Semi-continuous multi-step synthesis of lamivudine

Article abstract of DOI:10.1039/c7ob00480j

We report the first continuous flow synthesis of lamivudine, an antiretroviral drug used in the treatment of HIV/AIDS and hepatitis B. The key intermediate (5-acetoxy oxathiolane) was prepared by an integrated two step continuous flow process from l-menthyl glyoxalate hydrate in a single solvent, in 95% overall conversion. For the crucial glycosidation reaction, using pyridinium triflate as the novel catalyst, an improved conversion of 95% was obtained. The overall isolated yield of the desired isomer of lamivudine (40%) was improved in the flow synthesis compared to the batch process.

Full text of DOI:10.1039/c7ob00480j

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Semi-continuous multi-step synthesis of lamivudine DOI: 10.1039/C7OB00480J
Devender Mandala, Sravanthi Chada and Paul Watts*
Nelson Mandela Metropolitan University, University Way, Port Elizabeth, 6031,
South Africa.
Corresponding author: Paul.Watts@nmmu.ac.za
Abstract
We report the first continuous flow synthesis of lamivudine, an antiretroviral drug used in the
treatment of HIV/AIDS and hepatitis B. The key intermediate (5-acetoxy oxathiolane) was
prepared by an integrated two step continuous flow process from L-menthyl glyoxalate
hydrate in a single solvent, in 95% overall conversion. For the crucial glycosidation reaction,
using pyridinium triflate as the novel catalyst, an improved conversion of 95% was obtained.
The overall isolated yield of the desired isomer of lamivudine (40%) was improved in the
flow synthesis compared to the batch process.
Keywords
Continuous flow synthesis, lamivudine, N-glycosidation, micro reactor, pyridinium triflate.
1

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Introduction:
DOI: 10.1039/C7OB00480J
Continuous flow technology offers many potential advantages compared with traditional
batch manufacturing of pharmaceuticals, such as easier reaction optimization and greater
control of the process, improved safety and environmental profiles, as well as a reduced
1
-3
manufacturing footprint.
Pharmaceutical companies, fine-chemical producers, and
academia are pursuing continuous flow chemistry in the production of APIs and related
products with several interesting developments in this field are emerging. Recently, the safe
manufacturing of organic intermediates and APIs under continuous flow conditions has been
4
examined in a review by Kappe and co-workers. As pointed out by the authors, some
synthetic steps that were not permitted for safety reasons (e.g. use of potentially toxic or
explosive intermediates, reactions run under high pressures or above the boiling point of the
solvent) can now be performed under flow conditions with minimum risk. For these reasons,
flow chemistry can be seen as a novel technology that opens the way for new synthetic routes
of valuable molecules.
Nucleosides and their analogues are a well-established and important class of antiviral and
5
anticancer agents. Due to the broad applicability of these compounds, the commercial
development of more efficient methods for the synthesis of these drugs remains of high
importance. For example, despite a large number of methods in the literature used to prepare
antiretroviral drugs such as lamivudine 1 and emtricitabine 2 greener and more efficient
processes for the synthesis of such generic drugs is highly desired. Herein, we report the
synthesis of lamivudine 1 using flow chemistry; to the best of our knowledge, this is the first
general synthesis of lamivudine using continuous flow technology. Lamivudine displays
potent activity against HIV-1 and HIV-2 and in the last decade has been one of the most
6
successful drugs for the treatment of HIV as well as chronic hepatitis B. Since the chemical
structure of 1 has two chiral centres, it has four stereoisomers of which the (2R,5S)-isomer (as
illustrated in Figure 1) is the most potent in anti-HIV and anti-HBV activities, and
importantly its cytotoxicity on cells is lower than its enantiomer.
2

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DOI: 10.1039/C7OB00480J
N
NH
F
2
NH
O
O
N
2
HO
N
HO
N
O
S
O
S
1
(3TC)
2 (FTC)
Emtricitabine
Lamivudine
Figure 1 The structures of oxathiolane nucleoside, lamivudine 1 and emtricitabine 2.
Efficient syntheses of lamivudine, a drug that is still inaccessible to millions of people in
7
developing countries worldwide, holds great potential both scientifically and socially. This
potent nucleoside reverse transcriptase inhibitor (NRTI) is one of the preferred agents used in
8
combination therapy for the first-line treatment of HIV. Although there are quite a few
9
methods stated for the synthesis of lamivudine 1 as a single enantiomer, a novel more
efficient route which is suitable for large-scale local manufacture is still needed. Recently, we
1
0
developed an improved batch method for the synthesis of lamivudine and are now
transferring the results into a flow synthesis. South Africa does not have batch
manufacturing technology; if batch production was to be implemented the whole
infrastructure would have to be established. The fact the flow technology is now well
established (here and in many cases) to produce products in higher yield, and in many cases
at reduced cost, this is how the process would be implemented locally. The approach of large
scale manufacture has been reported in many industrial examples, so the issue of scale up in
1
-3
flow is not problematic.
Results & Discussion:
Continuous flow synthesis to 5-acetoxy oxathiolane:
A significant challenge in transferring the batch protocol into a continuous flow process was
related to the poor solubility of one of the starting materials, namely 1,4-dithiane-2,5-diol 4.
Initially, we tried to dissolve both starting materials (3 and 4) in acetonitrile and discovered
that although the menthyl glyoxalate 3 was freely soluble the 1,4-dithiane-2,5-diol 4 was not.
We found it necessary to heat the 1,4-dithiane 2,5-diol 4 solution to 40 °C prior to it being
fed into the microreactor channel. After preparation of both starting materials 3 (0.43 M) and
4
(0.22 M) in acetonitrile, they were pumped into a 2 mL microreactor using syringes (10
mL), with a flow rate of 0.05 mL/min each (20 min residence time). The microreactor
temperature was initially maintained at 110 °C for preliminary screening experiments (Figure
3

