.2.4. Synthesis of tubuvaline
2.2.5. Remove of Boc protected group
2.2.6. Synthesis of dipeptides
2.2.7. Synthesis of tripeptides
2.2.8. Synthesis of tubulysin derivatives
2.2.9. Synthesis of tubulysin analogues
2.2.10. Evaluate cytotoxic activity of tetrapeptide
5
CHAPTER 3. RESULTS AND DISCUSSIONS
3.1. Orientable synthesis of derivatives and analogue tubulysins
Scheme 3.1. Orientable synthesis of derivatives and analogue
tubulysins
3.2. Synthesis of tubuphenylalanine
Tubuphenylalanine acid (Tup) 47a was synthesized from Lphenylalanine methyl ester hydrochloride (68), through 7-step of
reactions process as described in scheme 3.2. First, the amino group of
68 was protected with tert-butoxycarbonyl group via the reaction with
Boc2O in the THF and NaHCO3 at room temperature for 16 h to obtain
the product 45 (94%). The conversion of ester 45 to aldehyde 52 were
6
effectuated through two steps reaction, by treated with NaBH4 in MeOH
at room temperature during 20h, the N-Boc-phenylalaninol (45a) was
obtained in 96% without purification. Next, conversion of hydroxy 45a
to aldehyde 52 was alternatively performed by refluxing 45a with
oxidative reagent IBX in EtOAc for 5h. Aldehyde 52 was obtained in
quantitative yield.
Scheme 3.2. Synthesis of Tup 47a
The Horner-Wadsworth-Emmons reaction of aldehyde 52 with
triethyl-2-phosphonopropionate and NaH in THF at 0oC to 25oC for 14h
provided α,β-unsaturated ester 42, with E-isomer as a mạjor product in
75% yield. Configuration of E-isomer of ester 42 was determined based
on data analysis of the 1H-NMR and NOESY spectrum, and comparison
with the spectrum of the substance has been published.
Transesterification of 42 to acid 69 by saponification, and
menthyl esterification furnished α,β-unsaturated menthyl ester what Pdcatalyzed hydrogenation afforded diastereomers 47a and 47b in ratio
4:1, which were easily separated by flash chromatography to provide
pure desired major diastereomer of 47a.
7
Analysis of 1H-, 13C-NMR spectra, and combined with published
of spectral comparison for confirmed structure of 47a.
3.3. Synthesis of ɤ-amino acid tubuvaline
3.3.1. Synthesis of 2-Bromo-4-((tert-butyldimethylsilyloxy)
methyl)thiazole (14)
2-bromethiazole 14 was prepared by reaction of thioure with
ethyl bromopyruvate under EtOAc refluxing for 4h (Scheme 3.6).
Conversion of 70 to 71 was proceeded by treatment with NaNO2,
CuSO4 and KBr in the presence of H2SO4 30% at 0 oC to room
temperature for 14h (Sandmeyer reaction), Ester 71 wass obtained with
65 % yield.
Scheme 3.6. Synthesis of 2-bromethiazole 14.
Ester 71 was reduced to hydroxy 72 that was protected with TBS by
treatment with TBSCl in DCM and presence of DMAP as a catalyst in
12h at room tempareture afforded 14 with 97% yield.
3.3.2. Synthesis of N-methyltubuvaline-OMe (79)
The synthesis of tubuvaline (Tuv) fragment was started from
Boc-Val-OH (Scheme 3.14). The homologation reaction of valine
amino acid was carried out by first treatment with ethyl chlorofomate in
TEA at 0oC for 2 h, followed by exposure to diazomethane in ether to
provide diazoketon 21. Under Wolff rearrangement conditions using
8
silver benzoate catalyst and HCl*NH(OMe)Me in triethylamine,
diazoketon 21 was converted to Weinred amide 22a in 84% yield.
Scheme 3.14. Synthesis of 79
The key step was involed in the coupling of compound 73 with
2-bromthiazole 14. Employment of strong base nBuLi in THF at -78 oC
gave the thiazolylketon 74 in 56% yield. Reduction of keton 74 to 15a
was used NaBH4 in MeOH/THF (1/1) or
LiAlH4 gave epimeric
alcohols 15a and 15b in ratio of 1:1. Alternatively, using the Corey,
Bakshi
and
Shibato
oxazaborolidine
((S)-(-)-2-methyl-CBS-
oxazaborolidine) catalyst in presence of BH3*Me2S, pure diastereomer
15a was obtained preferentially (15a/15b ≈ 85/15 in ratio). Acetylation
of 15a and subsequent desilylation using tetra-n-butylammonium
fluoride afforded hydroxy 76. According to reported previous protocols,
9
two-step oxidation of alcohol to carboxylic acid was performed by
treatment with NaOCl and subsequent with NaClO 2/NaH2PO4.
However, this synthetic approach did not give product in high yield, and
the pure product is needed to purify over column chromatography. In
our synthetic route, hydroxy 76 was exposed with IBX under EtOAc
reflux, followed by treatment by oxone in DMF at room temperature.
