Chemical Papers (2024) 78:3381–3387

https://doi.org/10.1007/s11696-023-03278-1

SHORT COMMUNICATION

One‑pot green synthesis of pidotimod in water

Truong‑Thanh Tung1,2 · Nguyen T. H. Yen1 · Pham B. Phuong1 · Nguyen C. Huy1 · Pham H. Ngoc1 · Dinh N. Minh1 ·
Pham D. Tung1 · Nguyen‑Hai Nam3

Received: 25 October 2023 / Accepted: 14 December 2023 / Published online: 23 January 2024
© The Author(s), under exclusive licence to the Institute of Chemistry, Slovak Academy of Sciences 2024

Abstract
Pidotimod is an immunostimulant drug that plays an important role in respiratory diseases. There are several methods for the
preparation of pidotimod but require complicated procedures. Herein, we report the one-pot, direct synthesis of the immu-
nostimulant drug pidotimod via the reaction of thiazolidine-4-carboxylic acid and L-pyroglutamic acid in water without the
protection step. The reaction runs well at room temperature and simple purification process. We believe that this result could
bring a new green method for application in the pharmaceutical industry.
Graphical abstract

Keywords Pidotimod · Green synthesis · Peptide · Synthesis · Aqueous media

Introduction

Pidotimod is an immunostimulant drug that has been used in

several countries as a supplement for the treatment of respir-
atory diseases (Beatrice et al. 2015; Gian and Chiara 2013;
In memorial to Professor John Nielsen, University of Copenhagen.
Ma et al. 2010; Susanna et al. 2015). Pidotimod prompts
* Truong‑Thanh Tung dendritic cell maturation, which releases pro-inflammatory
[email protected] mediators including TNF-α, resulting in the recruitment of
https://tunglab.com inflammatory cells (Cinzia et al. 2009). Pidotimod is able
1 to increase the activity of immunoglobulins including IgA,
Faculty of Pharmacy, PHENIKAA University, Yen Nghia,
Ha Dong, Hanoi 12116, Vietnam IgM, and IgG which resulted in adaptive immune responses
2 (Beatrice et al. 2015; Sonia et al. 2013). Recently, this
PHENIKAA Institute for Advanced Study (PIAS)
PHENIKAA University, Yen Nghia, Ha Dong, Hanoi 12116, compound is undergoing several clinical trials for treating
Vietnam COVID-19 (Désirée 2020; Pierachille 2021).
3
Department of Medicinal Chemistry, Hanoi University The demand for this drug is very high in many countries.
of Pharmacy, Hanoi, Vietnam However, the recent synthesis pathways of this drug require

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3382 Chemical Papers (2024) 78:3381–3387