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2
). The reaction mixture exited the reactor via a 10 PSI back pressure regulator, to afford
DOI: 10.1039/C7OB00480J
intermediate 5 in 88% conversion.
Figure 2 Flow diagram of the synthesis of 5-acetoxy oxathiolane.
After successfully completing the first step, the intermediate 5-hydoxy oxathiolane 5 (0.34
M) was reacted with pyridine and acetic anhydride in acetonitrile (2.17 M) at ambient
temperature using two Little Things Factory reactors (0.2 mL LTF-MX + 1.7 mL LTF-V)
totalling up to a reactor volume of 1.9 mL to provide the isomer mixture 6 in 95%
conversion, which upon recrystallization at -20 °C yielded the desired isomer 7 (48%).
With the knowledge that both steps were feasible, we turned our attention to the development
of a telescoped continuous flow synthesis for the two reaction steps (Figure 2). The first two
reagents (menthyl glyoxalate hydrate (0.43 M) and 1,4-dithaiane 2,5-diol (0.22 M) in
acetonitrile) were pumped into a 2 mL glass reactor (at 110 °C) and the outlet of this reaction
was connected to another LTF-MX reactor (0.2 mL, at RT) which was connected to another
syringe pump with pyridine and acetic anhydride in acetonitrile (2.17 M). The solution was
passed through a LTF-V (1.7 mL, at RT) reactor and the product 6 was collected. Such a
telescoping strategy is a very effective tactic for truncating a multistep synthesis, particularly
when it is to be performed in a continuous flow manner as it eradicates the need for isolation
and purification and allows for drastic changes in reaction conditions from step one to the
next.
In this combined two-step continuous flow process, reaction parameters such as
residence time, the temperature of the two reaction steps and concentration of the reagents
were subsequently investigated to order to maximize the efficiency of the process. We started
by determining the appropriate residence time for the continuous flow synthesis of compound
4

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6
from compounds 3 and 4 at predetermined reaction temperatures and concentrations of 3, 4
DOI: 10.1039/C7OB00480J
and acetic anhydride/pyridine.
1
00
9
8
7
6
5
4
3
0
0
0
0
0
0
0
0
5
10
15
20
25
Residence time (min)
Figure 3 Effect of residence time on conversion to 6 at 0.43 M of 3, 0.22 M of 4, 2.17 M of
Pyridine/Ac O and temperatures (reactor 1 at 90 °C, reactor 2 at 20 °C).
2
It was found that as the residence time increases, the conversion to 6 increased. The data in
the graph shows that it is possible to use reaction times greater than 9.7 min (Figure 3) to
achieve maximum conversion, however, the goal is to use as little time as possible in order to
gain maximum throughput from the reactor per unit time. As such if one transferred these
conditions into an industrial scenario you would select the minimum time of 9.7 min.
Having determined the optimum residence time for the two-step reaction being 9.7 min at the
above-mentioned conditions, we then decided to carry out a detailed temperature study on the
two reaction steps at this residence time; we started our investigation by varying the
temperature of the reaction from 70 °C to 120 °C.
The effect of temperature on the conversion to 6 is illustrated in Figure 4. In summary, it was
found that the maximum conversion (95%) was obtained at 90 °C. Then we investigated the
temperature of the second step, where the maximum conversion was observed in the range
2
0-30 °C (Figure 5); by-products were observed at higher temperatures.
5

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1
00
DOI: 10.1039/C7OB00480J
9
9
8
8
7
7
6
6
5
0
5
0
5
0
5
0
6
0
70
80
90
100
110
120
130
Reactor-1 temperature °C
Figure 4 Effect of reactor 1 temperature variations on the conversion to 6 at constant
residence time 9.7 min and at 0.43 M of 3, 0.22 M of 4 and 2.17 M of Pyridine/Ac O in
2
CAN.
1
00
9
8
7
6
5
4
3
0
0
0
0
0
0
0
1
5
20
25
30
35
40
45
50
Reactor-2 temperature °C
Figure 5 Effect of reactor 2 temperature variations on the conversion to 6 at constant
residence time 9.7 min, reactor 1 temperature 90 °C, at 0.43 M of 3, 0.22 M of 4 and 2.17 M
of Pyridine/Ac O in acetonitrile.
2
The residence time (9.7 min) and temperatures of reactors 1 (90 °C) and 2 (20 °C) were kept
constant; subsequently, we decided to investigate the concentrations of compound 4 and
pyridine/Ac O solutions in acetonitrile. We screened different concentrations of compound 4
2
(0.22 M to 0.41 M) but we found that the conversion was unchanged. Then we turned our
6