Acid 78 was obtained quantitative yield. Methylation of 78 was carried
out by treatment with diazomethane in ether at 0 oC for 12h providing
ester 79.
The structures were established by interpretation of their
spectral data, including HRMS, 1D-NMR (1H, 13C, DEPT 135), as well
as by comparison with literature data.
3.4. The removal of Boc protective group of Tuv and Tup
The products 79, 47a, 42 were processed with the TFA in the
presence of triisopropylsilane (TIPS) and water (TFA/TIPS/H2O =
95/2.5/2.5 in volume ratio) at 0 oC for 2h received the trifluoroacetic salt
80, 81, 82 with high performance (scheme 3.15).
Scheme 3.15. Deprotected of 42, 47a, 79
3.5. Synthesis of dipeptide
The reaction of Boc-N deprotected 80 with Boc-Ile-OH using
HATU as coupling reaction afforded dipeptide 84 in 54% yield.
(Scheme 3.17). Various coupling reagents, including DEPBT, PyBop,
10
HBTU and BOP-Cl always resulted in incomplete conversion and lower
yield in compared to using HATU.
Scheme 3.17: Synthesis of dipeptide 84, 85, 86
Hydrolysis of methyl ester 84 with 5% LiOH gave the
intermediate acid 85 in 90% yield. Acetylation of 85 in presence DMAP
as catalyst afforded 86 with high performance.
3.6. Synthesis of tripeptide
3.6.1. Synthesis of tripeptide 88
Dipeptide 88 was prepared from 84 by treatment 84 with
trifluoroacetic for 2h at 0oC, gave the intermediate salt. The reaction of
trifluoroacetic salt with N-methylpipecolic acid (Mep) using HATU as
coupling agent afforded tripeptide 88 in 55% yield. (Scheme 3.18).
Scheme 3.18. Synthesis of tripeptide 88
Structure of 88 was confirmed by analysis of the 1H-, 13C-NMR spectra.
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3.6.2. Synthesis of tripeptide 89
The coupling reaction of acid 86 with Boc-N-deprotected 82
using HATU in DMF and DIPEA for 12h at room tempareture obtained
tripeptide 89 in 60% yield.
Scheme 3.19: Synthesis of tripeptide 89
Analysis of the 1H-NMR spectroscopy of 89 show that singlet
signal of proton thiazole ring at 8.05 ppm (1H, H-9), signals of protons
phenyl ring at 7.29-7.21 ppm (5H). The doublet signal of the proton Eolefin (H-4) at 6.67 ppm (J = 9 Hz). Singlet signal of proton CH3-N at
3.01 ppm (3H). Signal of proton acetyl group (OAc) at 2.16 ppm (3H).
Singlet signals at 1.41 ppm of protons Boc group (9H).
3.6.3. Synthesis of tripeptide 90 and 90b
In a similar synthesis, dipeptides 85 and 86 were coupled with
Boc-N deprotected Tup (81) using HATU providing tripeptide 90b and
90 in 60-65% yield (Sheme 3.20).
Scheme 3.20: Synthesis of tripeptide 90 and 90b
Structure of 90, 90b were confirmed by analysis of the 1H,
spectra.
12
13
C-NMR
3.6.4. Synthesis of tripeptide 91 and 92
Similarly, from the dipeptide 85 and 86 coupling reaction with
phenylalanine methyl ester hydrochloride in the DMF and DIPEA at
room temperature for 14h (Sheme 3.21) afforded tripeptide 91 and 92 in
55-60% yield.
Sheme 3.21. Synthesis of tripeptide 91 and 92
Structures of 91 and 92 were confirmed by analysis of the 1H, 13C-NMR
spectra.
3.7. Synthesis of tubulysin derivatives
The synthesis of tetrepeptide tubulysin derivatives was prepared
from tripeptide 90 and 90b (Scheme 3.22). The deprotective reaction of
90 and 90b by treatment with TEA/TIP/H2O afforded trifluoroacetic
salts. Coupling of trifluoroacetic salts.with N-methylpipecolic acid
(Mep) using HATU as coupling reagent and DIPEA in DMF formed
tetrapeptide 93 and 93a. Safonification of menthyl ester group of 93 and
93a was performed upon treatment with 10% KOH in THF/H2O (2/1) at
45 oC for 48 h providing N-methyltubulysin V (94). Treatment of
hydroxy group of 94 with acetic anhydride in TEA at room temperature
overnight afforded N-methyltubulysin U (95) in 90% yield
The structures were established by interpretation of their
spectral data, including HRMS, 1D-NMR (1H, 13C, DEPT 135), as well
as by comparison with literature data.
13
Mass spectrometer HRMS-ESI of 95 was appeared molecule
ion peak m/z = 728.4051 [M+H]+, (calculated for C38H58N5O7S:
728.4057), this result is compliant with C 38H57N5O7S formula.