4–5 steps (Fig. 1) with the use of organic solvents, high tem- Optimization of two steps pathways
peratures, or other harsh conditions, which are harmful to the
environment (Jiangsu Sinopep 2016; Hu et al. 2009; Long Firstly, the thiazolidine-4-carboxylic acid was prepared from
et al. 2018; Zhang et al. 2015; Zhang et al. 2018; Zhejiang L-cysteine and formaldehyde (37%) in water by simple mix-
2011). For example, the four-step synthesis of pidotimod via ing. In a 5-mL round-bottom flask with a stir bar were added
the reaction of pyroglutamic acid and ester L-thiazolidine L-cysteine (100 mg, 1 equivalent), aq. 37% formaldehyde
4-carboxylic acid requires the presence of boronic acid and (0.065 mL, 1 equivalent), and H ­ 2O (2 mL). The mixture was
dichloromethane as solvent in the absence of water (Zheji- well-mixed at the speed of 200 rpm. After the 1 h, a small
ang 2011). In three published patents, pidotimod was syn- amount of ethanol (20% v/v) was added and the mixture was
thesized via three steps using expensive metal ion resin and kept at 0–5 °C for an additional 1 h. The precipitated thia-
non-eco-friendly solvent (such as DMF) (Jiangsu Sinopep zolidine-4-carboxylic acid was filtered and dried to afford a
2016; Hu et al. 2009; Zhang et al. 2015; Zhang et al. 2018). clean product (80 mg, 72%).
Another reported method reports four steps from L-cysteine The reported methods for this reaction require a heating,
(Zhang et al. 2018) or five steps for industrial scale (Long or organic solvent (Yu et al. 2011); however, we observed
et al. 2018). Common for all reported methodologies are that that the stirring speed plays a crucial role in this reaction.
they require four to five steps from L-cysteine and the pro- More specifically, the reaction can occur at room tempera-
tection of L-thiazolidine-4-carboxylic acid before coupling ture with constant stirring of 600 rpm (reaction was per-
reaction with pyroglutamic acid can take place (Jiangsu Sin- formed in 5-mL round-bottom flask, using 10 mm × 5 mm
opep 2016; Hu et al. 2009; Long et al. 2018; Magni et al. (with pivot ring) octagonal round white PTFE magnetic stir-
1994; Poli 1989a, 1989b; Poli 1990; Poli et al. 1996; Zhang ring bar (Sigma) to afford a good yield (93%, Table 1, entry
et al. 2015; Zhang et al. 2018; Zhejiang 2011). To the best 3). Increasing the amount of formaldehyde also increased the
of our knowledge, there is no existing method for one-pot reaction yield to 95% (Table 1, entry 8). However, increas-
and/or two-step pathways of this compound in the literature. ing the speed resulted in a slightly reduced yield (Table 1,
In our effort to green chemistry, herein, we report the entry 2–6). This observation can be explained by the effect
simple, green synthesis approach for one-pot synthesis of of stirring speed and the volatile properties of formaldehyde.
pidotimod at room temperature in water. Also, heating the reaction resulted in poor yield as reported
in Table 1, entry 4.

Fig. 1  Reported methods for the

synthesis of pidotimod (a) and
our approach for the one-pot
synthesis of pidotimod (b)
Chemical Papers (2024) 78:3381–3387 3383

Table 1  Optimization of the synthesis of thiazolidine-4-carboxylic acid

No. L-cysteine (equiv.) Formaldehyde Solvent Speed (rpm) Product (mg) Yield%*
(equiv.)

1 1 1 H2O 200 80.1 72

2 1 1 H2O 400 86.8 79
3 1 1 H2O 600 102.2 93
4 1 1 H2O 600** 37.4 34
5 1 1 H2O 800 84.6 77
6 1 1 H2O 1200 96.7 88
7 1 1 H2O, MeCN (2:1) 600 86.7 79
8 1 2 H2O 600 104.4 95

*Reaction was run at least 3 times. Yields are average values; ** heating up to 40 °C with closed cap. Reaction was performed in a 5-mL round-
bottom flask, using 10 mm × 5 mm (with pivot ring) octagonal round white PTFE magnetic stirring bar (Sigma)

Thiazolidine-4-carboxylic acid will be precipitated by more than 95% purities (Rf = 4.151 min) with two impurity
adding the ethanol correspond to a 20% (v/v) and keeping peaks at 2.719 and 3.547 min (Fig. 2).
the reaction mixture at 0–5 °C making the purification pro- Thiazolidine-4-carboxylic acid is a key intermediate in
cess very simple (if needed). In order to identify the purity our synthesis, and therefore, its identity was confirmed by
1
of the product, we performed the HPLC analysis using the H NMR. Figure 3 shows key proton-proton couplings and
Agilent Infinity 1260 system; column Agilent XDB-C18 their J-values.
(4.6 × 250 nm, 5 µm); column temperature: 40 °C; and Next, we optimized the novel method for direct coupling
solvent condition: from H ­ 2O to 5% MeOH over 10 min, reaction of thiazolidine-4-carboxylic acid and L-pyroglu-
0.8 mL/min flow rate, and UV detector (200 nm). HPLC tamic acid in water. This reaction normally requires a pro-
analysis of the crude sample showed that the compound is tection step due to the presence of an active carboxylic acid
group in both reactants. In this work, we proposed to use the

