Selective substitution of 31/42-OH in rapamycin guided by an in situ ir technique

Article abstract of DOI:10.3390/molecules19067770

An in situ IR technique was applied in the selective synthesis of the key intermediate for rapamycin derivatives, which made the reaction endpoint easily defined. This technology solved a bothersome problem in the preparation of rapamycin derivatives, and

Full text of DOI:10.3390/molecules19067770

Molecules 2014, 19, 7770-7784; doi:10.3390/molecules19067770
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Selective Substitution of 31/42–OH in Rapamycin Guided by
an in Situ IR Technique
Shuang Cao 1, Xinbo Zhou 2, Yuanshuai Yang 1, Wu Zhong 2,* and Tiemin Sun 1,*
1
Key Laboratory of Structure-Based Drug Design and Discovery, Shenyang Pharmaceutical
University, Ministry of Education, Shenyang 110016, China;
E-Mails: caoshuangknight@163.com (S.C.); yangmarshal88@163.com (Y.Y.)
2
Laboratory of Computer-Aided Drug Design & Discovery, Beijing Institute of Pharmacology and
Toxicology, Beijing 100850, China; E-Mail: hapwave@163.com
* Authors to whom correspondence should be addressed; E-Mails: zhongwu@bmi.ac.cn (W.Z.);
suntiemin@126.com (T.S.); Tel.: +86-10-66931634 (W.Z.); +86-24-23986398 (T.S.).
Received: 25 April 2014; in revised form: 26 May 2014 / Accepted: 27 May 2014 /
Published: 10 June 2014
Abstract: An in situ IR technique was applied in the selective synthesis of the key
intermediate for rapamycin derivatives, which made the reaction endpoint easily defined.
This technology solved a bothersome problem in the preparation of rapamycin derivatives,
and based on this technique, the 31-OH and 42-OH of rapamycin were chemically
modified by a series of quaternary ammonium salts to generate 11 compounds. The
solubility of all these compounds was remarkably improved (25,000 times higher than that
of rapamycin) and their structures were confirmed by MS, IR, 1D and 2D NMR techniques.
Keywords: rapamycin derivatives; in situ IR; selective substitution; aqueous solubility
1. Introduction
Rapamycin (Rapa, Figure 1) is a carboxylic lactone-lactam macrolide derived from the bacterium
Streptomyces hygroscopicus [1]. It was originally used as an immunosuppressant in the treatment of
organ rejection in transplant recipients, and then as an anti-cancer drug [2]. In recent years, some new
applications of rapamycin and rapamycin analogues have been disclosed, such as anti-aging, anti-HIV,
and so on [3]. Rapamycin has brought a new dawn to cancer treatment due to its special mechanism of

Molecules 2014, 19
7771
inhibiting cancer cell growth via inhibition of the mammalian target of rapamycin (mTOR), which is
the key factor in cell proliferation regulation [4].
Figure 1. Structure of rapamycin (Rapa).
Although Rapa has shown great potential in numerous ways, it also has two severe drawbacks,
namely poor aqueous solubility (2.6 μg/mL) [5] and low oral bioavailability (10%) [6]. To overcome
these problems, a number of Rapa derivatives have been developed, the majority of which are derived
from modifications on the 42-OH of Rapa. For example, Temsirolimus (CCI-779, Wyeth, Madison, NJ,
USA, 2007) [7], Everolimus (RAD001, Novartis, Basel, Switzerland, 2003), and Zotarolimus (ABT 578,
Abbott, Chicago, IL, USA, 2006) [2], These successful drugs suggest that the modification on the 42-OH
may be a more wise choice to optimize rapamycin, but the selective substitution of 42-OH is very
difficult because the structure of Rapa is very complicated and there are three -OHs in the structure thereof.
The traditional avenue for selective synthesis of 42-Rapa derivatives was first reported in US Patent
6277983 [8]. The crucial point of this method is preparation of the intermediate Rapa-31-OTMS
(Scheme 1), which is also the key intermediate for preparating 31-Rapa derivatives. However, in
practice, we found that though the yield of this preparation could reach up to 70% (column separation),
it is instable, and additionally, a lot of raw material and by-product are comingled with the end-product.
Scheme 1. Synthesis of Rapa-31-OTMS intermediate.
What contributes to this problem is the difficulty in defining a suitable reaction endpoint, in other
words, the amount of acid and the reaction time are difficult to control just right. On one hand, it is
necessary to use enough acid and the correct reaction time to convert Rapa-31,42-OTMS into
Rapa-31-OTMS. On the other hand, too much of them will lead to the decomposition of the
Rapa-31-OTMS into Rapa. Moreover, there exists a connection between the amount of acid and the
reaction time. The amount of acid can be reduced by extending the reaction time, and vice versa. These
problems manifest especially prominently in industrial production.
In view of the high cost of the raw material (Rapa) and the difficulty of accurate control in the
traditional method, a new technology is seriously required to solve these problems. Herein, we report