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DOI: 10.1039/C7OB00480J
attention to investigating the concentration of Ac O/pyridine solution (2.17 M to 1.07 M) and
2
found that over the range 1.73-2.17 M the maximum conversion (95%) remained constant
Figure 6).
(
1
00
9
9
8
8
7
7
6
6
5
0
5
0
5
0
5
0
1
1.2
1.4
1.6
1.8
2
2.2
2.4
Concentration of Pyridine/Ac O solution (M)
2
Figure 6 Effect of concentration variations of Pyridine/Ac O solution on the conversion to 6
2
at constant residence time 9.7 min temperatures (90 °C & 20 °C) at 0.43 M of 3 and 0.22 M
of compound 4.
In summary, the optimal condition for flow synthesis of 5-acetoxy oxathiolane 6 is 9.7 min
residence time, temperature (reactor one 90 °C, reactor two 20 °C) and concentrations
(
menthyl glyoxalate hydrate 0.43 M, 1,4-dithane 2,5-diol 0.22 M and Ac O/pyridine solution
2
1
0
1
.73 M). But when compared with the batch process (reaction time 12 h, temperature 110
°
C and concentrations of Ac O and pyridine solution 2.17 M) continuous flow gives better
2
results in shorter time. The above detailed optimization conditions, with yields, is shown
below (Scheme 1).
7

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DOI: 10.1039/C7OB00480J
S
S
OH
O
3
HO
4
O
O
O
Ac2O/Pyridine
OH
O
O
O
O
O
Acetonitrile, AcOH
0 min, 90 °C, 88%
OH Acetonitrile
4.7 min, 20 °C, 95%
O
1
S
S
OH
5
6:mixture of isomers
1% TEA in hexane
O
O
O
-20 °C, 36 h, 48%
O
7
O
S
(2R,5R)
Scheme 1 Synthesis of (2R,5R)-5-acetoxyoxathiolane 7.
N-Glycosidation reaction in flow synthesis:
1
1
Another substantial challenge in this work is the N-glycosidation reaction due to the poor
solubility and highly precipitating nature of the silylated nucleobase 8. The cytosine was
silylated with BSA in acetonitrile at 80 °C for about 1 hr in a batch process, after which the
solution became clear. This indicated the formation of silylated product 8. Syringe pumps
were subsequently used to deliver the solutions of nucleobase 8 (0.30 M) and a solution of
pure isomer 5-acetoxy oxathiolane 7 (0.30 M) and pyridinium triflate (0.30 M) in acetonitrile
(Figure 7). Firstly 5-acetoxy oxathiolane 7 and pyridinium triflate were pumped into a 0.2 mL
LTF-MS reactor at 20 °C and the product line was subsequently connected to a T-piece mixer
where the third syringe delivered the nucleobase 8, where they reacted at an elevated
temperature (80 °C) in PTFA tubing reactor (3.8 mL volume, 0.8 mm i.d.). The isolated
products were recrystallized to yield 9 (95%). One experimental detail of significance
regarding silylated nucleobase 8; in order to prevent precipitation and subsequent clogging at
the syringe we used thermal controller wrapping to keep the syringe warm (55-60 °C).
8

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Figure 7 Flow diagram of glycosylation reaction.
After preliminary confirmation of the glycosidation reaction in flow, we further explored the
effect of residence time, temperature, and concentration on the conversion of 9. A residence
time investigation on the conversion of 9 was chosen as a first study, where all the reagents
concentrations were maintained at 0.30 M and temperature fixed at 80 °C. In this reaction, it
was found that as the residence time increases, the conversion of 9 increased (Figure 8). The
maximum conversion was observed at a residence time of between 8.4 and 12.6 min.
1
00
9
9
8
8
7
7
6
6
5
0
5
0
5
0
5
0
4
6
8
10
12
14
Residence time (min)
Figure 8 Effect of residence time on conversion to 9 at all reagents concentrations 0.30 M
and temperature 80 °C in acetonitrile.
9

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Next, we turned our attention to studying the temperature effect on the conversion to 9. The
DOI: 10.1039/C7OB00480J
concentrations of all reagents were kept constant throughout the study at 0.30 M and the
minimum residence time of 8.4 min was selected. Effect of temperature on the conversion to
9
(Figure 9) shows that the conversion reaches a maximum at 80 to 85 °C.
1
00
9
9
8
8
7
7
6
6
5
5
5
0
5
0
5
0
5
0
5
0
6
0
65
70
75
80
85
90
Temperature °C
Figure 9 Effect of temperature variations on the conversion to 9 at constant residence time
.4 min & concentrations of all reagents 0.3 M in ACN.
8
Finally, we turned our attention towards the effect of reagent concentrations on the
conversion to 9. Firstly we chose silyl product 8 concentrations from 0.20 M to 0.37 M at the
optimum residence time (8.4 min) and the temperature (80 °C) was kept constant. As the
concentration of silyl product, 8 increases the conversion increased (Figure 10).
Interestingly, when the concentration of pyridinium triflate catalyst decreased from 0.30 M to
0
.20 M the conversion was not affected (98%), however, the further reduction in a
concentration below 0.20 M did result in lower conversions (Figure 11). In summary, the
optimal condition for this step of the flow synthesis was 8.4 min residence time, temperature
(80 °C) and concentrations (5-acetoxy oxathiolane 7 0.30 M, silyl cytosine 8 0.30 M and
pyridinium triflate 0.20 M). As compared to batch process in flow, we reduced the amount of
catalyst needed and shortened the reaction time giving a higher yield of product.
1
0

