Alternate synthesis of HSP90 inhibitor AT13387

Article abstract of DOI:10.1080/00397911.2014.902068

AT13387 is a novel HSP90 inhibitor, and it is currently used in clinical trials for the treatment of refractory gastrointestinal stromal tumors (GIST). In this article, two synthetic routes are described for synthesizing the title compound via [2 + 2 + 2]

Full text of DOI:10.1080/00397911.2014.902068

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Alternate Synthesis of HSP90 Inhibitor
AT13387
Cuirong Liang a , Lingling Gu a , Yang Yang b & Xin Chen a
a School of Pharmaceutical and Life Sciences, Changzhou University ,
Changzhou , Jiangsu , China
b School of Petrochemical Engineering, Changzhou University ,
Changzhou , Jiangsu , China
Accepted author version posted online: 24 Apr 2014.Published
online: 01 Jul 2014.
To cite this article: Cuirong Liang , Lingling Gu , Yang Yang & Xin Chen (2014) Alternate
Synthesis of HSP90 Inhibitor AT13387, Synthetic Communications: An International
Journal for Rapid Communication of Synthetic Organic Chemistry, 44:16, 2416-2425, DOI:
10.1080/00397911.2014.902068
To link to this article: http://dx.doi.org/10.1080/00397911.2014.902068
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Synthetic Communications1, 44: 2416–2425, 2014
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2014.902068
ALTERNATE SYNTHESIS OF HSP90 INHIBITOR
AT13387
Cuirong Liang,1 Lingling Gu,1 Yang Yang,2 and Xin Chen1
1School of Pharmaceutical and Life Sciences, Changzhou University,
Changzhou, Jiangsu, China
2School of Petrochemical Engineering, Changzhou University,
Changzhou, Jiangsu, China
GRAPHICAL ABSTRACT
Abstract AT13387 is a novel HSP90 inhibitor, and it is currently used in clinical trials for
the treatment of refractory gastrointestinal stromal tumors (GIST). In this article, two
synthetic routes are described for synthesizing the title compound via [2 þ 2 þ 2] cycloaddi-
tion of dipropargylamine derivatives and propargyl alcohol as a key step. The alternate
synthetic methods are economical and suitable for multigram-scale preparation of
AT13387 and the related isoindoline derivatives.
Keywords AT13387; cycloaddition; HSP90; inhibitors; synthesis
INTRODUCTION
Heat shock protein 90 (HSP90) is one of the most common heat-related
proteins, and it is important in signal transduction pathways associated with steroid
hormone receptors, tyrosine kinases, and serine=threonine kinases. It also performs
autoregulation for the heat shock response in cell cycle and proliferation.[1] Numer-
ous studies have indicated that HSP90 plays a key role in the regulation of tumor cell
growth and survival and is an important part in many oncogenic pathways.[2] During
the past decade, HSP90 has received considerable research interest as a drug target,
and HSP90 inhibitors have been investigated as chemotherapeutic agents. HSP90
inhibitors can disrupt the normal protein-stabilizing function of HSP90 and induce
apoptosis of tumor cells including hormone-dependent and nondependent prostate
cancer cells.[3] Currently known HSP90 inhibitors include antibiotics of the
ansamycin family such as geldanamycin (GA)[4] and its derivatives 17-AAG[5] and
Received January 9, 2014.
Address correspondence to Xin Chen, School of Pharmaceutical and Life Sciences, Changzhou
University, Changzhou, Jiangsu 213164, China. E-mail: xinchen@cczu.edu.cn
2416