Molecules 2014, 19
7772
an application of in situ IR in selective synthesis of the key intermediate for Rapa derivatives. Based
on this method, a series of mono-substituted Rapa derivatives with quaternary ammonium groups were
obtained, which all had excellent aqueous solubility.
2. Results and Discussion
2.1. In Situ IR
Through analysis of a series of features in the IR spectrum of Rapa-31-OTMS (Figure 2), the
intensity of the absorption peak at 1.055 cm−1, a characteristic peak attributed to -Si-O(C) [9–12], was
selected as marker to monitor the levels of Rapa, Rapa-31,42-OTMS and Rapa-31-OTMS indirectly
(Figure 3) [13]. The structure of Rapa-31,42-OTMS and Rapa-31-OTMS had been identified by
1H-NMR (Supplementary Material).
Figure 2. Expanded solid state FT-IR spectra for Rapa, Rapa-31-OTMS and Rapa-31,42-OTMS.
It can be seen from the profile (Figures 3 and 4) that the peak intensity at 1055 cm−1 increases
markedly from a to b, indicating that Rapa is quickly converted to Rapa-31,42-OTMS. After adding
H2SO4 at point b, the peak gradually decreased and then remained stable from c to d. This result
indicates that Rapa-31,42-OTMS is slowly transformed into Rapa-31-OTMS and then the level of
Rapa-31-OTMS remains mostly unchanged, while the sharp decrease of the absorption peak from d to e
indicates that Rapa-31-OTMS begin to convert back to Rapa. The long reaction time and continuously
rising acidity lead to the quick hydrolysis of Rapa-31-OTMS after the breakthrough point (d). From
the in situ IR data, we concluded that the reaction endpoint lay between c and d with a stable yield of
88% (column separation). In situ IR has the advantages of being highly sensitive, continuous and real-time,
which make it more useful than TLC and HPLC, especially for a complicated reaction. This
technology could give explicit instructions for process optimization and quality control in the
preparation of Rapa-31-OTMS, especially in industrial production.

Molecules 2014, 19
Figure 3. 3D FT-IR spectrum fragments for the preparation of Rapa-31-OTMS from Rapa.
7773
The reaction was monitored from 0 to 230 min. (a) Addition of TMSCl and imidazole
(00:05:00). (b) Addition of 0.5 N H2SO4 (00:25:00 to 03:25:00). (a) to (b) Rapa is totally
converted to Rapa-31,42-OTMS. (b) to (c) Rapa-31,42-OTMS is mostly converted into
Rapa-31-OTMS. (d) to (e): Rapa-31-OTMS is totally converted into Rapa.
Figure 4. Kinetic profile (peak height vs. time) of the intensity of the absorption peak
at 1055 cm−1 (the characteristic peak attributed to -Si-O(C)) for the reaction of preparation
of Rapa-31-OTMS from Rapa. (a) Addition of TMSCl and imidazole (00:05:00).
(b) Addition of 0.5 N H2SO4 (00:25:00 to 03:25:00).
2.2. Synthesis
After determining the preparation and control method, the technique was immediately applied in the
synthesis of new Rapa derivatives. The choice of substituent groups is another key point to this
subject. To avoid the toxicity and side effects produced by metabolism of substituent groups,

Molecules 2014, 19
7774
N-(carboxymethyl) thiazolium (or pyridine) derivative side chains were introduced into 31-OH and 42-OH
by esterification. The quaternary ammonium salt derivatives developed by our team were nontoxic and
showed good water-solubility, and all of them were themselves drugs (AGEs cross-link breakers).
In the paper, the traditional synthesis method was optimized by merging two reactions into one
(Scheme 2), so that the reaction steps in the preparation of 42-Rapa derivatives were decreased from 5
to 3 (overall yield 48%). The reaction merging strategy could also be implemented in the synthesis of
31-Rapa derivatives, in which the number of steps could be decreased from 7 to 4 (overall yield 15%).
These selective synthesis strategies were achieved by tactfully taking advantage of the subtle
differences in reactivity between the two positions, whereby that of the 42-OH is greater than the 31-OH,
as well as the greater reactivity of the tert-butyldimethylsilyl (TBDMS) ether which is stronger than
that of the trimethylsilyl (TMS) ether [14–16]. Smaller steric hindrance may be the main reason for the
high reaction activity of the 42-OH position.
Scheme 2. Synthesis routes of 42-Rapa derivatives (a) and 31-Rapa derivatives (b).
O
Br
O
42
O
Br
① TMSCl
Br
①
RAPA
RAPA
a.
b.
RAPA
②
H2SO4
② H2SO4
31 OTMS
intermediate I
O
Br
R
42
O
R
RAPA
42 OTBDMS
①
① TBDMSCl
TMSCl
Rapa
Rapa
Rapa
②
H2SO4
② H2SO4
31 OTMS
Br
O
R
Br
①
Rapa
Rapa
O
O
Br
② H2SO4
R
31
O
31
O
Br
N
intermediate II
HO
N
N
N
N
N
R =
S
S
Finally, 11 compounds were produced (Table 1), which were all quaternary ammonium salts.
According to the reaction point, these compounds can be classified into two series, 42-Rapa
derivatives and 31-Rapa derivatives.
2.3. Structure
For these complicated compounds, the determination of the linkage position of the substituent
groups is difficult but indispensable. Here, a method for confirming the attachment position and
number of substituent groups was developed based on the corresponding NMR spectra.
In order to verify the structures of these 11 derivatives, 2D NMR data including HSQC,
HMBC, COSY, TOCSY and ROESY were utilized to assign 1H and 13C-NMR signals (Supplementary