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1
00
DOI: 10.1039/C7OB00480J
9
9
8
8
7
7
6
6
5
0
5
0
5
0
5
0
0
.18
0.23
0.28
0.33
0.38
Concentration of 8 (silyl cytosine) (M)
Figure 10 Effect of concentration variations of silyl product 8 on the conversion to 9 at
constant residence time 8.4 min temperature (80 °C) at 0.30 M of all reagents.
1
00
9
9
8
8
7
5
0
5
0
5
0
.05
0.10
0.15
0.20
0.25
0.30
0.35
Concentration of PyOTf (M)
Figure 11 Effect of concentration variations of pyridinium triflate on the conversion to 9 at
constant residence time 8.4 min temperature (80 °C) at 0.30 M of other reagents.
Reduction of a nucleoside precursor (9) with NaBH in flow:
4
As the final step in the synthesis, we successfully deprotected the menthyl group using
12
sodium borohydride in the presence of K HPO in methanol and water (Figure 12). The
2
4
preliminary reaction was done with the nucleoside precursor 9 (0.26 M) dissolved in
1
1

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methanol and K HPO (0.78 M) dissolved in water, and both solutions were pumped into the
2
4
0
.2 mL LTF-MS microreactor at room temperature and the outlet of this reactor connected to
another 0.2 mL LTF-MS, where a solution of NaBH (0.52 M) in water and 0.1 mL of 25%
4
NaOH solution was added. This mixture went through the 1.1 mL LTF-VS and the product
was collected to afford the crude product, which upon recrystallization yielded the final
product 1 (94 %, chiral purity 99.81%).
Figure 12 Reduction of nucleosides 9 with NaBH4.
Having developed the preliminary conditions in flow, we then extended the investigation
towards the residence time, temperature and optimum concentrations of all reagents.
Residence time investigations revealed that the maximum conversion was observed at a
residence time of just 3.3 min (100%).
1
2

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1
00
DOI: 10.1039/C7OB00480J
9
9
8
8
7
7
6
6
5
0
5
0
5
0
5
0
1
3
5
7
9
11
Residence time (min)
Figure 13 Effect of residence time on conversion to 1 at concentrations of (9: 0.26 M,
K HPO : 0.78 M & NaBH : 0.52 M) and temperature 20 °C.
2
4
4
We then performed a detailed reaction temperature study on this reaction at known residence
time. Complete conversion was observed at temperatures of 20 °C and above (Figure 14).
1
00
9
9
8
8
7
7
6
5
0
5
0
5
0
5
0
5
10
15
20
25
30
35
Temperature °C
Figure 14 Effect of temperature variations on the conversion to 1 at residence time 3.3 min
and concentrations of (9: 0.26 M, K HPO : 0.78 M & NaBH : 0.52 M).
2
4
4
At final we were keen to investigate the concentration studies on conversion of 1 from
compound 9 at a residence time 3.3 min and temperature 20 °C. Firstly, the concentration of
K HPO from 0.78 M to 0.26 M studied. At 0.78 gives the maximum conversion then it
2
4
1
3

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remains up to 0.65 M, then it slowly dropped (figure 15). After these parameters in hand, we
DOI: 10.1039/C7OB00480J
have investigated the concentration range of NaBH from 0.52 M to 0.26. The observations
4
are shown in graph (figure 16) and it gives the maximum conversion at 0.52 M and it was
sustained until 0.39 M.
1
00
9
9
8
8
7
7
6
6
5
0
5
0
5
0
5
0
0
.25
0.35
0.45
0.55
0.65
0.75
0.85
Concentration of K HPO (M)
2
4
Figure 15 Effect of concentration variations of K HPO on the conversion to 1 at residence
2
4
time 3.3 min temperature (20 °C) and concentrations of (9: 0.26 M & NaBH : 0.52 M).
4
1
00
9
9
8
8
7
7
5
0
5
0
5
0
0
.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
Concentration of NaBH (M)
4
Figure 16 Effect of concentration variations of NaBH on the conversion to 1 at residence
4
time 3.3 min temperature (20 °C) and concentrations of (9: 0.26 M & K HPO : 0.65 M).
2
4
1
4