SYNTHESIS OF AT13387
2417
17-DMAG,[6] the antifungal agent radicicol,[7] the antibiotic novobiocin,[8] and a
class of purine-based structures, such as PU3.[9] An additional novel HSP90 inhibitor
AT13387 was discovered by Astex Pharmaceuticals through fragment-based drug
design strategy.[10,11] The drug is currently in clinical trials for the treatment of
refractory gastrointestinal stromal tumors (GIST).
As a part of our efforts to investigate structure–activity relationships (SAR) for
HSP90 inhibitors, we needed to synthesize multigram quantities of AT13387. From
the retrosynthetic analysis, AT13387 can be obtained via coupling of resorcylic acid
A and the piperazino-isoindoline C (Scheme 1). Resorcylic acid A is readily obtained
from 2,4-dihydroxybenzoic acid,[12] whereas the availability of the piperazino-
isoindoline C, the key to the whole synthesis, is limited; it must be prepared via
multistep synthetic protocol. Previously, Astex Pharmaceuticals had reported the syn-
thesis of AT13387.[12,13] They synthesized C through cycloaddition of 1-propargyl-4-
methylpiperazine D and N-Boc-dipropargylamine E. However, when following the
reported protocol, we obtained a very poor yield (less than 20%) for the desired iso-
indoline C, which was contaminated with the unreacted D. Because C and D have
almost the same Rf values by the thin-layer chromatography (TLC) analysis, it
was extremely difficult to separate the product from the unreacted material by
standard chromatographic methods.
Patel and Barrett recently reported a new method of synthesizing the
piperazino-isoindoline C.[14] First, sodium borohydride and boron trifluoride were
used to reduce 4-bromo-phthalimide H, and then the amino functionality in the
resulting 5-bromo-isoindoline was Boc-protected to give N-Boc-5-bromo-isoindoline
G (Scheme 1). In the following step, potassium 4-methyl-piperazino-methyl-
trifluoroborate F, which was obtained from potassium bromomethyl trifluoroborate
and N-methylpiperazine, was subjected to Suzuki–Miyaura cross coupling with
isoindoline G to afford the N-Boc piperazino-isoindoline in 56% overall yield. The
coupling reaction was catalyzed by palladium(II) acetate in the presence of the
ligand, dicyclohexyl-2-(2,4,6-triisopropylphenyl)phenylphosphine (XPhos). Despite
a reasonable overall yield, the lack of starting material availability and prohibitive
Scheme 1. AT13387 and its retrosynthetic analysis.

2418
C. LIANG ET AL.
cost of the ligand XPhos make the reported procedure unsuitable for large-scale
preparation of AT13387.
Our objective was to develop a straightforward preparative-scale method for
synthesizing AT13387 with high chemical purity in sufficient quantities to permit
laboratory bioassays and animal studies. Here we report convenient synthesis of
the title molecule from commercially available and relatively inexpensive materials.
RESULTS AND DISCUSSION
By modifying the known procedure,[15] N-Boc-dipropargylamine 3 was
obtained from propargylamine in two steps with 52% overall yield (Scheme 2). In
the presence of (PPh3)3RhCl (the Wilkinson’s catalyst), 3 and propargyl alcohol were
cyclized in EtOH to afford isoindoline alcohol 4, but the yield was only 44%.[16]
Therefore, the reaction conditions (solvents, catalysts, and temperature) needed to
be optimized, and the results are summarized in Table 1. At room temperature
and using EtOH as solvent, either CpꢀRuCl(PPh3)2 or CpRuCl(PPh3)2 failed to
catalyze the cycloaddition (Table 1, entries 2 and 3). When the reaction was run
at elevated temperature (70 ꢁC), CpꢀRuCl (PPh3)2 gave moderate yield of 4 (53%;
entry 4), but CpRuCl(PPh3)2 showed no activity (entry 5). It seems that solvent effect
has obvious impact on the cycloaddition. For example, when tetrahydrofuran (THF)
was used as the solvent, (PPh3)3RhCl catalyzed the cycloaddition much faster (4 h vs
24 h in EtOH) and afforded 4 in 70% yield (entry 7); CpꢀRuCl (PPh3)2 also worked
well in THF and at room temperature to give 4 in 58% yield (entry 6). The
Wilkinson’s catalyst is effective in dimethylformamide (DMF; 58% yield; entry 10)
and in dimethoxyethane (DME; 60% yield; entry 12). The reaction temperatures also
had some effects on the cycloaddition. For example, in the presence of the
Wilkinson’s catalyst and using THF as the solvent, the cyclization did not occur
at 0 ꢁC (entry 8). At the elevated temperature (70 ꢁC), 61% of 4 was obtained (entry
9). Based on the results described, the optimized conditions for the [2 þ 2 þ 2]
cycloaddition of N-Boc-dipropargylamine 3 and propargyl alcohol are (PPh3)3RhCl
as the catalyst, THF as the solvent, and room temperature. If CpꢀRuCl (PPh3)2 is
chosen as the catalyst, the cyclization should be performed in THF or DMF and
at ambient temperature.
With the optimized conditions for synthesizing isoindoline alcohol 4 in hand,
we turned our attention to the remaining steps in the synthetic sequence. Horiuchi
et al.[16] oxidized 4 with MnO2 in CCl4, and obtained isoindoline aldehyde 5 in
Scheme 2. Synthesis of the piperazino-isoindoline 7. Reagents and conditions: (a) (Boc)2O, THF, rt, 4 h,
90%; (b) propargyl bromide, NaH, THF, rt, 8 h, 76%; (c) propargyl alcohol, (PPh3)3RhCl, THF, rt, 4 h,
70%; (d) MnO2, DCM, rt, 4 h, 84%; (e) 1-methylpiperazine, NaBH(OAc)3, AcOH, DCM, rt, 3 h, 90%; and
(f) HCl, DCM, rt, 4 h, 100%.