Molecules 2014, 19
7775
Material). For some particular groups, such as the -OH, the combination of hydrogen-deuterium
exchange and HMBC technique were used to assign their chemical shifts. Specifically, the 13-OH, 31-OH
and 42-OH signals were first located by the hydrogen-deuterium exchange method, and then
accurately assigned at 6.44, 5.25 and 4.57 ppm by relevant HMBC correlations, respectively (Table 2).
As shown in Figure 5, the three characteristic peaks could serve as markers to locate the position
where the substituent group was linked to Rapa.
Table 1. Structures and solubility for compounds 1–11.
Aqueous
Solubility
(mg/mL)
Times Increased of
Aqueous Solubility
(Comp. vs. Rapa)
Comp.
R1
H
R2
H
Rapa
0.0026
4.2
1
1
2
H
H
1615
11
0.028
3
4
H
H
H
H
0.025
6.7
10
2577
4
5
0.01
6
6
2308
8962
3846
10269
20231
24038
7
H
H
H
H
H
23.3
10
8
9
26.7
52.6
62.5
10
11

Molecules 2014, 19
Table 2. 1H (600 MHz) and 13C (150 MHz) NMR chemical shifts (δ, ppm) of Rapa, 1 and
7776
7 in DMSO-d6.
Rapa
Comp. 1
δH
Comp. 7
C
δC
δH
5.46
6.15
6.22
6.37
6.11
—
δC
δC
δH
5.46
6.12
6.25
6.37
6.20
—
3.63
1
2
3
4
5
6
7
8
139.26
130.38
132.28
126.99
126.94
137.81
82.22
39.91
66.18
29.60
31.08
34.76
98.97
—
198.84
166.95
43.45
24.42
20.32
26.39
50.72
169.15
73.55
39.50
207.48
45.16
124.93
137.09
—
75.70
85.52
210.47
39.37
39.22
35.15
33.34
38.36
32.50
35.41
83.71
73.55
—
139.28
130.37
132.33
126.94
126.87
137.83
82.19
39.92
66.17
29.65
30.27
34.77
98.97
—
198.76
166.97
43.46
24.33
20.31
26.40
50.81
169.24
73.57
39.50
207.47
45.18
124.73
137.06
—
75.77
85.41
210.31
39.36
39.22
35.11
33.27
37.98
31.81
34.93
79.85
78.14
—
5.46
6.15
6.22
6.37
6.11
138.73
130.64
132.23
127.18
126.94
138.03
81.91
40.03
66.00
29.54
31.02
34.57
98.91
—
—
3.61
3.61
1.24,1.83
4.01
1.80,1.16
0.83,1.51
2.01
1.21,1.83
4.06
1.80,1.17
0.97,1.61
2.01
1.08,1.84
4.03
1.16,1.88
0.82,1.49
1.99
9
10
11
12
13
13-OH
15
16
18
19
20
21
22
23
25
26
27
28
29
30
31-OH
31
32
33
34
35
36
37
38
39
40
41
42
42-OH
43
44
—
—
6.49
—
—
—
6.45
6.44
—
—
198.21
167.00
43.67
24.41
20.40
26.27
51.25
169.46
73.56
40.03
207.03
45.07
124.30
138.03
—
—
—
3.41,3.15
1.55,1.26
1.65,1.38
2.08,1.56
4.93
3.42,3.15
1.28,1.56
1.65,1.38
1.59,2.08
4.93
—
4.97
2.79,2.38
—
2.99,3.43
1.28,1.57
1.43,1.69
1.60,2.20
4.93
—
4.87
2.34,2.66
—
—
4.98
2.72,2.37
—
3.25
5.08
3.24
5.08
—
5.23
4.06
3.97
—
2.43
3.20
4.81
—
—
—
5.25
4.01
3.94
78.51
80.89
208.09
39.92
40.02
35.05
33.57
38.24
32.42
35.26
83.68
73.15
—
5.35
4.54
—
2.48
—
2.39
1.39,1.02
2.20
1.66
1.03,0.94
1.23
1.88,0.58
2.81
3.15
4.57
1.73,1.15
1.52
1.39,1.02
2.22
1.67
0.99,1.06
1.35
2.05,0.73
3.15
4.67
—
2.01,2.21
1.55
1.07,1.83
2.24
1.59
0.92,0.99
1.22
0.51,1.87
2.76
3.10
4.56
32.86
31.08
35.11
26.40
32.75
31.02
1.16,1.74
1.56

Molecules 2014, 19
Figure 5. Expanded 1H-NMR spectra (600 MHz, DMSO-d6) for compounds Rapa, 1 and 7.
7777
An in-depth analysis of these NMR data revealed more information about the structures. Based on
these NMR data (Table 2), a curve was drawn to record the chemical shifts (ppm) of different protons,
and the profiles of Rapa, compounds 1 and 7 were put together for comparison (Figure 6). By this way,
some structure information was deduced. The chemical shifts of most positions in compounds 1 and
7 are nearly identical, except for those of positions 39 to 44 (region A) and positions 29 to 36 (region B)
in three cases (Figure 6). This phenomenon implied that the chemical environments of most of the
protons were unchanged, furthermore, we inferred that most of the atoms in compounds 1 and 7
maintain a similar chemical environment as those in Rapa. Thus, the change of chemical shifts in
region A and B could be used to locate the substituents directly.
Figure 6. The curves (chemical shifts vs. positions) recording the chemical shifts (ppm) for
different positions of protons of Rapa (blue curve), 1 (red curve) and 7 (green curve).
Regions A and B were marked by blue and red, respectively.