Scheme 3.22. Synthesis of tubulysin derivatives 93a,93,94,95
The analysis of the 1H-NMR spectrum of 95 show that the
signal of proton thiazole ring at 8.10 ppm (H-9), the signals of the
proton phenyl ring at 7.26-7.17 ppm (5H). The doubtlet-doublet
signals at 5.73-5,70 ppm features of the proton H-11. The proton's
singlet signal of CH3-N on Tuv at 3.10 ppm (H-17), and signal of
CH3-N on pipecolinic (H-6') at 2.40 ppm. In addition, the singlet
signals characteristic of group CH3-acetyl at 2.15 ppm. The 1H-NMR
spectrum of 95 show that similar with the 1H-NMR spectrum of
previously published in reference [11, 48, 49].
3.8. Synthesis of tubulysin analogues
3.8.1. Synthesis of tubulysin analogues with isoleucine substitution by
leucine
Tetrapeptide 96 was prepared from tripeptide 87 through out
five-step reactions (Scheme 3.24). Fist, tripeptide 87 was removed Boc
protection by TFA, next, coupling reaction of trifluoroacetic salt with
N-methylpipecolic acid using HATU in DMF to furnish intermediate
14
tripeptide. Safonification of tripeptide and coupling reaction with
trifluoroacetic salt 81 formed tetrapeptide 96a. Acetylation 96a gave
tetrapeptide 96 in overal 45% yield.
Scheme 3.24: Synthesis of tetrapeptide 96
The structure of 96 was defined by analysis of their spectral
data, including HRMS, 1D-NMR (1H, 13C, DEPT 135).
3.8.2. Synthesis of tubulysin analogues with with the replacements of
amino acid at the top of N-terminal
From tripeptide 90 through out four-step reactions (Scheme
3.25), afforded tetrapeptide 97, 98. Tripeptide 90 was removed Boc
protected and coupling reaction with 3-methylpicolinic acid and
isoquinoline acid using HATU in DMF gave intermediate tetrapeptides
that were safonified with KOH in THF/H2O, followed by acetylated
reaction with acetic anhydride to provide tetrapeptides 97 and 98 .
15
Scheme 3.25. Synthesis of tetrapeptide 97, 98
Structure of 97 and 98 were confirmed by analysis of HRMS, 1H-, 13CNMR spectra. 1H-NMR spectra of 97 and 98 have similar signal
spectrum of 90. Beside, in the 1H-NMR spectra of 97 and 98 had
appeared signals of proton nitrogen heterocyclic of 3-methylpicolinic
and isoquinoline.
In similar strategy, tetrapeptide 99, 100, 101 were prepared
from tripeptide 90 and 90b by treatment with TFA/TIPs/H 2O and
coupling reaction with 5-methylpyrazin cacrboxylic acid and Nallylpipecolic acid providing 99, 100 and 101 in 55-60% yield. (Sheme
3.26 and 3.27)
Scheme 3.26. Synthesis of tetrapeptide 99
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Scheme 3.27. Synthesis of tetrapeptide 100, 101
The structure of the 99, 100 and 101 were confirmed by
analysis of the HRMS-ESI, 1H-, 13C-NMR spectra.
3.8.3. Synthesis of tubulysin analogues with the replacements of amino
acid at the top of C-terminal.
From tripeptide 89, coupling reaction with N-methylpipecolic
acid afforded tetrapeptide 102 in 60% yield. Saponification of ethyl
ester with 5% LiOH in THF/H2O at room temperature provided acid
103 in 92% yield (Scheme 3.28)
Scheme 3.28: Synthesis of tetrapeptide 102 and 103
In another strategy, owing to the complexity of the synthesis of
Tup amino acid, a replacement of this amino acid by 4-(2aminoethyl)benzen-sulfonamide was carried out. Saponification of 88
with
LiOH
in
THF/H2O
and
17
coupling
reaction
with
4-(2-
aminoethyl)benzen-sulfonamide using HATU in DMF followed by
acetylation with acetic anhydride in TEA afforded 104 in the best yield
(Scheme 3.29).
Scheme 3.29. Synthesis of tetrapeptide 104
The structure of the 102, 103 and 104 were confirmed by
analysis of the HRMS-ESI, 1H-, 13C-NMR spectra.
3.8.4. Synthesis of tubulysin analogues with the replacement of amino
acids at the top of N-and C-terminal
3.8.4.1. Synthesis of tubulysin analogues from tripeptide 89
Coupling reaction of trifluoroacetic salt of 89 with 5methylpyrazin-1-carboxylic and isoquinoline -1-carboxylic acid using
HATU in DMF afforded tetrapeptide 105 and 106 in 55-65% yield
(Scheme 3.30).
Scheme 3.30. Synthesis of tetrapeptide 105, 106
Next, saponification of ethyl ester of 106 with 5% LiOH in
THF/H2O at room temperature for 12h provided acid 107 in quantitative
yield (93%) (Scheme 3.31)
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