SYNTHESIS OF AT13387
2419
Table 1. Optimization of [2 þ 2 þ 2] cycloaddition of N-Boc-dipropargylamine 3 with propargyl alcohol
Entry
Solvent
Catalysta
Temperature (time)
Isolated yield (%)
1
2
EtOH
EtOH
EtOH
EtOH
EtOH
THF
(PPh3)3RhCl
20 ꢁC (24 h)
20 ꢁC (24 h)
20 ꢁC (24 h)
70 ꢁC (24 h)
70 ꢁC (24 h)
20 ꢁC (9 h)
20 ꢁC (4 h)
0 ꢁC (12 h)
70 ꢁC (2 h)
20 ꢁC (4 h)
20 ꢁC (8 h)
20 ꢁC (4 h)
20 ꢁC (24 h)
44
NRb
NR
53
CpꢀRuCl(PPh3)2
CpRuCl(PPh3)2
CpꢀRuCl(PPh3)2
CpRuCl(PPh3)2
CpꢀRuCl(PPh3)2
(PPh3)3RhCl
3
4
5
Trace
58
70
6
7
THF
THF
THF
8
(PPh3)3RhCl
(PPh3)3RhCl
(PPh3)3RhCl
CpꢀRuCl(PPh3)2
(PPh3)3RhCl
CpꢀRuCl(PPh3)2
NR
61
9
10
11
12
13
DMF
DMF
DME
DME
58
57
60
<30
aCatalyst: CpꢀRuCl(PPh3)2 ¼ pentamethylcyclopenta-dienylbis-(triphenylphosphine)ruthenium(II) chloride;
CpRuCl(PPh3)2 ¼ cyclopentadienylbis-(triphenylphosphine)ruthenium(II) chloride; (PPh3)3RhCl ¼ tris
(triphenylphosphine)rhodium(I) chloride.
bNR, no reaction.
66%. When we tried the same reaction but used DCM instead of CCl4 as the solvent,
the yield was increased to 84%. In the following step, reductive amination of alde-
hyde 5 with methyl piperazine and sodium triacetoxyborohydride[17] gave N-Boc
piperazino-isoindoline 6 in 90% yield. Upon treatment with HCl gas, the Boc group
of 6 was removed, and the piperazino-isoindoline HCl salt 7 was obtained in quan-
titative yield (Scheme 2).
It is interesting to note that the 13C NMR spectra of the N-Boc isoindoline
derivatives (4, 5, and 6) clearly show a pair of peaks for almost every carbon atom.
For example, in the 13C NMR spectra of isoindoline alcohol 4 (in CDCl3, 125 MHz
NMR), every carbon atom (except for the methyl and carboamide groups) has two
peaks with almost 1:1 ratio (the partial spectrum for C3, C4, C6, C8, and C9 is shown
in Fig. 1(a); the full spectra are in the Supplementary Information). Most likely, this
is because the restricted rotation around the C-N amide bond generates two confor-
mational isomers (called conformers or rotamers),[18] such as cis-4 and trans-4
(Fig. 1). Some similar restricted amide rotations have been reported in the literature.
For example, Quintanilla-Licea et al. employed one-dimensional nuclear Overhauser
effect (1D-NOE) to investigate the restricted C-N bond rotation of N-formyl-o-
toluidine and found the cis-isomer predominates in the equilibrium mixture.[19]
The restricted amide rotation of N,N-diethyl-m-toluamide (m-DEET) and N,N-
diethyl-o-toluamide (o-DEET) was recently studied by using dynamic NMR,
two-dimensional exchange spectroscopy (2D EXSY), and molecular mechanics
calculation.[20]
The rotational isomerism of compound 4 (and similar compounds) is tempera-
ture dependent. When the inner temperature in the NMR spectrometer was raised to
333 K (60 ꢁC), the doublet peaks for C8, C9 (at 51.51–51.77 ppm) and C6 (125.62–
125.64 ppm) became singlet peaks, and the two doublets for C3 and C4 (134.83–
136.91 ppm) changed into two broad peaks [Fig. 1(c)]. At 333 K, all the 13C chemical
shifts of 4 have been shifted upfield. There were many examples for the upfield