Molecules 2014, 19
7778
Despite two years of efforts, we had never been able to obtain single crystals of these quaternary
ammonium salt derivatives, so we tried to simulate the 3D structure of typical compounds 1 and 7.
Given the structure of rapamycin was extremely complicated, we attempted to construct this 3D
structure based on the X-ray single crystal data of rapamycin reported by Swindel [16–18].
By comparison of the NMR data, we decided to simulate the configuration of region A, region B
and the side chain using the molecular modeling software SYBYL 6.5, and at the same time, maintain
most of the 3D structure of rapamycin (Figure 7). The accuracy of this simulated 3D structure was
confirmed by ROESY NMR experiments. In compound 1, the existence of the side chain caused the
cyclohexane ring to bend downward, leading to many distinct NOE effects of H41 to H21, H29, H31,
H37, H38, H39, H40 and H42 (Figure 7a). In compound 7, the molecule folds the side chain in region
B placing it very closd to the cyclohexane ring in region A, and this situation could be proved easily by
observing the NOE effect between H31c and 42-OH. Moreover, the ROESY correlations between H36 and
H29, H32, H34 were clearly presented because of the obvious fold in the C26 to C36 region (Figure 7b).
Figure 7. (a) Key partial simulated 3D structure of compound 1 (region A) and key
ROESY correlations (red arrows); (b) Key partial simulated 3D structure of compound 7
(region B) and key ROESY correlations (red arrows).
All the above mentioned results not only proved that the reaction took place at the right positions,
but also provided useful information regarding the three dimensional structures of these compounds,
which might have a significant value in the field of compound design and structure simulation (Figure 7).
2.4. Aqueous Solubility
The aqueous solubility for all 11 compounds was improved remarkably (Table 1), and some (10 and 11)
of them were even up to 25,000 times more soluble than Rapa. After summing up the effects of
different substituents, a rule about the effect of substitution on aqueous solubility emerged. Generally
speaking, the aqueous solubility of 31-Rapa derivatives is obviously superior to that of 42-Rapa
derivatives.

Molecules 2014, 19
7779
3. Experimental
3.1. Physical Measurements
Melting points were determined using an RY-1 apparatus. MS spectra were obtained using an
Applied Biosystems API-150EX LC/MS mass spectrograph. NMR data was recorded on a Varian
Inova 600 MHz spectrometer operating at a proton frequency of 599.6 MHz and a carbon frequency of
150.8 MHz. All NMR spectra were acquired at 27 °C on a sample of 20 mg of rapamycin derivatives
dissolved in 0.2 mL of DMSO-d6, with TMS as an internal standard. 3D-FTIR spectra were obtained
from a ReactIR™ iC10 instrument equipped with a MCT Detector and a DiComp (Diamond) probe.
3.2. Synthesis
Preparation of Rapa-42-ester of 2-(4,5-dimethylthiazolium-3-yl)acetic Acid (1)
Step 1: Rapa (5 g, 5.5 mmol) and imidazole (3.74 g, 55 mmol) were dissolved in ethyl acetate (50 mL)
and the solution was stirred at 0 °C, then a solution of trimethylchlorosilane (2.98 g, 27.5 mmol) in
ethyl acetate (5 mL) was added into the reaction solution within 30 min. After the Rapa was
transformed into Rapa-31,42-bis-OTMS, dilute sulfuric acid solution (0.5 mol/L, 10 mL) was slowly
added dropwise to the reaction mixture within 3 h. The organic layer was washed three times with
water. The organic layer was concentrated and dried over MgSO4. The residue was purified by
chromatography on silica gel (EA/PE v/v = 1:5) to obtain Rapa-31-OTMS (4.7 g, 4.8 mmol, yield 88%).
Step 2: Rapa-31-OTMS (4.7 g, 4.8 mmol) and pyridine (3.7 g, 48 mmol) were added to dichloromethane
at −10 °C, then, a solution of bromoacetyl bromide (4.8 g, 24 mmol) in dichloromethane (40 mL) was
added into the reaction solution within 30 min. The solution was further stirred for 10 min, and
warmed to 0–5 °C in 10 min. Then, dilute sulfuric acid (1 mol/L, 8 mL) was added to the reaction
mixture over 1 h. This mixture was stirred for 30 min. The organic layer was washed three times with
water. The combined organic layers were concentrated and dried over MgSO4. The residue was
purified by chromatography on silica gel (EA/PE v/v = 1:8) to obtain Rapa-42-ester of bromoacetic
acid (intermediate I) (3.82 g, 3.7 mmol, yield 77%).
Step 3: Intermediate I (3.82 g, 3.7 mmol) and 4,5-dimethylthiazole (5 equiv., 2.09 g, 18.5 mmol) were
added to acetone (30 mL) at 20 °C, then, the reaction mixture was heated to 60 °C for 3 h. The solution
was concentrated to dryness and the residue was purified by chromatography on silica gel (MeOH/DCM
v/v = 1:5) to obtain compound 1 (3.61 g, yield 85%). m.p. 135–138 °C; FTIR (KBr): νmax/cm−1 = 3422
(OH) and 2931 (-CH3), 1723 (C=O), 1643 (C=O), 1451, 1091 (-C-O-C-); MS:1067.9 [M-Br]+; 1H-NMR
(δ ppm): 10.03 (s, 1H, -S-CH=N-), 6.49 (s, 1H, 13-OH), 6.37 (m, 1H, 4-C), 6.21 (m, 1H, 3-C), 6.14 (m,
2H, 2-C), 5.62 (m, 2H, -CH2-), 5.46 (m, 1H, 1-C), 5.23 (s, 1H, 31-OH), 5.08 (s, 1H), 4.97 (s, 1H), 4.93
(s, 1H), 4.68 (s, 1H), 4.06 (s, 2H), 4.01 (s, 1H). 13C-NMR (δ ppm): 210.31, 207.47, 198.76, 169.24,
158.14, 141.91, 138.28, 130.37, 98.97. Anal. calcd for C58H87N2O14SBr: C 60.67, H 7.64, N 2.44, Br
6.96%. Found: C 60.49, H 7.66, N 2.45, Br 6.93%.