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In summary, the optimized conditions for the flow synthesis to lamivudine 1 from nucleoside
DOI: 10.1039/C7OB00480J
precursor 9 are a residence time 3.3 min, temperature (20 °C) and concentrations (nucleoside
precursor 9 0.26 M, K HPO 0.65 M and NaBH 0.39 M) (Scheme 2).
2
4
4
O
O
O
OAc
NH2
NH2
NHSiMe3
N
S
batch process
BSA, ACN
7
O
N
N
N
O
Pyridiniumtriflate, ACN
8.4 min, 80 °C, 95%
O
O
N
H
Me
3
SiO
N
O
80 °C, 1 h.
S
8
9
NH2
K HPO /H O
N
2
4
2
NaBH /MeOH
N
O
4
3
.3 min, 20 °C, 94%
O
1
S
HO
(3TC)
Scheme 2 Synthesis of lamivudine 1.
Conclusion
In summary, we have developed the protocol for the synthesis of lamivudine in semi-
continuous flow synthesis and also reported optimized conditions for each step. The
continuous flow synthesis of 5-acetoxy oxathiolane which is an important tool for synthesis
of lamivudine and also achieved using pyridinium triflate as glycosidation reagent with
higher yields and desired isomer are highlights of this work. By telescoping the condensation
(step 1) and acetylation steps into a single, continuous and uninterrupted reactor network,
thereby avoiding the need to isolate and purity of the intermediate product, the 5-acetoxy
oxathiolane has been significantly streamlined. High yields, reduced reaction times,
temperatures and reduced volumes of reagents enhanced safety and the ease of scalability is
improved in the continuous flow synthesis as compared with traditional batch synthesis. A
more comprehensive investigation towards the large scale process in continuous flow
synthesis of lamivudine is ongoing and will be disseminated when implemented.
Acknowledgements
We wish to thank the National Research Fund (NRF) and NMMU for their financial support.
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Experimental Section
General: Solvents and starting materials were taken from Aldrich and Alfa Aesar companies,
were used as obtained without further purification. The syringe pumps (Chemyx Fusion
Classic syringe pumps) and syringes (1 mL, 5 mL, and 10 mL, SGE Luer lock gas tight glass
syringes) were purchased from Supelco Company. PTFE tubing refers to
Polytetrafluoroethylene tubing. PTFE and Tygon MHLL tubing were purchased from IDEX
Health & Sciences. Used different microreactors such as: LTF-MX (Internal volume 0.2 mL
regularly used for small volumes 0.1-10 mL/min/channel and mix intensive chemicals), LTF-
MS (Internal volume 0.2 mL regularly used for higher volumes 0.5-20 mL/min/channel and
insensitive against blocking), LTF-V (Internal volume 1.7 mL used for reaction residence
time) and LTF-VS (Internal volume 1.1 mL used for reaction residence time for mix
intensive chemicals) were purchased from Little Things Factory-Germany.
The GC- FID analysis was performed using HP-5 MS capillary column (30 mm × 0.25 mm
i.d.) and ultra-high purity nitrogen carrier gas. The samples were analyzed by using the
following method; injector temperature 250 °C, nitrogen flow rate 0.1 mL/min, oven
1
temperature 80 °C for 3 min and then ramped to 320 °C at 50 °C min− and final hold-up
time 5 min. Chiral HPLC analysis was performed on Cyclobond I 2000 (4.6 x 250 mm)
column using 30:70 acetonitrile: buffer (0.025 M ammonium acetate solution).
1
13
19
H-NMR, C NMR, F, NOESY and HSQC spectra were recorded on a Bruker AvanceIII-
00 MHz spectrometer (400 and 100 MHz, respectively) in CDCl , DMSO-d and CD OD.
4
3
6
3
1
Chemical shifts in H NMR spectra were reported in parts per million (ppm) on the ∂ scale
from an internal standard of residual CHCl in CDCl (7.26 ppm), DMSO in DMSO-d (2.50
3
3
6
ppm) or MeOH in MeOD (3.31 ppm). Data are reported as follows: chemical shift,
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet and
br = broad, dd = doublet of doublet, td = triplet of doublet), coupling constant in hertz (Hz),
13
and integration. Chemical shifts of C NMR spectra were reported in ppm from the central
peak of CDCl (77.16 ppm), DMSO-d (39.52 ppm) or MeOD (49.15 ppm) on the ∂ scale.
3
6
1
3
C signals with identical chemical shifts for more than one carbon are specified, and
overlapping peaks for multiple carbons are indicated by a shift range. Melting points were
recorded using a Mel-Temp melting point apparatus and are uncorrected. Combustion
analyses were performed using a CHNS analyzer. Infrared (IR) spectra were recorded on a
Perkin-Elmer Model 2000 FT-IR. Optical rotations were measured at 25 °C in the stated
1
6

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solvent and matched with standard samples. All data generated from these experiments was
DOI: 10.1039/C7OB00480J
plotted using Microsoft Excel to illustrate the trends observed.
(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl) 5-hydroxy-1,3-oxathiolane-2-carboxylate 5
Figure 17: Microreactor setup for the synthesis to 5-hydroxy oxathiolane 5
A solution of L-menthyl glyoxalate hydrate (0.43 M) in acetonitrile was prepared in reagent
bottle A. While reagent bottle B was with a solution containing 1,4-dithiane 2,5-diol (0.22
M) in acetonitrile, and 0.05 mL of acetic acid. This solution B was heated at a temperature
between 40-45 °C for 10 min to get the clear solution prior to performing the reaction. Both
solutions from reagent bottles A and B transferred into 10 mL syringes and pumped through a
T-piece mixing point to ensure proper mixing and into a 2 mL glass microreactor plate heated
at 90 °C at 10 min residence time. A 10 PSI back pressure regulator was fitted onto the fluid
line after the reactor plate so as to keep the system pressurized thus enabling the use of
acetonitrile above its boiling point (figure 17). The reaction mixture was then collected in a
collection vial equipped with a septum (to prevent solvent evaporation). After completion of
the reaction, the solvent was evaporated and the reaction mixture was cooled to 0-5 °C. 1% of
triethylamine in heptane solution drop wise was thereafter added (The addition of TEA in n-
heptane/hexane the rapid interconverison among the stereoisomers happened). The mixture
was stirred at 0-5 °C for 2-3 h and the formation of a precipitate was observed. The isolated
1
7