2420
C. LIANG ET AL.
Figure 1. Partial 13C NMR spectra of 4 in different solvents and at different temperatures: (a) in CDCl3,
at 293 K; (b) in DMSO-d6, at 293 K; and (c) in DMSO-d6, at 333 K.
chemical shifts at the elevated temperature in the literature.[21,22] As further evidence
for the existence of the rotational isomerism caused by the restricted C-N amide
bond rotation, when the Boc group in N-Boc piperazino-isoindoline 6 was removed,
the 13C NMR spectrum of the resulting piperazino-isoindoline 7 showed no doublet
peaks for any carbon atoms.
The next phase for synthesizing AT13387 was to couple piperazino-isoindoline
7 with 2,4-bis(benzyloxy)-5-isopropenylbenzoic acid 9 (Scheme 3); the latter was

SYNTHESIS OF AT13387
2421
Scheme 3. Synthetic route of AT13387. Reagents and conditions: (a) 7, HOBt, EDC ꢃ HCl, DIEA,
DMF, rt, 10 h, 87%; and (b) H2, 10% Pd=C, MeOH, rt, 12 h, 80%.
prepared from methyl 2,4-dihydroxybenzoate 8 by following the known
procedure.[12] In the presence of EDC=HOBt, 7 was coupled 9 to give the resorcylate
10 in 87% yield. In the final step, under catalytic hydrogenation conditions (H2, 10%
Pd=C), 10 was transformed into the target molecule in 80% yield and with 98%
1
purity (HPLC analysis). The identity of the final product was confirmed by the H
NMR, 13C NMR, and high-resolution mass spectrometry (HRMS). The synthetic
route described is convergent, is easy to operate under the mild reaction conditions,
and has been adopted to prepare multigram quantities of AT13387 in improved
overall yield and with good purity.
The feasibility of the Wilkinson’s catalyzed [2 þ 2 þ 2] cycloaddition of N-Boc-
dipropargylamine 3 and propargyl alcohol has prompted us to try another approach
for synthesizing the isoindoline intermediate 10 based on the availability of acid 9
(Scheme 4). First, acid 9 was coupled with propargylamine followed by alkylation
with propargyl bromide to give amide dialkyne 12 in good yield. Next, under the
similar reaction conditions used to make isoindoline 4, 12 was subjected to the
[2 þ 2 þ 2] cycloaddition with propargyl alcohol in THF, affording isoindoline
intermediate 13 in 72% yield. In the next step, alcohol 13 was oxidized with MnO2
in DCM but resulted in unsatisfactory yield (ꢂ50%). When 13 was reacted with
methanesulfonyl chloride, mesylate 14 was obtained in 63% yield. In the presence
Scheme 4. Another synthetic route for intermediate 10. Reagents and conditions: (a) 2-propynyl
amine, HOBt, EDC ꢃ HCl, DIEA, DMF, rt, 14 h, 99%; (b) propargyl bromide, NaH, DMF, rt, 17 h,
98%; (c) propargyl alcohol, (PPh3)3RhCl, THF, rt, 4 h, 72%; (d) MsCl, Et3N, DCM, rt, 13 h, 63%; and
(e) 1-methylpiperazine, K2CO3, DMF, rt, 12 h, 90%.