Molecules 2014, 19
7780
Rapa-42-ester of 2-(4-methylthiazolium-3-yl)acetic Acid (2). The procedure described above for the
preparation of compound 1 was applied to intermediate I and 4-methylthiazole to afford the title
compound 2 (yield 89%). m.p. 130–133 °C; FTIR (KBr): νmax/cm−1 = 3422 (OH) and 2933 (-CH3),
1720 (C=O), 1643 (C=O), 1451, 1103 (-C-O-C-); MS:1053.8 [M-Br]+; 1H-NMR (δ ppm): 10.16 (d,1H,
-S-CH=N-), 8.06 (s, 1H, -S-CH=), 6.45 (s,1H, 13-OH), 6.38 (m, 1H, 4-C), 6.22 (m, 1H, 3-C), 6.15 (m,
1H, 2-C), 5.62 (s, 2H, -CH2-), 5.46 (m, 1H, 1-C), 5.24 (s, 1H, 31-OH), 5.08 (d, 1H), 4.97 (s, 1H), 4.93
(s, 1H), 4.68 (m, 1H), 4.00 (s, 2H), 3.97 (s, 1H). 13C-NMR (δ ppm): 214.58, 178.64, 169.24, 164.76,
146.53, 120.99, 98.40, 67.14. Anal. calcd for C57H85N2O14SBr: C 60.36, H 7.55, N 2.47, Br 7.04%.
Found: C 60.28, H 7.57, N 2.48, Br 7.00%. (89% yield).
Rapa-42-ester of 2-(pyridinium-1-yl)acetic Acid (3). The procedure described above for the preparation
of compound 1 was applied to intermediate I and pyridine to afford the title compound 3 (yield 87%).
m.p. 135–137 °C; FTIR (KBr): νmax/cm−1 = 3408 (OH) and 2934 (-CH3), 1738 (C=O), 1638 (C=O),
1
1452, 1104 (-C-O-C-); MS:1034.1 [M-Br]+; H-NMR (δ ppm): 9.07 (d, 1H, pyridine), 9.02 (m, 1H,
pyridine), 8.71 (m, 1H, pyridine), 8.24 (m, 2H, pyridine), 6.45 (s, 1H, 13-OH), 6.41 (m, 1H, 4-C), 6.22
(m, 1H, 3-C), 6.14 (m, 1H, 2-C), 5.80 (m, 5H), 5.67 (m, 2H, -CH2-), 5.44 (m, 1H), 5.25 (s, 1H,
31-OH), 5.08 (m, 1H). 13C-NMR (δ ppm): 211.92, 208.23, 193.37, 169.24, 160.10, 145.63, 139.71,
130.17, 98.46. Anal. calcd for C58H85N2O14Br: C 62.52, H 7.69, N 2.51, Br 7.17%. Found: C 62.44, H
7.71, N 2.53, Br 7.13%.
Rapa-42-ester of 2-(3-methylpyridinium-1-yl)acetic Acid (4). The procedure described above for the
preparation of compound 1 was applied to intermediate I and 3-methylpyridine to afford the title
compound 4 (yield 82%). m.p. 138–140 °C; MS:1048 [M-Br]+; FTIR (KBr): νmax/cm−1 = 3422 (OH)
1
and 2933 (-CH3), 1742 (C=O), 1641 (C=O), 1452, 1103 (-C-O-C-); H-NMR (δ ppm): 9.07 (d, 1H,
pyridine), 9.02 (m, 1H, pyridine), 8.71 (m, 1H, pyridine), 8.14 (m, 2H, pyridine), 6.45 (s, 1H, 13-OH),
6.41 (m, 1H, 4-C), 6.22 (m, 1H, 3-C), 6.14 (m, 1H, 2-C), 5.80 (m, 5H), 5.67 (m, 2H), 5.44 (m, 1H),
13
5.25 (s, 1H, 31-OH), 5.08 (m, 1H). C-NMR (δ ppm) 214.60, 208.32, 169.25, 166.67, 165.55,
139.01, 98.41, 80.42. Anal. calcd for C59H87N2O14Br: C 62.81, H 7.77, N 2.48, Br 7.08%. Found: C
62.80, H 7.79, N 2.49, Br 7.05%.
Rapa-42-ester of 2-(3-hydroxypyridinium-1-yl)acetic Acid (5). The procedure described above for the
preparation of compound 1 was applied intermediate I and 3-hydroxypyridine to afford the title
compound 5 (yield 80%). m.p. 146–148 °C; FTIR (KBr): νmax/cm−1 = 3426 (OH) and 2933 (-CH3),
1
1721 (C=O), 1641 (C=O), 1452, 1102 (-C-O-C-); MS:1050 [M-Br]+; H-NMR (δ ppm): 12.08 (s, 1H,
pyridine-OH), 8.60 (m, 1H, pyridine), 8.53 (m, 1H, pyridine), 8.02 (m, 2H, pyridine), 6.45 (m, 2H,
13-OH), 6.38 (s, 1H, 4-C), 6.22 (m, 1H, 3-C), 6.12 (m, 2H, 2-C), 5.60 (m, 2H), 5.48 (m, 1H), 5.25 (s,
13
1H, 31-OH), 5.08 (m, 1H). C-NMR (δ ppm): 214.41, 208.35, 169.29, 166.66, 164.97, 157.62,
139.53, 133.25, 98.43. Anal. calcd for C58H85N2O14Br: C 61.64, H 7.58, N 2.48, Br 7.07%. Found: C
61.56, H 7.60, N 2.49, Br 7.04%.
Rapa-42-ester of 2-(4-methylpyridinium-1-yl)acetic Acid (6). The procedure described above for the
preparation of compound 1 was applied to intermediate I and 4-methylpyridine to afford the title
compound 6 (yield 84%). m.p. 157–160 °C; FTIR (KBr): νmax/cm−1 = 3425 (OH) and 2933 (-CH3),