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solid was filtered and washed with cold n-heptane to give a (1R,2S,5R)-2-isopropyl-5-
DOI: 10.1039/C7OB00480J
methylcyclohexyl 5-hydroxy-1,3-oxathiolane-2-carboxylate 5. Yield- 88%, white colour
1
solid, Rf = 0. 4 (Ethyl acetate : Hexane 1:1), H-NMR in CDCl (400 MHz): ∂ 0.78 (d, J =
3
6
.84, 3H), 0.91(m, 6H), 1.09-1.00 (m, 2H), 1.51-1.42 (m, 2H), 1.70 (d, J = 11.84, 1H), 2.02
(bd, J = 8.76, 2H), 3.15 (dd, J = 4.5, 11.2, 1H), 3.32-3.29 (m, 1H), 4.74 (s, 1H), 5.57 (d, J =
13
8
.76, 1H), 5.96 (d, 1H). C-NMR in CDCl (100 MHz): ∂ 16.27, 20.69, 23.30, 26.16, 31.42,
3
3
4.11, 38.46, 40.35, 46.86, 46.07, 80.20, 101.22, 103.20, 172.18. FT-IR (Neat): 3456, 2956,
2
864, 1731, 1457, 1387, 1288, 1196, 1041, 986. Anal.calcd for C H O S: C 58.30, H 8.39,
14
24
4
S 11.12. Found: C 58.46, H 8.35, S 10.91.
(2R,5R)-((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl) 5-acetoxy-1,
3-oxathiolane-2-
carboxylate 7:
Figure 18: Microreactor setup for the synthesis to 5-acetoxy oxathiolane 7
Reagent bottle A was filled with a solution of (1R,2S,5R)-2-isopropyl-5-methylcyclohexyl 5-
hydroxy-1,3-oxathiolane-2-carboxylate 5 (0.34 M) dissolved in acetonitrile and reagent bottle
B was filled with a stock solution of acetic anhydride and pyridine (1.73 M) in acetonitrile at
room temperature. Both solutions from reagent bottles A and B transferred into 10 mL
syringes and pumped with two separate Chemyx fusion pumps into 0.2 mL LTF-MX
microreactor then passed through 1.7 mL LTF-V microreactor to increase the residence time
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8

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DOI: 10.1039/C7OB00480J
(
4.7 min) at room temperature. The output stream was collected in closed cap vial (figure 18).
After the reaction completion was confirmed by TLC (20% ethyl acetate in hexane) & GC,
the reaction mixture was evaporated under rotary pressure, quenched with dilute HCl (25
mL). The reaction mixture was extracted with ethyl acetate (3 x 25 mL) washed with water
(
50 mL), brine solution (50 mL) and dried over anhydrous Na SO . On concentration of this
2 4
organic layer under the reduced pressure, a brown colored solid comprising a mixture of
diastereomers 6 (95%) was obtained.
The crude product (780 mg) was dissolved in triethylamine (0.1 mL) and hexane (30 mL).
The solution was kept at -20 °C for 36 h after which the desired product (2R,5R)-((1R,2S,5R)-
2
3
-isopropyl-5-methylcyclohexyl) 5-acetoxy-1,3-oxathiolane-2-carboxylate 7 was obtained
75 mg, yield-48%, white colour solid, R = 0.52 (Ethyl acetate : Hexane 3:7), M. P. 99-102
f
1
°
C (Lit 101-102 °C). H-NMR in DMSO-d (400 MHz): ∂ 0.72 (d, J = 6.8, 3H) 0.98-0.81 (m,
6
7
H), 1.03 (t, 2H), 1.47-1.36 (m, 2H), 1.63 (d, J = 11.76, 2H), 1.89 (bd, J = 12.32, 2H), 2.04
1
3
(
s, 3H), 3.38-3.23 (m, 2H), 4.63 (td, J = 3.88, 1H), 5.76 (s, 1H), 6.78 (d, J = 3.8, 1H). C-
NMR in DMSO-d (100 MHz): ∂ 16.19, 20.71, 21.50, 23.24, 26.15, 31.37, 34.13, 37.33,
6
4
1
7
0.40, 47.00, 76.10, 79.90, 99.85, 168.58, 169.64. FT-IR (Neat): 3463, 2959, 2932, 2865,
745, 1730, 1463, 1385, 1287, 1169, 1040, 986, 876. Anal.calcd for C H O S: C 58.16, H
1
6
26
5
.93, S 9.70. Found: C 57.90, H 8.20, S 9.83.
Continuous flow synthesis to 5-acetoxy oxathiolane 7 from L- methyl glyoxalate hydrate
3
:
Figure 19: Complete setup for continuous flow synthesis to 5-acetoxy oxathiolane 7
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DOI: 10.1039/C7OB00480J
Three reagent solutions (stock solutions A, B, and C) were prepared as explained below.
Stock solution A was prepared by dissolving L-menthyl glyoxalate hydrate (0.43 M) in
acetonitrile.
Stock solution B contained 1,4-dithiane 2,5-diol (0.22 M) and acetic acid (0.05 mL) in
acetonitrile. This was first heated to 40 °C for 10 min to form a clear solution prior to use.
Stock solution C comprised of acetic anhydride and pyridine (1.73 M) in acetonitrile.
All three stock solutions were kept in 100 mL glass reagent bottles.
Stock solutions A and B were taken into 10 mL syringes and pumped through the Chemyx
pump at a flow rate of 0.1 mL/min each, these were mixed at T-piece mixing point and
further pumped into a 2 mL glass microreactor plate (reactor-1) which is preheated at 90 °C.
The reactor-1 outlet was connected to a 10 PSI back pressure regulator. The output stream of
reactor 1 (step 1 product 5) was connected to 0.2 mL LTF-MX reactor (This LTF-MX
microreactor was kept in cooling bath to cool the first step solution) which is already
connected to another Chemyx Fusion syringe pump with pyridine and acetic anhydride in
acetonitrile (1.73 M). These solutions were passed through another LTF-V (1.7 mL, at RT)
reactor (reactor-2) to increase the residence time at room temperature and the product was
collected in a closed cap vial (figure 19). After the reaction completion was confirmed by
TLC (30% EtOAc in hexane) & GC, the solvent was evaporated. The reaction mixture was
quenched with ice-cold water (10 mL) and extracted using ethyl acetate. The organic layer
was washed with dilute HCl (10 mL), dried over anhydrous Na SO and concentrated under
2
4
the reduced pressure to give gave 1.25 g (87.4%) compound 6 as brown color solid with the
mixture of four diastereomers. A crude sample of compound 6 (1.0 g) was dissolved in a little
amount of triethylamine (0.1 mL) and hexane (30 mL). This was kept at -20 °C for 36 h after
which the desired product (2R,5R)-((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl) 5-acetoxy-
1
,3-oxathiolane-2-carboxylate 7 was obtained. (450 mg, 45% overall for two steps).
Procedure for the glycosidation reaction in flow:
2
0