2422
C. LIANG ET AL.
of K2CO3, 14 was reacted with 1-methylpiperazine to produce 10 in 90% yield. This
synthetic route has fewer steps and with satisfactory overall yield in comparison to
the known procedures reported in the literature.
In summary, the conditions for the [2 þ 2 þ 2] cycloaddition of N-Boc-dipro-
pargylamine 3 with propargyl alcohol have been optimized, and based on the results,
two synthetic routes were developed for synthesizing the HSP90 inhibitor AT13387.
The described synthetic methods are economical and suitable for relatively
large-scale preparation of the title compound and the related isoindoline derivatives.
EXPERIMENTAL
tert-Butyl-5-hydroxymethyl-1,3-dihydro-2H-isoindole-2-carboxylate
4: General Procedure
Catalyst (0.03 equiv) was added to a stirred solution of 3 (1.0 equiv) and pro-
pargyl alcohol (3.0 equiv) in a solvent (Table 1) at rt. After the mixture was stirred at
the given temperature for certain period of time, the solvent was concentrated to dry-
ness in vacuo, and the crude product was purified by flash column chromatography
to give 4 as white solid. When the [2 þ 2 þ 2] cycloaddition of 3 with propargyl
alcohol was performed in THF at ambient temperature and in the presence of
1
(PPh3)3RhCl, 4 was obtained in 70% yield. Mp 105–106 ꢁC; H NMR (500 MHz,
CDCl3) d 7.15–7.24 (m, 3H), 4.66 (s, 2H), 4.55 (br, 4H), 2.75 (brs, 1H), 1.50
(s, 9H); 13C NMR (125 MHz, CDCl3) d 155.28, 141.29 (þ141.26, rotamer) 138.16
(þ137.83), 137.05 (þ136.79), 126.94 (þ126.86), 123.29 (þ123.11), 121.96
(þ121.83), 80.52 (þ80.49), 65.59 (þ65.54), 52.79 (þ52.70), 52.47 (þ52.34), 29.17;
HRMS=ESI: m=z [MþNa]þ calcd. for C14H19NNaO3: 272.1257; found: 272.1254.
[2,4-Bis(benzyloxy)-5-isopropenylphenyl]-[5-(4-methyl-piperazin-
1-ylmethyl)-1,3-dihydro-Isoindol-2-yl]-methanone (10)
From 9. Compound 7 (715 mg, 2.1 mmol, 1.0 equiv) was added to a mixture of
9 (786 mg, 2.1 mmol), HOBt (312 mg, 2.3 mmol, 1.1 equiv), and EDC ꢃ HCl (442 mg,
2.3 mmol, 1.1 equiv) in anhydrous DMF (30 mL), and the mixture was stirred at rt
for 0.5 h. Then DIEA (1.4 mL, 8.4 mmol, 4.0 equiv) was added at 0 ꢁC, and the mix-
ture was stirred at the same temperature for 0.5 h. The mixture was allowed to warm
to ambient temperature, and the stirring was continued for an additional 10 h. Water
(30 mL) was added, and the resulting mixture was extracted with EtOAc
(3 ꢄ 100 mL). The combined organic layers were washed with water and brine and
dried (Na2SO4). The crude product was purified by flash column chromatography
on silica gel (DCM=MeOH ¼ 20:1) to afford 10 as pale yellow solid (1.08 g,
1.8 mmol, 87%). Mp 43–44 ꢁC; 1H NMR (500 MHz, CD3OD) d 7.08–7.42 (m,
14H), 6.83 (d, J ¼ 3.35 Hz, 1H), 5.13 (s, 4H), 5.07 (d, J ¼ 11.65 Hz, 2H), 4.83 (s,
3H), 4.58 (s, 2H), 3.5 (d, J ¼ 20.25 Hz, 2H), 2.47 (s, 8H), 2.25 (d, J ¼ 10.05 Hz,
3H), 2.07 (s, 3H); 13C NMR (125 MHz, CD3OD) d 170.74, 159.28 (þ159.27,
rotamer), 156.16 (þ156.15), 144.38, 138.41 (þ138.30), 138.24, 138.09 (þ138.06),
137.98, 137.39, 136.93, 136.38, 130.29 (þ130.14), 129.62, 129.59, 129.53, 129.39,
129.05, 129.00 (þ128.97), 128.56, 128.39 (þ128.30), 127.56, 125.06 (þ124.83),