Molecules 2014, 19
7781
1742 (C=O), 1643 (C=O), 1452, 1102 (-C-O-C-); MS:1048.1 [M-Br]+; 1H-NMR (δ ppm): 8.89 (d, 1H,
pyridine), 8.05 (m, 2H, pyridine), 6.45 (s, 1H, 13-OH), 6.41 (m, 1H, 4-C), 6.22 (m, 1H, 3-C), 6.12 (m, 2H,
2-C), 5.61 (m, 3H), 5.46 (m, 1H), 5.25 (m, 1H, 31-OH), 5.08 (d, 1H), 4.93 (m, 2H). 13C-NMR (δ ppm):
214.65, 208.23, 192.96, 169.26, 160.09, 145.61, 139.70, 130.17, 98.46. Anal. calcd for
C59H87N2O14Br: C 62.81, H 7.77, N 2.48, Br 7.08%. Found: C 62.73, H 7.80, N 2.49, Br 7.05%.
Preparation of Rapa-31-ester of 2-(4,5-dimethylthiazolium-3-yl)acetic Acid (7)
Step 1: Rapa (5 g, 5.5 mmol) and imidazole (3.74 g, 55 mmol) were dissolved in ethyl acetate (50 mL)
and the solution was stirred at 0 °C, then a solution of trimethylchlorosilane (2.98 g, 27.5 mmol) in
ethyl acetate (5 mL) was added into the reaction solution within 30 min. After the Rapa was
transformed into Rapa-31,42-bis-OTMS, dilute sulfuric acid (0.5 mol/L, 10 mL) solution was slowly
added dropwise into the reaction mixture within 3 h. The organic layer was washed three times with
water. The organic layer was concentrated and dried over MgSO4. The residue was purified by
chromatography on silica gel (EA/PE v/v = 1:5) to obtain Rapa-31-OTMS (4.6 g, 4.7 mmol, yield 88%).
Step 2: To a mixture of Rapa-31-OTMS (4.6 g, 4.7 mmol) and imidazole (3.3 g, 48 mmol) in ethyl
acetate (40 mL) at 0 °C, a solution of tert-butyldimethylsilylchlide (TBDMSCl) (3.54 g, 24 mmol) in
ethyl acetate (10 mL) was added within 30 min. After all the reagent was transformed into
Rapa-31-OTMS-42-OTBDMS, dilute sulfuric acid (0.5 mol/L, 8 mL) was added into the solution
within 1 h. The reaction solution was washed with water three times and the organic layer was
concentrated and dried over MgSO4. The residue was purified by chromatography on silica gel (EA/PE
v/v = 1:5) to obtain Rapa-42-OTBDMS (2.41 g, 2.4 mmol, yield 50%).
Step 3: Rapa-42-OTBDMS (2.41 g, 2.4 mmol) and pyridine (1.89 g, 24 mmol) were added into
acetone (50 mL) at −10 °C. A solution of bromoacetyl bromide (5 equiv) in acetone (5 mL) was added
into this mixture within 30 min and further stirred for 10 min. Then, the reaction was heated to 0–5 °C
in 10 min, and dilute sulfuric acid (2 mol/L, 5 mL) was added into the solution in 30 min. The solution
was concentrated to dryness and the residue was purified by chromatography on silica gel (EA/PE
v/v = 1:8) to obtain Rapa-31-ester of bromoacetic acid (intermediate II) (0.94 g, 0.9 mmol, yield 38%).
Step 4: Intermediate II (0.94 g, 0.9 mmol) and 4,5-dimethylthiazole (5 equiv., 0.51 g, 4.5 mmol) were
added to acetone (10 mL) at 20 °C, then, the reaction mixture was heated to 60 °C for 3 h. The solution
was concentrated to dryness and the residue was purified by chromatography on silica gel
(MeOH/DCM v/v = 1:5) to obtain compound 7 (0.72 g, 0.63 mmol, yield 70%). m.p. 125–127 °C;
FTIR (KBr): νmax/cm−1 = 3421 (OH) and 2933 (-CH3), 1720 (C=O), 1643 (C=O), 1452, 1104 (-C-O-C-);
MS:1067.7 [M-Br]+; 1H-NMR (δ ppm): 10.02 (d, 1H, -S-CH=N-), 6.45 (s,1H, 13-OH), 6.37 (m, 1H, 4-C),
6.25 (m, 1H, 3-C), 6.20 (m, 1H, 5-C), 6.15 (m, 1H, 2-C), 5.70 (m, 2H), 5.45 (m, 1H), 5.33 (s, 1H),
5.08 (s, 1H), 4.93 (s, 1H), 4.81 (m, 1H), 4.56 (m, 3H, 42-OH). 13C-NMR (δ ppm): 208.09, 207.03,
198.21, 169.46, 157.92, 142.01, 138.03, 130.64, 98.91. Anal. calcd for C58H87N2O14SBr: C 60.67,
H 7.64, N 2.44, Br 6.96%. Found: C 60.53, H 7.65, N 2.46, Br 6.93%.
Rapa-31-ester of 2-(4-methylthiazolium-3-yl)acetic Acid (8). The procedure described above for the
preparation of compound 7 was applied to intermediate II and 4-methylthiazole to afford the title