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DOI: 10.1039/C7OB00480J
Figure 20: Microreactor setup for glycosidation reaction:
The preparation of silylated cytosine (8) was prepared by batch process (cytosine 1.0 eq and
BSA 2.25 eq in acetonitrile at 80 °C for 1 hour) and this solution was made up into 0.30 M
with acetonitrile in an oven dried, screw-cap volumetric flask.
On the other hand, a solution of (2R,5R)-((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl) 5-
acetoxy-1,3-oxathiolane-2-carboxylate 7 (0.30 M) and pyridiniumtriflate (0.20 M) in
acetonitrile in a separate oven dried, screw-cap volumetric flasks was prepared.
All three prepared solutions were then transferred into 10 mL SGE glass syringes (nucleobase
8
was placed in the syringe wrapped around with the heating tape and maintained at 60 °C
during the reaction to prevent the clogging in the syringe). Initially, the two syringes having
solutions of compound 7 and pyridinium triflate were pumped into 0.2 mL LTF-MX for
mixing then the outlet of this reactor and third syringe pump having nucleobase 8 were
connected at T-piece mix joint where they were mixed and further pumped through the PTFE
tubing (3.8 mL, 1.6 mm (1/16”) o.d. and 0.8 mm i.d.) at a flow rate of 0.15 mL/min each
(residence time 8.4 min). And the PTFE tubing was dipped into an oil bath to keep at 80 °C,
the outlet of this tube connected to 10 PSI back pressure regulator. The reaction mixture was
then collected into a vial equipped with a septum (to prevent solvent evaporation, figure 20).
The solvent in the collected reaction mixture was evaporated under reduced pressure and the
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residue obtained was purified by recrystallization in hexane: ethyl acetate: methanol (2:2:1)
DOI: 10.1039/C7OB00480J
to get the clear white solid 1R,2S,5R)-2-isopropyl-5-methylcyclohexyl(2R,5S)-5-(4-amino-2-
oxo-1,2-dihydro-1-pyrimidinyl)-1,3-oxathialane-2-carbaxylate 9. Yield-95%, white solid, Rf
1
=
0.35 (MeOH: CH Cl 1:9), M. P. 203-206 °C (Lit 202-203 °C), H-NMR in CDCl (400
2
2
3
MHz): ∂ 0.78 (d, J = 6.84, 3H), 0.95-0.89 (m, 6H), 1.10-1.04 (m, 2H), 1.45 (t, , J = 11.82,
2
1
7
1
9
1
H), 1.72 (bd, J = 11.36, 2H), 2.06 (d J = 11.76, 1H), 1.96 (t, J = 6.64, 1H), 3.13 (dd, J = 6.6,
1.80, 1H), 3.54 (dd, J = 4.06, 11.76, 1H), 4.77 (td, J = 4.04, 1H), 5. 47 (s, 1H), 5.92 (d, J =
1
3
.48, 2H), 6.47 (t, J = 5.36, 1H), 8.36 (d, J = 6.72, 1H), C-NMR in CDCl (100 MHz):
3
6.09, 20.72, 21.93, 23.24, 26.09, 31.46, 34.06, 36.32, 40.75, 47.07, 76.70, 78.49, 90.22,
4.86, 141.87, 155.18, 165.22, 169.75. Anal.calcd for C H N O S: C 56.67, H 7.13, N
1
8
27
3
4
1.01, S 8.41. Found: C 56.59, H 7.18, N 10.98 S 8.26.
Reduction of nucleoside precursor (9) by flow synthesis:
Figure 21: Flow setup for the synthesis to lamivudine
Using three oven dried 10 ml SGE glass syringes, a suspension of nucleoside precursor 9
0.26 M) in methanol (syringe A), a prepared solution containing potassium hydrogen
phosphate (0.65 M) in water (syringe B) and sodium borohydride (0.39 M) in water +0.1 mL
5% w/w sodium hydroxide solution (syringe C) was fed into a microreactor plates with the
aid of three Chemyx Fusion syringe pumps. The first two Fluid lines (syringe A and B)
(
2
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2