Fig. 2  HPLC of reaction mix-

ture of thiazolidine-4-carboxylic
acid (Rf = 4.151 min) and two
impurity peaks at 2.719 and
3.547 min
3384 Chemical Papers (2024) 78:3381–3387

added, and the mixture was stirred for 2 h to afford the final
product. Pidotimod was obtained via crystallization by add-
ing 10% v/v of MeCN to the reaction mixture. The overall
step-by-step reaction is demonstrated in Fig. 4.
As shown in Table 2, using the excess amount of coupling
reagent EDC.HCl may reduce the reaction yield (Table 2,
entry 2 & 3). In addition, heating the mixture resulted in
poor yield and decomposition (as observed in TLC, Table 2,
entry 4). This observation can be explained by the hydrolysis
of reactants and products in the presence of a strong base
and water.

One‑pot synthesis of pidotimod

The one-pot synthesis of pidotimod was performed by a

combination of the above-optimized reactions. More spe-
cifically, the intermediate thiazolidine-4-carboxylic acid
was prepared from L-cysteine and formaldehyde in water at
Fig. 3  Key NMR couplings of thiazolidine-4-carboxylic acid room temperature. Then, the reaction mixture (solution A)
was used directly for the next step. In another round-bottom
flask, pyroglutamic acid was treated with sodium hydroxide
salt version of pyroglutamic acid as a directing group and in water, and then EDC.HCl was added and stirred for an
pre-activated by water-soluble coupling reagent. As reported additional 5 min (solution B). Finally, solution A was added
previously, EDC.HCl was discovered by our group to be the dropwise to solution B and stirred until completion (via
best reagent (Tung and John 2021). Firstly, pyroglutamic TLC). Pidotimod was obtained via crystallization by add-
acid (50 mg, 1 equiv) was treated with aq. 4 M sodium ing 10% v/v of MeCN to the reaction mixture (See procedure
hydroxide (0.10 mL, 1 equiv) at 5 °C for 5 min in 1 mL in the reference). The results are summarized in Table 3.
water, followed by EDC.HCl (74 mg, 1 equiv) and stirred for As shown in Table 3, pidotimod was one-pot synthesized
an additional 5 min to afford the activated version of acid. from various scales (100–500 mg) of L-cysteine with moder-
Then, thiazolidine-4-carboxylic acid (51.5 mg, 1 equiv) was ate yield (2-step yield). Double time was observed for the

Fig. 4  Proposed step-by-step

reaction of direct coupling of
thiazolidine-4-carboxylic acid
and pyroglutamic acid in water
Chemical Papers (2024) 78:3381–3387 3385

Table 2  Direct coupling of thiazolidine-4-carboxylic acid and pyroglutamic acid in water

No. Thiazolidine-4-carboxylic acid mg Pyroglutamic acid (equiv) EDC.HCl (equiv) Product (mg) Yield%*
(equiv)

1 50 mg (1) 1 1 77.5 82
2 50 mg (1) 1 2 68.0 72
3 50 mg (1) 1 3 67.2 71
4 50 mg (1) 1 1 31.2 33**

*Reaction was run at least 3 times. Yields are average values

**Heating. Reaction was performed in a 5-mL round-bottom flask, using 10 mm × 5 mm (with pivot ring) octagonal round white PTFE magnetic
stirring bar (Sigma); the product was isolated by crystallization

Table 3  One-pot synthesis of pidotimod*

No L-cysteine Reaction time Solvent Stirring speed Product (mg) Yield**

1 100 mg 2 H 2O 600 137.2 68

2 300 mg 4 H 2O 600 375.1 62
3 500 mg 4 H 2O 800 595.1 59

*The reaction product was isolated by crystallization in the mentioned yield, and with identical purity as is seen in the NMR and HPLC data
presented in the supporting document
**One-pot yield, reaction was run at least 3 times. Yields are average values

larger amount of starting material (Table 3, entry 2 &3). In

addition, the stirring speed was increased to 800 (Table 3,
entry 3) due to the large amount of product precipitated dur-
ing the course of reaction. We are currently applying this
protocol to the flow reactor system for 50–100 g scales to
overcome the limitation of the reaction (data not shown),
and the detailed experiment will be published in due course.