Molecules 2014, 19
7782
compound 8 (yield 62%). m.p. 130–132 °C; FTIR (KBr): νmax/cm−1 = 3420 (OH) and 2932 (-CH3),
1
1746 (C=O), 1642 (C=O), 1452, 1106 (-C-O-C-); MS:1054 [M-Br]+; H-NMR (δ ppm): 10.16 (d,1H,
-S-CH=N-), 8.05 (s, 1H, S-CH=C), 6.45 (s, 1H, 13-OH), 6.37 (m, 1H, 4-C), 6.25 (m, 1H, 3-C), 6.18
13
(m, 1H, 5-C), 5.70 (s, 2H), 5.46(m, 1H), 5.35 (s, 1H), 4.55 (d, 2H, 42-OH). C-NMR (δ ppm):
214.58, 178.64, 169.24, 164.76, 146.53, 120.99, 98.40, 67.14. Anal. calcd for C57H85N2O14SBr: C
60.36, H 7.55, N 2.47, Br 7.04%. Found: C 60.28, H 7.57, N 2.48, Br 7.00%.
Rapa-31-ester of 2-(3-methylpyridinium-1-yl)acetic Acid (9). The procedure described above for the
preparation of compound 7 was applied to intermediate II and 3-methylpyridine to afford the title
compound 9 (yield 82%). m.p. 138–140 °C; FTIR (KBr): νmax/cm−1 = 3424 (OH) and 2932 (-CH3),
1723 (C=O), 1641 (C=O), 1452, 1190 (-C-O-C-); MS:1048.1 [M-Br]+; 1H-NMR (δ ppm): 8.88 (d, 1H,
pyridine), 8.80 (d, 1H, pyridine), 8.57 (m, 1H, pyridine), 8.12 (m, 1H, pyridine), 6.45 (m, 1H, 13-OH),
6.40 (m, 1H, 4-C), 6.23 (m, 1H, 3-C), 6.17 (m, 1H, 5-C), 5.81 (d, 1H), 5.74 (m, 2H), 5.47 (m, 1H),
13
5.28 (s, 1H), 4.58 (s, 1H, 42-OH). C-NMR (δ ppm): 212.91, 208.19, 169.30, 166.92, 164.60,
143.79, 139.41, 130.17, 98.29. Anal. calcd for C59H87N2O14Br: C 62.81, H 7.77, N 2.48, Br 7.08%.
Found: C 62.73, H 7.79, N 2.50, Br 7.04%.
Rapa-31-ester of 2-(pyridinium-1-yl)acetic Acid (10). The procedure described above for the
preparation of compound 7 was applied to intermediate II and pyridine to afford the title compound 10
(yield 59%). m.p. 140–142 °C; FTIR (KBr): νmax/cm−1 = 3425 (OH) and 2932 (-CH3), 1723 (C=O), 1640
1
(C=O), 1452, 1092 (-C-O-C-); MS: 1034 [M-Br]+; H-NMR (δ ppm): 8.96 (d, 2H, pyridine), 8.72 (m,
1H, pyridine), 8.22 (m, 2H, pyridine), 6.44 (s, 1H, 13-OH), 6.40 (m, 1H, 4-C), 6.24 (m, 1H, 3-C), 6.17
(m, 2H, 5-C), 5.87 (m, 1H), 5.79 (m, 1H), 5.48 (m, 1H), 5.29 (s, 1H), 4.95 (m, 2H), 4.81 (s, 1H), 4.52
(s, 1H, 42-OH). 13C-NMR (δ ppm): 208.14, 169.29, 166.94, 146.65, 140.02, 126.43, 98.29. Anal. calcd
for C58H85N2O14Br: C 62.52, H 7.69, N 2.51, Br 7.17%. Found: C 62.44, H 7.71, N 2.52, Br 7.14%.
Rapa-31-ester of 2-(4-methylpyridinium-1-yl)acetic Acid (11). The procedure described above for the
preparation of compound 7 was applied to intermediate II and 4-methylpyridine to afford the title
compound 11 (yield 65%). m.p. 145–148 °C; FTIR (KBr): νmax/cm−1 = 3424 (OH) and 2932 (-CH3),
1723 (C=O), 1644 (C=O), 1452, 1092 (-C-O-C-); MS:1048.1 [M-Br]+; 1H-NMR (δ ppm): 8.77 (d, 2H,
pyridine), 8.03 (d, 2H, pyridine), 6.44 (s, 1H, 13-OH), 6.40 (m, 1H, 4-C), 6.23 (m, 1H, 3-C), 6.16 (m,
2H, 5-C), 5.80 (d, 1H), 5.70 (d, 1H), 5.44 (m, 1H), 5.29 (m, 1H), 5.29 (s, 1H), 4.52 (s, 1H, 42-OH).
13C-NMR (δ ppm): 212.72, 169.35, 164.95, 160.41, 145.50, 139.79, 129.23, 98.17, 84.23. Anal.
calcd for C59H87N2O14Br: C 62.81, H 7.77, N 2.48, Br 7.08%. Found: C 62.67, H 7.79, N 2.49, Br 7.05%.
4. Conclusions
The in situ IR technique was successfully applied in the selective synthesis of the key intermediate
for preparing Rapa derivatives, which made the determination of the reaction endpoint easier so as to
make the reaction high yield (88%) and controllable. In total 11 mono-substituted Rapa derivatives
(six 42-Rapa derivatives and five 31-Rapa derivatives) were synthesized, and all of them had a
quaternary ammonium salt structures. The aqueous solubility of all these compounds was obviously
improved. Among them, the aqueous solubility of compounds 10 and 11 jumped to 62 mg/mL, nearly