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leading to mixing and passed through the 0.2 mL LTF-MX microreactor. The outlet of this
DOI: 10.1039/C7OB00480J
reactor and the third syringe pipe from another Chemyx Fusion syringe pump which contains
the NaBH solution were added together at 0.2 mL LTF-MS and further into 1.1 mL LTF-VS
4
microreactor at room temperature (3.3 min residence time). The product was collected at the
end using closed cap vial (figure 21). The reagent solutions were each fed into the system at a
flow rate of 0.15 mL/min residence time 3.3 min. The collected solution was quenched with
dil. HCl (pH 4-4.5), extracted with diethyl ether to remove the menthol. The pH of the
aqueous layer was adjusted to 7 using saturated NaHCO solution. This was then filtered and
3
evaporated under reduced pressure. The crude compound was purified by recrystallization
using hexane: EtOAc: methanol (2:2:1) at 65 °C, slowly taken into ambient temperature to
give the pure compound of lamivudine 1. Yield-94%, white colour solid, Chiral purity
1
9
9.81%, R = 0.30 (MeOH: CH Cl 1:9), M. P. 162-165 °C (Lit 158-160 °C), H-NMR in
f
2
2
DMSO-d (400 MHz): ∂ 3.04 (dd, J = 4.92, 11.68, 1H), 3.40 (dd, J = 5.48, 11.68, 1H), 3.73
6
(dd, J = 5.32, 10.44, 2H), 5.17 (t, J = 4.48, 1H), 5.30 (t, J = 5.76, 1H), 5.73 (d, J = 7.48, 1H),
1
3
6
.20 (t, J = 5.16, 1H), 7.23-7.18 (br, 2H), 7.81 (d, J = 7.44, 1H); C-NMR in DMSO-d (100
6
MHz): 36.74, 63.35, 86.28, 87.03, 94.40, 141.43, 155.18, 166.13; Anal.calcd for
C H N O S: C 41.91, H 4.84, N 18.33, S 13.99. Found: C 41.88, H 4.71, N 18.19 S 13.
8
11
3
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DOI: 10.1039/C7OB00480J
Notes and References:
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. (a) B. Gutmann, D. Cantillo, C. O. Kappe, Org. Biomol. Chem., 2016, 14, 853-857; (b) B.
P. Mason, K. E. Price, J. L. Steinbacher, A. R. Bogdan, D. T. McQuade, Chem. Rev. 2007,
1
07, 2300−2318.
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. (a) K. Geyer, T. Gustafsson, P. H. Seeberger, Synlett, 2009, 15, 2382-2391; (b) R. L.
Hartman, H. F. Jensen, Lab Chip, 2009, 9, 2495-2507; (c) C. Wiles, P. Watts, Eur. J. Org.
Chem., 2008, 10, 1655-1671.
3
. (a) M. W. Bedore, N. Zaborenko, K. F. Jensen, T. F. Jamison, Org. Process Res. Dev.,
010, 14, 432-440; (b) P. Poechlauer, J. Manley, R. Broxterman, B. Gregertsen, M.
2
Ridemark, Org. Process Res. Dev. 2012, 16, 1586−1590.
4
5
. B. Gutmann, D. Cantillo and C. O. Kappe, Angew. Chem. Int. Ed, 2015, 54, 6688-6729.
. (a) C. K. Chu, D. C. Bake, Nucleosides, and Nucleotides as antitumor and antiviral agents,
Plenum, New York, 1993; (b) D. Mandala, W. A. Thompson, P. Watts, Tetrahedron, 2016,
7
6
7
2, 3389-3420.
. G. Romeo, U. Chiacchio, A. Corsaro, P. Merino, Chem. Rev., 2010, 110, 3337-3370.
. Access campaign, medicins sans frontiers, “untangling the web of antiretroviral price
th
reductions” 17 edn., July 2014.
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. (a) R. C. Rizzo, M. Udier-Blagovic, D. P. Wang, E. K. Watkins, M. B. Kroeger Smith, R.
H. Smith, J. Triado-Rives, W. L. Jorgensen, J. Med. Chem., 2002, 45, 2970-2987; (b) J. W.
Corbett, S. S. Ko, J. D. Rodgers, L. A. Gearhart, N. A. Magnus, L. T. Bacheler, S. Diamond,
S. Jeffrey, R. M. Klabe, B. C. Cordova, Med. Chem., 2000, 43, 2019-2030.
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. (a) J. Li, L-X. Gao, M-X. Ding, Synth. Commun., 2002, 32, 2355–2359; (b) R. P. C.
Cousins, M. Mahmoudian, P. M. Youds, Tetrahedron: Asymmetry, 1995, 6, 393-396; (c) J.
Milton, S. Brand, M. F. Jones, C. M. Raynor, Tetrahedron: Asymmetry, 1995, 6, 1903–1906;
(d) D. C. Humber, M. F. Jones, J. J. Payne, M. V. Ramsay, B. Zacharie, H. Jin, A. Siddiqui,
C. A. Evans, H. L. A. Tse, T. S. Mansour, Tetrahedron Lett., 1992, 33, 4625–4628. (e) H.
Jin, A. Siddiqui, C. A. Evans, H. L. A. Tse, T. S. Mansour, M. D. Goodyear, P. Ravenscroft,
C. D. Beels, J. Org. Chem., 1995, 60, 2621–2623; (f) M. F. Caso, D. D’Alonzo, S. D’Errico,
G. Palumbo, G. Annalisa, Org. Lett., 2015, 17, 2626-2629.
10. D. Mandala, P. Watts, ChemistrySelect, 2017, 2, 1102-1105.
11. A. Snidy, M. W. Bedore, T. F. Jamison, Angew. Chem. Int. Ed. 2011, 50, 2155 –2158.
12. M. D. Goodyear, M. L. Hill, J. P. West, A. J. Whitehead, Tetrahedron Letters, 2005, 46
8535–8538.
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DOI: 10.1039/C7OB00480J
Mandala table of contents entry
We report the first semi-continuous flow synthesis of lamivudine, an antiretroviral drug used in the
treatment of HIV/AIDS.
O
N
NH
2
HO
N
O
S