SYNTHESIS OF AT13387
2423
123.79 (þ123.57), 119.91, 115.81, 100.48, 71.84 (þ71.78), 71.64, 63.52, 55.67
(þ55.65), 54.61 (þ54.53), 53.47, 53.02 (þ52.93), 45.95, 23.63; HRMS=ESI: m=z
[MþNa]þ calcd. for C38H41N3NaO3: 610.3040; found: 610.3047.
From 14. Compound 14 (950 mg, 1.6 mmol) was added to a mixture of
1-methylpiperazine (192 mg, 1.9 mmol, 1.2 equiv) and K2CO3 (267 mg, 1.9 mmol,
1.2 equiv) in anhydrous DMF (20 mL), and the mixture was stirred at rt for 12 h.
Then the mixture was diluted with water (30 mL) and extracted with DCM
(3 ꢄ 100 mL). The combined organic layers were washed with water and brine and
dried (Na2SO4). After the solvent was removed in vacuo, the resulting material
was subjected to flash column chromatography (DCM=MeOH ¼ 15:1) to give 10
as pale yellow solid (850 mg, 1.4 mmol, 90%). The NMR spectra of the product were
identical to those obtained from 9.
[2,4-Bis(benzyloxy)-5-isopropenylphenyl]-[5-(hydroxymethyl)-
1,3-dihydro-isoindol-2-yl]-methanone (13)
Propargyl alcohol (250 mg, 4.4 mmol, 4.0 equiv) was added dropwise to a
solution of 12 (500 mg, 1.1 mmol) in THF (15 mL), followed by the addition of
tris(tri- phenylphosphine) rhodium(I) chloride (30 mg, 0.032 mmol, 0.03 equiv). The
reaction was stirred at rt for 4 h, and then the solvent was removed in vacuo to give a
red oil, which was subjected to flash column chromatography (petroleum ether=
EtOAc ¼ 1:3) to give 13 as solid (405 mg, 0.8 mmol, 72%). Mp 162–164 ꢁC; 1H
NMR (500 MHz, CDCl3) d 7.37–7.29 (m, 5H), 7.24–7.03 (m, 9H), 6.56 (s, 1H),
5.10 (s, 2H), 5.04 (s, 4H), 4.87 (s, 2H), 4.61–4.65 (m, 4H), 2.92 (br, 1H), 2.10 (s,
3H); 13C NMR (125 MHz, CDCl3) d 168.38, 157.50, 154.73 (þ154.70, rotamer),
142.65, 141.09 (þ140.88), 136.92, 136.65, 136.58 (þ136.57), 136.53, 135.69 (þ135.52),
128.68 (þ128.66), 128.61 (þ128.57), 127.98 (þ127.94), 127.16, 126.96 (þ126.94),
126.45 (þ126.23), 122.87 (þ122.39), 121.45 (þ121.02), 119.56 (þ119.51), 115.49,
99.55, 77.07 (þ77.06), 64.71 (þ64.68), 53.41 (þ53.32), 51.97 (þ51.86), 23.29;
HRMS=ESI: m=z [MþNa]þ calcd. for C33H31NNaO4: 528.2145; found: 528.2152.
FUNDING
We are grateful for the financial support from the National Science
Foundation of China (21272029), the 333 High-Level Talent Training Program of
Jiangsu Province of China, the Major Technological Innovation-Supporting
Program of Jiangsu Province of China (BE2012376), the Priority Academic Program
Development (PAPD) of Jiangsu Higher Education Institutions, and the Inter-
national Collaboration Program of Changzhou City (CZ20130024).
SUPPORTING INFORMATION
Supplemental data for this article can be accessed on the publisher’s website.

2424
C. LIANG ET AL.
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