Molecules 2014, 19
7783
25,000 times that of Rapa. The spatial configuration of these Rapa derivatives was simulated by the
molecular modeling software SYBYL 6.5, which showed a good agreement with the 2D NMR data.
This provides valuable information to guide further compound design and synthesis.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/6/7770/s1.
Acknowledgments
This work was supported by the Program for Innovative Research Team of the Ministry of
Education and Program for Liaoning Innovative Research Team in University, and by Grant
No. 2011BAI18B01 to Wu Zhong from the National Key Technology R & D Program.
Author Contributions
Shuang Cao, Wu Zhong and Tiemin Sun designed research; Shuang Cao, Xinbo Zhou and
Yuanshuai Yang performed research and analyzed the data; Shuang Cao wrote the paper. All authors
read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Gomez-Pinillos, A.; Ferrari, A.C. mTOR signaling pathway and mTOR inhibitors in Cancer
therapy. Hematol. Oncol. Clin. N. 2012, 26, 483–505.
2. Zhou, H.Y.; Huang, S.L. Current development of the second generation of mTOR inhibitors as
anticancer agents. Chin. J. Cancer 2012, 31, 8–18.
3. Lv, X.; Ma, X.; Hu, Y. Furthering the design and the discovery of small molecule ATP-competitive
mTOR inhibitors as an effective cancer treatment. Expert Opin. Drug Discov. 2013, 8, 991–1012.
4. Laplante, M.; Sabatini, D.M. mTOR signaling in growth control and disease. Cell 2012, 149,
274–293.
5. Simamora, P.; Alvarez, J.M.; Yalkowsky, S.H. Solubilization of rapamycin. Int. J. Pharm. 2001,
213, 25–29.
6. Crowe, A.; Bruelisauer, A.; Duerr, L. Absorption and intestinal metabolism of SDZ-RAD and
rapamycin in rats. Drug Metab. Dispos. 1999, 27, 627–632.
7. Gu, J.X.; Ruppen, M.E. Proline CCI-779, Production of and Uses Therefor, and Two-step
Enzymatic Synthesis of Proline CCI-779 and CCI-779. US Patent 20050234086A1, 12 April 2005.
8. Shaw, C.C. Treating Rapamycin with Chlorotrimethylsilane in an Inert Solvent in the Presence of
a Suitable Base to Provide Rapamycin 31,42-bis-Trimethylsilyl ether; Treating with Dilute Acid
to prOvide rapamycin 31-Trimethylsilyl Ether. US Patent 6277983, 21 August 2001.
9. Niznansky, D.; Rehspringer, J.L. Infrared study of SiO 2 sol to gel evolution and gel aging.
J. Non-Cryst. Solids 1995, 180, 191–196.

Molecules 2014, 19
7784
10. Yokogawa, H.; Yokogawa, M. Hydrophobic silica aerogels. J. Non-Cryst. Solids 1995, 186, 23–29.
11. Latthe, S.S.; Nadargi, D.Y. TMOS based water repellent silica thin films by co-precursor method
using TMES as a hydrophobic agent. Appl. Surf. Sci. 2009, 225, 3600–3604.
12. Orcel, O.; Hench, L.L. Effect of formamide additive on the chemistry of silica sol-gels II. gel
structure. J. Non-Cryst. Solids 1988, 105, 223–231.
13. Cao, S.; Zhou, X.B.; Zhang, H. Novel cross-link breaker based on zwitterion structure: Synthesis,
structure and druggability studies. Eur. J. Med. Chem. 2013, 68, 89–95.
14. Gupta, N. Process for the Preparation of Temsirolimus and Its Intermediates. US Patent
20110184167A1, 28 July 2011.
15. Lee, K.C. Process for Preparation of Temsirolimus. US Patent 20100249415A1, 30 September 2010.
16. Swindells, D.C.; White, P.S.; Findlay, J.A. The X-ray crystal structure of rapamycin, C51H79NO13.
Can. J. Chem. 1978, 56, 2491–2492.
17. Xie, L.; Huang, J.; Zuo, J. 41-Azido-41-de-oxy-rapamycin. Acta Crystallogr. E 2012, 68 (Pt. 4),
o1206–o1207.
18. Xie, L.; Zuo, J.; Yang, G. 40-De-oxy-40(S)-iodo-rapamycin. Acta Crystallogr. E 2012, 68 (Pt. 8),
o2331.
Sample Availability: Samples of the compounds 1–11 are available from the authors.
© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).