Fig. 5  Shows structural variations cause rotamers phenomenon in

Comparison of synthetic and authentic 13
C-NMR
pidotimod

The synthetic material was compared to the authentic pidoti- The 1H and 13C comparison is listed in Table 4 and Fig. 6
mod via TLC (see supporting information) and NMR. (see supporting information for the copy of NMR).
Interestingly, there are a small number of rotamer peaks In summary, we have successfully synthesized pidotimod
observed in 13C-NMR of our synthesized pidotimod and from L-cysteine via a one-pot synthesis pathway at room
authentic pidotimod (see supporting information). This phe- temperature in only water (see workup procedure below and
nomenon is due to the transition state between amide rotam- supporting information). The main novelties of our reported
ers in NMR solvent (Fig. 5), which has been widely studied method are that compound can be prepared from very cheap
(Crimella et al. 1994; Magni et al. 1994; Xiaorui et al. 2015). material L-cysteine via a one-pot two-step pathway using
3386 Chemical Papers (2024) 78:3381–3387

Table 4  1H and 13C NMR No. Synthetic Authentic Synthetic Authentic
of synthetic and authentic 1 13
pidotimod (SML1335, Sigma- H-NMR (ppm) C-NMR (ppm)
Aldrich)
1 7.82 (s, 1H) 7.82 (s, 1H) 177.31 177.30
2 4.84–4.88 (m, 1H) 4.85–4.88 (m, 1H) 170.95 170.95
3 4.26–4.61 (m, 3H) 4.26–4.61 (m, 3H) 170.25 170.25
4 3.10–3.13 (dd, J = 4 Hz, 1H) 3.11–3.13 (dd, J = 4 Hz, 1H) 61.12 61.12
5 2.33–2.38 (dd, J = 8.5 Hz, 1H) 2.33–2.38 (dd, J = 8.5 Hz, 1H) 53.80 53.80
6 2.09–2.12 (m, 4H) 2.09–2.12 (m, 4H) 47.26 47.26
7 31.76 31.76
8 28.82 28.82
9 23.85 23.85

Fig. 6  Comparison of 1H and 13C NMR of synthetic and authentic pidotimod

only water as solvent. In addition, the direct coupling of precipitated pidotimod. Characterization data: thiazolidine-
thiazolidine-4-carboxylic acid and pyroglutamic acid to form 4-carboxylic acid: 1H NMR (500 MHz, DMSO-d6) δ (ppm):
pidotimod, without the protection of the carboxylic groups, 4.22 (d, J = 9 Hz, 1H, Hd), 4.03 (d, J = 9 Hz, 1H, He), 3.83
was established for the first time. We believe that this report (t, J = 6.5 Hz, 7 Hz, 1H, Ha), 3.09 (dd, J = 7 Hz, 10 Hz,
will provide an alternative application for drug manufactur- 1H, Hc), 2.83 (dd, J = 6.5 Hz, 10 Hz, 1H, Hb). Pidotimod:
ing with respect to environmental protection. Mp = 195–197 °C; Rf = 0.55 (DCM/MeOH = 5/1); EI-MS
m/z: 242.28 [M-2H]; IR (KBr) (cm-1): 3276 (0H), 2880,
Workup procedure for one‑pot reaction 1705, 1656, 1625, 1250; 1H NMR (500 MHz, DMSO-
d6) δ 7.82 (s, 1H), 4.84–4.88 (m, 1H), 4.26–4.61 (m, 3H),
The final reaction mixture was diluted 10% v/v of MeCN, 3.10–3.13 (dd, J = 4 Hz,1H), 2.33–2.38 (dd, J = 8.5 Hz, 1H),
keeping the reaction mixture at 0–5 °C to afford clean 2.09–2.12 (m, 4H).
Chemical Papers (2024) 78:3381–3387 3387

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